i

PHOSPHOROUS MANAGEMENT FOR BIOFORTIFICATION OF

ZINC IN MAIZE (ZEA MAYS L.) GROWN ON CALCAREOUS

SOILS

By

MUHAMMAD IMRAN

M.Sc. (Hons.) Soil Science

2012–ag–220

A dissertation submitted in partial fulfillment of the requirements for

the degree of

DOCTOR OF PHILOSOPHY

in

Soil Science

DEPARTMENT OF SOIL SCIENCE

FACULTY OF AGRICULTURAL SCIENCES AND TECHNOLOGY

BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN (60800)

PAKISTAN.

2017

ii

To,

The Controller of Examinations,

Bahauddin Zakariya University,

Multan.

We, the supervisory committee, certify that the contents and form of thesis submitted by Mr.

Muhammad Imran, Regd. No. 2012–ag–220 have been found satisfactory and recommend that it be

processed for evaluation by the External Examiner(s) for the award of degree.

SDUPERVISOR Dr. Abdur Rehim

Assistant Professor

Department of Soil Science

Bahauddin Zakariya University, Multan

iii

DECLARATION

I hereby declare that the contents of the thesis “Phosphorous Management for Biofortification of

Zinc in Maize (Zea mays L.) Grown on Calcareous Soils” are product of my own research and no

part has been copied from any published source (except references, standard mathematical or generic

models / equations / formulate / protocols, etc.). I further declare that this work has not been

submitted for award of any other degree. The university may take action if the information provided

is found inaccurate at any stage. I am aware that in case of any default, I will be proceeded against as

per HEC plagiarism policy.

Muhammad Imran

iv

Dedicated To

My Parents

Hearts that are full of unconditional love

“A constant source of inspiration and Duaa, not only

during the entire period of my studies

but also throughout my life...”

v

ACKNOWLEDGEMENTS

I bow my head, with all the humility and modesty, before Allah Almighty, the Creator of everything,

Who imbibed in man a thirst for knowledge and desire to explore the unknown for the benefit of

mankind; for granting me courage, patience, and perseverance to undertake and accomplish this prime

task of research work which could not be possible without His Blessings and Grace. May peace and

Blessings of Allah Almighty be upon His all Prophets including Muhammad (PBUH), His last

messenger, who is the fountain of knowledge and guidance for the salvation of mankind in this world

and the hereafter.

I feel paucity of words and phrases to express my deepest and eternal gratitude to my supervisor, Dr.

Abdur Rehim, Assistant Professor, Department of Soil Science, Bahauddin Zakariya University,

Multan. He was very kind and generous in his auspicious help, scholarly guidance, great encouragement

and affectionate support that was a colossal source of inspiration and solace for me in the quest of my

PhD program. At the same time, I don‟t have words to utter for Dr. Shahid Hussain, Assistant

Professor, Department of Soil Science, Bahauddin Zakariya University, Multan. He always motivated me

during my PhD and I don‟t forget his valuable suggestions in my whole dissertation.

I extend my sincere thanks to my respectable Chairman, Dr. Muhammad Zafar ul Hye, Associate

Professor, Department of Soil Science and other faculty members: Dr. Muhammad Abid, Dr. Niaz

Ahmed, Dr. M. Arif Ali, Dr. M.F. Qayyum and Dr. Muhammad Aon for their moral support,

innovative teaching and positive criticism to improve this draft. I would also like to pay my thanks to Dr.

Bernard Wehr, The School of Agriculture and Food Science, The University of Queensland, Australia

for their consistent help in improving my experimental plans.

I owe a heartfelt debt of gratitude to my Friends; Rizwan Yaseen and Nadeem Sarwar who endured all

the strains and stress during the course of my study while their hearts were beating with prayers for my

success. I dedicate this dissertation to my most affectionate parents, sweet sisters, and cute nephews and

nieces to cheer my life during the highs and lows in this course. Last but not the least, I am grateful to

Dr. Iram Imran; my better half, for motivating me and keeping my morale high.

My laboratory colleagues and friends include Khurram Shahzad, Asif ur Rehman, Sadiq Naveed

Kalrou and Amjad Bashir, who helped me in crop harvesting and experimental setup, deserve my

heartfelt thanks for their unconditional support. I can never forget the sweet memories of the time spent

with them.

I am also thankful to International Plant Nutrition Institute (USA) for a partial support granted

through IPNI Research Scholar Award 2015.

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May Allah Almighty infuse me with the energy to fulfill their inspirations and expectations and modify

my competence. May Allah bless them all with lives full of happiness and contentment (Aameen).

Muhammad Imran (2012–ag–220)

vii

List of Publications and Award from the Dissertation Research

Publications in Referred Journals

1. Imran, M., A. Rehim, N. Sarwar and S. Hussain. 2016. Zinc Bioavailability in Maize Grains

in Response of Phosphorous-Zinc Interaction. Journal of Plant Nutrition and Soil Science

179:60-66. DOI:10.1002/jpln.201500441.

2. Imran, M., A. Rehim, S. Hussain, M.Z. Hye and H. Rehman. 2016. Efficiency of zinc and

phosphorous applied to open-pollinated and hybrid cultivars of maize. International Journal

of Agriculture and Biology. 18:1249-1255. DOI: 10.17957/IJAB/15.0239.

3. Imran, M. and A. Rehim. 2017. Zinc Fertilization Approaches for Agronomic

Biofortification and Estimated Human Bioavailability of Zinc in Maize Grain. Archives of

Agronomy and Soil Science. 63: 106-116. DOI:10.1080/03650340.2016.1185660.

Abstracts in Conferences Proceedings

1. Imran, M., A. Rehim and S. Hussain. 2016. Zinc fertilization for biofortification and

estimated zinc bioavailability in maize grain. p 37. Abstract published in 16th

international

congress of soil science on healthy soils for food security held on 15-17 March at University

of Arid, Agriculture, Rawalpindi, Pakistan.

2. Imran, M., A. Rehim, N. Sarwar and R. Yaseen. 2016. Zinc Bioavailability in Maize Grains

in Response of Phosphorous-Zinc Interaction. p 123. Abstract Published in 2nd

international

conference on malnutrition on Malnutrition Kills! Can we see the writing on the wall? held

on 7-8 April at Bahauddin Zakariya University, Multan, Pakistan.

3. Imran, M., A. Rehim and S. Hussain. 2016. Modeling temporal variations in adsorption of

applied zinc and phosphorous in alkaline-calcareous soils. pp 79. Abstract published in

international joint conference for the New Zealand Society of Soil Science and Soil Science

Australia on Soil, a balancing act down under held on 12-16 December at Queenstown, New

Zealand.

Popular Article

1. Imran, M. and A. Rehim. 2015. Zinc Biofortification of Maize crop by Managing

Phosphorous. The Pakistan Times. December 12, 2015

Award

1. Selected for prestigious International Plant Nutrition Institute (IPNI) Research

Scholar award for the year 2015 from USA

viii

Table of Contents

No. Title Page

Acknowledgments v

List of Publications from the Thesis Research vii

List of Figures x

List of Tables xi

Abstract xii

CHAPTER 1 Introduction 1

CHAPTER 2 Review of Literature 6

2.1 Extent of Zinc Deficiency in Soil 6

2.2 Extent of Zinc deficiency in Pakistan 7

2.3 Zinc Forms and Behavior in soils 8

2.4 Zinc Pools in Soils 10

2.5 Functions of Zinc in Plants and Humans 11

2.6 Zinc Deficiency in Plants 12

2.7 Factors Affecting Zinc Bioavailability 13

2.7.1 Soil pH 13

2.7.2 Organic Matter 14

2.7.3 Calcareousness (CaCO3 Contents) 15

2.7.4 Salt Affected Soils 15

2.7.5 Clay Minerals 16

2.8 Zinc Bioavailability Estimation in Human Diet 17

2.9 Zinc Fertilization Approaches to Maize 18

2.10 Physiological Functions of Zinc in Plants 21

2.10.1 Photosynthesis 22

2.10.2 Zinc in Enzyme Function 22

2.10.3 Superoxide Dismutase 22

2.10.4 Zinc in Carbohydrate Metabolism 23

2.10.5 Zinc in Protein Metabolism 23

2.10.6 Zinc in Membrane Integrity 23

2.11 Mechanism of Zinc Uptake in Plants 24

2.12 Zinc Translocation in Plants 25

2.13 Secretions of Chelating Agents and Organic Acids 26

2.14 Genotypic Variation of Zinc in Plants 26

2.15 Zinc Uptake Efficiency in Plants 26

2.16 Extent of Soil Deficient in Phosphorous 27

2.17 Phosphorous Utilization Efficiency in Plants 28

2.18 Zinc Interaction with Phosphorous 28

CHAPTER 3 Zinc adsorption characteristics of three calcareous soils at varying

phosphate level; an incubation study

31

3.1 Abstract 31

3.2 Introduction 31

3.3 Materials and Methods 33

3.4 Results 36

3.5 Discussion 43

3.6 Conclusions 46

ix

CHAPTER 4 Efficiency of Zinc and Phosphorous applied to open-pollinated and

hybrid cultivars of maize

47

4.1 Abstract 47

4.2 Introduction 47

4.3 Materials and Methods 49

4.4 Results 51

4.5 Discussion 58

4.6 Conclusions 63

CHAPTER 5 Comparison of Zinc and Phosphorous rates in maize hybrids and

non-hybrids for Zinc biofortification under field conditions

64

5.1 Abstract 64

5.2 Introduction 64

5.3 Materials and Methods 66

5.4 Results 69

5.5 Discussion 75

5.6 Conclusions 80

CHAPTER 6 Effectiveness of different Zinc application methods in maize for

grain Zinc biofortification

81

6.1 Abstract 81

6.2 Introduction 81

6.3 Materials and Methods 84

6.4 Results 87

6.5 Discussion 92

6.6 Conclusions 96

CHAPTER 7 Summary 97

Literature cited 101

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LIST OF FIGURES

Figure No. Figure Caption Page

No. Figure 2.1 Extent of soil zinc deficiency in the world 07

Figure 2.2 Dynamics of Zinc in the Soil 10

Figure 2.3 Release and fixation of zinc in soil–plant system 16

Figure 2.4 Approaches for zinc biofortification of major cereal grain 21

Figure 3.1 Temporal changes on P adsorption under different Zn and P

rates.

39

Figure 3.2 Temporal changes on Zn adsorption under different Zn and P

rates.

40

Figure 3.3 Lineweaver-Bulk expression of P adsorption data in three

different textured soils

42

Figure 3.4 Lineweaver-Bulk expression of Zn adsorption data in three

different textured soils

44

Figure 4.1 Zinc and Phosphorus uptake of maize cultivars affected by

applied P and Zn levels.

54

Figure 4.2 Utilization efficiency of Zn and P in maize cultivars affected by

applied P and Zn levels.

56

Figure 4.3 Agronomic Zn and P efficiency of maize cultivars at control

(without) and recommended (with) rates of P and Zn.

57

Figure 4.4 Physiological Zn and P efficiency of maize cultivars at control

(without) and recommended (with) rates of P and Zn.

60

Figure 4.5 Apparent Zn and P recovery efficiency of maize cultivars at

control (without) and recommended (with) rates of P and Zn.

61

Figure 5.1 Cob length (A) and grain yield (B) of maize genotypes Neelam

and DK-6142 after different Zn and P application

70

Figure 5.2 Straw yield (A) and 1000-grain weight (B) of maize genotypes

Neelam and DK-6142 after different Zn and P application

71

Figure 5.3 Phosphorus concentrations in grains (A) and stover (B) of maize

genotypes Neelam and DK-6142 after different Zn and P

application

72

Figure 5.4 Zn concentrations of grain (A) and stover (B), and Zn content

per seed (C) of maize genotypes (Neelam and DK-6142) at

different Zn and P rates.

74

Figure 5.5 Phytate concentrations of grains (A) and phytate content per

seed (B) of maize genotypes Neelam and DK-6142 after

different Zn and P application

76

Figure 5.6 Grain [phytate] : [Zn] ratio (A) and estimated Zn bioavailability

(B) in grains of maize genotypes Neelam and DK-6142 after

different Zn and P application

79

Figure 6.1 Phytate concentration (6.1a) and Grain [phytate]:[Zn] ratio

(6.1b) in grain of maize crop and differentially treated with Zn

91

Figure 6.2 Grain protein concentration (6.2a) and Estimated Zn

bioavailability (6.2b) in grain of maize crop and differentially

treated with Zn

93

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LIST OF TABLES

Table No. Table Caption Page

No. Table 2.1 Potential solutions to overcome zinc deficiency in humans 19

Table 3.1 Physico-chemical properties of soils used in the incubation trial 34

Table 3.2 Analysis of variance for P and Zn at varying Zn and P levels 35

Table 3.3 Parameters of Lineweaver-Bulk and Michaelis-Menten models

for adsorption of Zn and P on differently textured soils

38

Table 4.1 Physical and chemical properties of soil used in the experiment 49

Table 4.2 Effect of Zn and P applications on growth attributes of four

maize cultivars

52

Table 4.3 Effect of Zn and P applications on Zn and P concentration in

four maize cultivars

53

Table 5.1 Physical and chemical soil properties 67

Table 6.1 Physical and chemical properties of soil used in the experiment 84

Table 6.2 Effect of various Zn treatments on the growth attributes of

maize plant

87

Table 6.3 Effect of various Zn treatments on the yield attributes of

maize plant

89

Table 6.4 Effect of various Zn treatments on the nutritional attributes

in maize grain

90

xii

ABSTRACT

Zinc (Zn) and phosphorus (P) deficiencies are widespread in most of the alkaline calcareous soils of

Pakistan. These both caused the significant reduction in their bioavailability and uptake by the plants

from the soil. Most of the applied Zn and P are adsorbed on soil colloidal surfaces short after their

applications in the soil. As these both are essential plant nutrients and interact with each other in

soil–plant systems. Therefore, such interactions may cause deficiency of one of the nutrients

interacting with each other if interactions are antagonistic. However, papers about positive

interaction between Zn–P are also present, but the exact mechanism is still unknown. Excessive P

fertilizer to the soil decreased plant bioavailable Zn from the soil. There is the dire need to increase

the Zn concentration in maize grain by managing P fertilization. Maize (Zea mays L.) is generally

low in bioavailable Zn; agronomic biofortification with Zn is an easy and rapid solution to combat

Zn deficiency problems in humans. For this purpose, series of experiments were conducted focusing

on these major issues: 1) fixation and retention capacities of Zn and P in different textured soils; 2)

Efficiencies of Zn and P applied to open–pollinated and hybrid maize cultivars; 3) phosphorous

management to increase Zn bioavailability in maize grain; 4) changes in phytate and Zn human

bioavailability over different Zn and P fertilization rates; 5) better Zn fertilization approach for

maximum plant growth, yield and grain Zn density; 6) to improve nutritional status of maize grain.

In first experiment, different levels of Zn (0, 4, 12 mg kg−1

soil) and P (0, 40, 120 mg kg−1

soil) were

applied in all possible combinations to three differently textured soils (clay, loam and loamy sand) in

plastic lined pots. The soils were incubated and soil samples were extracted at different time

intervals (20, 40, 60, 90 and 120 days) at 25+5 °C. Adsorption of Zn and P in these soils was

measured by AB–DTPA method. Adsorption of Zn and P depended on their applied rate, inherent

soil physico–chemical properties and time. Percent adsorption of added Zn and P increased with

increasing clay content and this adsorption behavior of soil was best described by Michaelis-Menten

equation. Most of the fixation of Zn and P took place within 65–75 days after their addition in soils.

Labile Zn in soil was reduced by P application but labile P was not as significantly affected by Zn

application. In second experiment, a study was conducted to check the interactive effects of different

levels of Zn (0, 9 mg kg-1

soil) and P (0, 40 mg kg-1

soil) on growth, mineral acquisition and nutrient

efficiencies of 04 maize cultivars (DK–6142, P1543, Neelam and Afghoi) grown in pots. Maize

cultivars significantly differed for growth, nutrient uptake and nutrient efficiencies in all treatments.

Combined Zn+P fertilization significantly increased dry matter production, nutrient acquisition and

xiii

efficiencies as compared with unfertilized control. Agronomic, physiological and recovery

efficiencies of P and Zn were increased in Neelam, Afghoi and DK-6142 if the other nutrient was

supplied to soil. Afghoi and DK–6142 were more responsive in agronomic, physiological, nutrient

and apparent Zn and P recovery efficiency than other varieties. With the application of the other

nutrient to P1543, physiological Zn and P efficiencies were decreased, agronomic Zn and P

efficiencies remained almost unchanged and recovery efficiency of Zn and P was increased. On

average, Neelam, Afghoi and DK-6142 were more efficient for the applied nutrients than P1543.

Efficiency of nutrients did not relate to open–pollinated or hybrid cultivars rather it varied with

genetic makeup which ultimately effect fertilizer use efficiency. In third trial, a field experiment was

conducted to investigate the interactive effect of Zn (0, 16 kg ha–1

) and P (0 and 60 kg ha–1

) on

growth, yield and grain Zn concentration of two best maize genotypes obtained from the previous

experiment i.e. Neelam (open–pollinated) and DK–6142 (hybrid) grown in field. Growth and yield

of both maize genotypes were increased significantly (P≤0.05) by the application of Zn and P

treatments compared with control, but Zn+P was more effective than their sole application. As

compared to control, combined application of Zn+P increased grain Zn and P concentrations by 52%

and 32%, respectively, averaged for the two genotypes. Sole application of P decreased grain Zn

concentration and Zn bioavailability by 10% and 11% respectively, over unfertilized control.

Application of P and Zn particularly in combination decreased the grain [phytate] : [Zn] molar ratio

and increased the estimated human Zn bioavailability in grains based on a trivariate model of Zn

absorption in both maize genotypes. In last experiment, different Zn fertilization approaches were

used for maize grain agronomic biofortification and human Zn bioavailability in maize grain. Zinc

was applied in pots to best Zn responsive maize cultivar DK–6142 as foliar spray (0.5% w/v Zn

sprayed 25 days after sowing and 0.25% w/v at tasseling stage), surface broadcasting (16 kg Zn ha–

1), subsurface banding (16 kg Zn ha

–1 at the depth of 15 cm), surface broadcasting + foliar and

subsurface banding + foliar in comparison to an unfertilized control. As compared to unfertilized

control, all treatments significantly (P≤0.05) increased growth, yield and nutritional attributes in

maize grain. Grain Zn and protein concentrations were correlated with each other and ranged from

22.3 to 41.9 mg kg−1

and 9 to 12 %, respectively. Zinc fertilization also significantly reduced grain

phytate and increased grain Zn concentration. Zinc fertilization, especially broadcasting and

subsurface banding combined with foliar spray decreased grain [phytate] : [Zn] molar ratio to 28 and

21 and increased Zn bioavailability by trivariate model of Zn absorption to 2.04 to 2.40,

xiv

respectively. Therefore, Zn biofortified maize grain by proper P fertilization was suggested for

human consumption to overcome malnutrition problems in humans. As a whole, this project supply

adequate Zn concentration in maize grain by proper P fertilization and their importance to grain

yield and nutritional quality of maize grain under suitable application approaches.

1

Chapter 1

INTRODUCTION

Among all micronutrient deficiencies in the world, Zinc (Zn) deficiency is most prominent

influencing maize crop growth (Kabata-Pendias, 2001; Rehman et al., 2012). Meanwhile, it is

required for optimum plant growth and to be supplied by fertilizers to crop plants. Now these days,

various Zn fertilizers applications have gained a dominant concern to ameliorate Zn deficiency

problems in the soils. Usually, Zn fertilization to alkaline calcareous soils is quite useless and is of

instant concern because it forms complex with other nutrients present in the soil and not available to

crop plants (Rattan and Sharma, 2004). Zinc movement as well as uptake by plants from the soil is

influenced by soil reaction (pH), organic matter (OM), Zn concentration in the soil and types of soil

etc. Root growth of plants in the soil influenced the Zn bioavailability due to decrease in light

intensity and soil temperature (Chang et al., 2007; Alloway, 2008).

Zinc bioavailability is also dependent on soil textural class, calcium carbonate (CaCO3), clay

mineralogy, organic matter content and salt affected soils etc. (Sharma et al., 2004). Zinc absorption

by plant roots is also influenced by low moisture content in the soils (Cakmak et al., 1999). For

example, Zn problems are directly correlated with soils having high pH and alkaline calcareous soils

of Pakistan (Alloway, 2008; Alam et al., 2010). About thirty fold decrease in soil Zn bioavailability

was observed by changing the soil pH from 6 to 7 which in turn effect the plant Zn concentrations

(Cakmak et al., 1999).

Aluminosilicate clays affect Zn sorption due to clay mineral impacts on cation exchange

capacity (CEC) and also Zn sorption behavior in the soils (McBride, 1989). Zinc deficiencies occur

in calcareous soils receiving high and frequent applications (Zahedifar et al., 2010) of phosphorous

(P). There are four major Zn fractions in soils; the water soluble which is found in soil solution, an

organically bound pool of Zn+2

that are chelated or adsorbed/complexed by organic matter, and an

exchangeable pool that is loosely bound to soils due to electrical charges to soil particles (Alloway,

2008). During a 15 to 60 day incubation trial, that most of the soil Zn was found in the residual

fraction that is associated with soil minerals (Obrador et al., 2003).

2

Total concentration of Zn in the soil depends upon the chemical characteristics of its raw

material present in the soil mineralogy. Different forms of Zn are found in the soil like water-

soluble, exchangeable, complex with organic matter / clay colloids. Approximately, 80 mg Zn kg-1

of

soil is found on earth crust; however commonly its concentration is about 10-300 mg Zn kg-1

in soil

having an average value of 50 mg Zn kg-1

of soil (Alloway, 2008).

As a result of low dietary Zn intake, Zn deficiency affected about 50% population of the

world. Low concentration of bioavailable Zn in human residents by the intake of major cereal food

grain is the main cause of high occurrence of Zn insufficiency, particularly in the developing

populations of the world. Zinc concentration is very low in cereal grain to fulfill the requirements of

human‟s daily need (Cakmak, 2008). Furthermore, planting/growing of staple crops in Zn

insufficient soils can accelerate the problems of Zn insufficiency and decreases the crop yield and

quality. Plants are also vulnerable to stress factors by soil Zn insufficiency (Alloway, 2008).

From biological point of view, Zn is also an important element. One most important Zn

physiological role in human body is synthesis of protein and in metabolism reactions. About 2800

human proteins are able to bind with Zn that forms about 10% proteome of humans (Andreini et al.,

2006). It is believed that nearly a major portion of transcription factors are made from 40% of the

zinc binding proteins. These derived transcription factors from Zn binding proteins are very much

used for gene regulation. The other 60% significant portions of Zn binding proteins are present in the

form of enzymes and proteins that take part in the transportation of ions (Andreini et al., 2006).

Apart from these above described functions, Zn is important for biological membranes

functional and structural integrity (Cakmak, 2000). Change in Zn homeostasis/reduction in human

result a lot of disturbances in cellular and immune system dysfunctions, impairments in body,

susceptible to infections, development of mental retardation and children stunted growth

development (Black et al., 2008). Main reason of child decease in the developing countries were due

to Zn deficiency in their diet and caused of about 450,000 deceases in children ≤5 years of age,

which relates to 4.4% of the children deceases around the globe (Black et al., 2003).

To increase Zn concentration in grain (e.g. agronomic biofortification), Zn fertilization

approaches by surface broadcasting on the soil surface or by foliarly sprays on leaves looks very

useful approaches. In recent times, Harvest Plus program was started worldwide by Zn fertilizer

3

project by the name Harvest Zinc project (www.harvestzinc.org). As described in Harvest Zinc

project (www.harvestzinc.org), Use of Zn fertilizers are required for agronomic biofortification of

major cereal grain aimed that sufficient concentration of bioavailable Zn should be present in soil

solution equilibrium and retaining sufficient Zn transportation in grain throughout plant reproductive

stage. However, surface broadcasting of Zn fertilizers or foliar Zn spray is the major approaches to

increase Zn density in grain. Zinc sulfate (ZnSO4), because of its high solubility in water and less

cost is mostly used in the agricultural fields as compared to other sources of Zn (Cakmak, 2008).

Increasing soil or foliar Zn approaches have many benefits for crop growth. Fertilization of

Zn to plants that are grown to Zn insufficient soils is a good approach to reduce phosphorous (P)

utilization and its accumulation in other plant parts (hence reducing phytate). So, it resulted in better

Zn bioavailability in human digestive system from cereal grain. Apart from this, high Zn content

grains have better ability to tolerate unfavorable environmental circumstances as compared to low

Zn in grain (Mirvat et al., 2006; Cakmak, 2008).

Now these days, increase in grain Zn concentration is an immense task all over the world. To

improve Zn concentration in cereal grains and in human diet, Zn fertilization and plant breeding

approaches are mainly used in the world. These approaches are economical and inexpensive than

other solutions to increase grain Zn density. An international organization (Harvest Zinc and Harvest

Plus Biofortification challenge program) ambition is to improve the quality of cereal grains with Zn

by Zn fertilization or plant breeding techniques (Pfeiffer and McClafferty, 2007). Very similar

strategies have also been implemented for many cereal growing areas of several countries such as

Australia, India and Pakistan etc.

Phytic acid (PA) is an antinutrient that binds with Zn; reduces Zn bioavailability in staple

food crops. Major storage compound of P in cereals is in the form of PA. Reducing bioavailability of

Zn from foods obstructs Zn utilization in human body. Conversely, the destructive influence of PA

(poorly bioavailable of dietary Zn absorption) should be maintained by positive effects of PA on the

health of humans (e.g. decrease the cancer occurrence in humans; Vucenik and Shamsuddin 2003;

Somasundar et al. 2005). Apart from human health, PA in seeds has also of vital significance for

germination of seeds as well as seedling growth (Oltmans et al., 2005; Guttieri et al., 2006).

4

Soil pH affects P solubility and plant uptake (Ortas and Rowell, 2000). Phosphate adsorption

capacity increases as pH decreases (Bolan and Hedley, 1990). Moreover, at higher pH values, lower

P sorption can be obtained at the initial portion of the isotherm. An incubation study on three

calcareous soils of the UK indicated that decreasing soil pH could increase the P solubility in the soil

(Ortas and Rowell, 2000). Laboratory studies also have indicated that P sorption could vary with pH

(Zhou et al., 2005). They believed that P sorption decreases as pH increases and as result, the surface

charge becomes more negative. Soil pH is still considered to be the most important soil factor

affecting P availability from phosphate rock additions (Ramirez, et al., 2001).

In soils having low Zn concentrations, increased in shoot P transportation increased more

leaves P density and may cause P toxicity in the plant (Khorgamy and Farnis, 2009; Salimpour et al.,

2010). Problems of P toxicity occurred in Zn deficient soils and it was not experienced in other

micronutrient insufficiencies. In roots, permeability of plasma membrane is more in Zn deficient

soils as compared to P deficient soils (Hu et al., 1996; Bukvić et al., 2003).

Phosphorus restricts Zn density in the plants and Zn density decreased by increasing more P

fertilization in the soil (Das et al., 2005a; Salimpour et al., 2010). Soil properties can also be

influenced by high P rates that could affect the bioavailability of Zn to crop plants. Phosphorous

fertilization can also cause change in soil chemical properties; hence change Zn dynamics in the soil

in various fractions. A lot of experiments were done on Zn-P interactions in soil-plant system. For

example, Mandal and Mandal (1990) described that increase in P fertilization in soil decreases Zn

bioavailability to crops. Severities of such type of Zn deficiencies are more in soils having

low/negligible amount of bioavailable Zn in soil (Norvell et al., 1987).

5

Despite its key importance in Zn availability to humans, little work has been done on the

management of P for biofortification of Zn in maize genotypes for their Zn uptake, which may be

important for the following reasons.

To find out the fixation and retention capacities of Zn and P in different textured soils with

respect to time

Efficiency of zinc and phosphorous applied to open-pollinated and hybrid cultivars of maize

Management of P to increase Zn bioavailability in maize grain

To find out the changes in phytate and Zn human bioavailability over different Zn and P

fertilization rates

To find out the better Zn fertilization approach for maximum plant growth, yield and grain

Zn density.

6

Chapter 2

REVIEW OF LITERATURE

Zinc (Zn) is one of the most important mineral element for all living things. About 300 enzymes in

biological systems are controlled by Zn element. In all the regions of the world ranging from arid to

semi-arid climate, all cereal growing areas are Zn deficient (Nube and Voortman, 2006). Different

cultivars respond in a different way to Zn fertilization. However, single nutrient requirement is

different for different crops. Nutrient requirements are also different with change in genotype and

species and it also occurs in the same species (Furlani et al., 2005). Apart from it, P-Zn interactions

in major cereal grains are still uncertain, particularly in exhaustive farming community having

maximum P fertilization and ignoring Zn. If these (P-Zn interaction) are not managed properly

then these cause deficiency of each other. Here is the brief summary of this chapter as given

below.

2.1. Extent of Zinc Deficiency in Soil

Among micronutrients deficiencies in the soil, Zn is major micronutrient deficient in our soils and

effect crop production around the globe particularly in Bangladesh, India and Pakistan (Alloway,

2009). Next to nitrogen (N) and phosphorous (P), Zn is insufficient plant element in high pH

Pakistani soils (Rashid and Ryan, 2008). About 50-70% soils of Pakistan are Zn deficient (Hamid

and Ahmad, 2001). Fifty percent soils of the world are also deficient in Zn and it creates the

problems for agricultural crops (Alloway, 2009). Soil Zn deficiency was observed in the worlds

about 49 countries (Alloway, 2004). Developed countries like USA and others; it is also most

prevalent problems of third world countries (White and Zasoski, 1999). An experiment was

accompanied under FAO programme to check micronutrient elements prominence in both developed

and third world countries. All the 30 countries that were including in that experiment were Zn

deficient (Sillanpää 1982).

7

Figure 2.1. Extent of soil zinc deficiency in the world (Alloway, 2009)

2.2. Extent of Zinc Deficiency in Pakistan

In arid to semiarid regions of Pakistan, Zn deficiency is related by salinity/sodicity and

calcareousness of soil (Ghafoor et al., 2004; Rafiq, 2005). Agricultural soils of Pakistan as well as

several other worlds‟ countries are calcareous (Alloway, 2009). Proton activity is directly correlated

8

with Zn2+

activity in these soils. So changes in soil pH also affect the Zn solubility in these soils

(Kiekens, 1995).

Nature of Pakistani agricultural soils are alkaline (high pH) and calcareous with more

susceptibility to Zn deficiency. It might also be due to naturally low Zn bioavailability from the soils

(Tinker and Lauchli, 1984). Zn forms precipitate/complex in these soils and become unavailable to

crop plants (Khoshgoftar et al., 2004). Salt stress in Pakistani arid as well as semi-arid regions

exaggerates more Zn deficiency in these soils (Alloway, 2009).

In 1966, identification of Zn insufficiency was first recognized in Pakistani paddy fields and

could cure “hadda” disorder of paddy rice (Yoshida and Tanaka, 1969). After that, it was concluded

that 86% Pakistani soils were Zn deficient (Kausar et al., 1976). In another experiment, it was

concluded that 23% KPK soil samples were deficient in bioavailable Zn (Khattak and Parveen,

1986). After that above conclusions, additional investigation exposed that about 70% Pakistani

farming soils were insufficient in bioavailable Zn (Hamid and Ahmad, 2001). Zinc insufficiency was

described earlier in many cereals, fiber and fruit tress (Rashid, 2006). Occurrences of prevalent Zn

insufficiencies resulted in stunted plant growth, delaying in crop maturity and yield of crops (Rashid

and Fox, 1992). Hence, higher fertilization of Zn (300 kg ZnSO4·7H2O ha-1

) might be the best

solution for Zn biofortification to alleviate malnutrition problems in human nutrition (Wang et al.,

2015).

2.3. Zinc Forms and Behavior in Soils

Zinc concentration in the soil is influenced by parent material (PM) and is most important for Zn

bioavailability in the soil. Basalt and gabbro rocks (called as volcanic rocks) have the maximum Zn

concentration (about 70–130 mg Zn kg–1

soil) as well as the average Zn concentrations is present in

carbonated rocks and sandstone (~20 and ~16 mg Zn kg-1

soil), respectively. In soil, Zn is found

comparatively in higher concentration (Sauchelli, 1969), beside higher in concentration in soil, the

bioavailable Zn is found in very minute concentration to meet the crop requirements (10–300 mg Zn

kg–1

in soil; Mengel and Kirkby, 1978; Broadley et al., 2012;. The optimal limit of bioavailable Zn

to plants from the soil, by 0.005 M DTPA method (Bansal et al., 1990) is about 0.75-1.5 mg Zn kg–1

9

soil. Zinc is bioavailable to crop plants from the soils in the form of Zn2+

and ZnOH+, labile Zn (that

can easily be desorbed) and Zn complexes with soluble organic materials (Kiekens, 1995).

Zinc bioavailability to crop plants is influenced by many soil and environmental factors.

High CaCO3, HCO3–

, Mg, Ca and Na content in irrigation water, low total Zn content in soil, high

pH (greater than 8), high OM (>3%), water logging (reduced conditions), high P prominence in soil,

texture having more sand percentage and imbalance use of fertilizers (NPK) influence the Zn

bioavailability by the crops. High yielding cultivars (hybrids cultivars) further aggravate the process

of Zn deficiency in the soil by maximum Zn mining from these soils (Lindsay, 1972a; Mortvedt et

al., 1991; Tandon, 1995; Cakmak, 1998; Alloway, 2009).

Zinc adsorption/desorption dynamics in soil is also dependent on above mentioned soil

characteristics that plays a critical role in regulating the Zn content in the soil equilibrium, hence

effect Zn bioavailability to crops plants (Shuman, 1975; Takkar and Sidhu, 1979). High P

fertilization may increase Zn sorption on soils colloids. This increase might be due to increase in

negative charge on clay colloid (Bolland et al., 1977).

Adsorption-desorption processes in the soil are influenced by Zn bioavailability in the soil as

well as its solution and solid phase partitioning (Gangloof et. al., 2005; Khoshgoftarmanesh et al.,

2006). The mechanism amid adsorption-desorption kinetics of Zn have been comprehensively

deliberated. In numerous papers, Langmuir model was mainly used to explain Zn adsorption on soil

colloid (Maftoun and Karimian, 1989). However, Freundlich model was good to describe Zn

adsorption by from diluted solutions. But a very little work was done on that model to show Zn

adsorption from diluted solutions. Commonly, Zn fixation in soils was described by retention studies

having high soil Zn concentration. But, none of the studies were conducted on balancing Zn solution

on adsorption models with low Zn concentrations. A very little work is done on that topic and a

limited knowledge is accessible for agro-climatic zones of Pakistani soils where these insufficiencies

are described extensively (Rashid et al., 1997).

10

2.4. Zinc Pools in Soil

Several organic and inorganic forms of Zn in the soil influence plant Zn bioavailability. It is present

in the soil as a) complexed/free ion in soil equilibrium b) adsorbed as specifically/nonspecifically

cations c) occluded as carbonates/hydrous oxides ions in soil d) as residues in living organisms or

biological system and e) primary or secondary clay colloid arrangement of minerals (Reed and

Martens, 1996). On the other hand, another scientist Viets (1962) deliberated that Zn is found in 5

biochemical pools as adsorbed on clay colloid, exchangeable with other elements, soluble in water

soluble and co-precipitated by primary or secondary minerals. Zinc sources soluble in water or labile

Zn portions are the direct sources of Zn bioavailability to crops (Li and Shuman, 1997). However,

tightly bound fraction of Zn (residual) acts as a non-labile (not available to plants) Zn source in the

soil (Clevenger and Mullins, 1982; Xian, 1989).

Figure 2.2. Dynamics of zinc in the soil (modified from Havlin et al., 2005)

11

Various Zn chemical forms are directly related with amount and nature of Zn found in the soil and

other factors like soil CaCO3 content, soil textural class, soil organic matter and soil reaction

(Sharma et al., 2004). Immediate pool of Zn to plants in the soil is in the soil solution and it is the

direct source of root Zn uptake by plants (Marschner, 1995; Barber, 1995). Particular nutrient

replacement from soil solution is made by the nutrient which is bound in various forms of solid

phase on the soil. Diverse solid fractions of soil influence Zn distribution and provide the nutrients to

plants from Zn solid phase. Various Zn pools in various soils were determined by several

consecutive extraction procedures (McLaren and Crawford, 1973; Shuman 1979; Tessier et al.,

1979; Lyengar et al., 1981; Shuman, 1985; Salbu et al., 1998; Ahnstrom and Parker, 1999).

Different Zn fractions are found in the soils by different procedures/protocols (Sims and Johnson,

1991). Hence for the better understanding, the best approach should be adopted that has the capacity

to remove that particular nutrient from that fraction more efficiently than other protocols

(Nascimento et al., 2007).

2.5. Functions of Zinc in Plants and Humans

Several important functions in plants and humans are controlled by Zn. Zinc is only metal from all

other metals that exists in six enzyme classes (hydrolases, isomerases, ligases, lyases

oxidoreductases and transferases) and acts as protein interface, structural, catalytic/co-catalytic

functioning (Broadley et al., 2007). Bio-membrane functioning and structure are controlled by Zn.

Maximum amino acids (AA) and phenolics are exuded from plant roots from Zn deficient soil

environment (Bell and Dell, 2008; Broadley et al., 2012). Reactive oxygen species (ROS)

detoxification and regeneration are also dependent on Zn concentration (Cakmak, 2000). Oxidative

degradation of auxin (plant growth hormone) is also caused by deficiency of Zn. Apart from these

functions; gene regulation, DNA transcription, RNA polymerase and protein synthesis in plants are

also controlled by Zn. In Zn insufficiency, reduced RNA level could depress the protein synthesis in

plants (Broadley et al., 2007).

Photosynthetic rate is also reduced in plants by Zn deficiency but the mechanism behind this

is still unclear. For example in maize plants (C4 plant), carbonic anhydrase is Zn-dependent enzyme

that provide HCO3–

required in photosynthesis process to phosphoenol pyruvate carboxylase (Bell

12

and Dell, 2008; Broadley et al., 2012). But in rice and wheat plants (C3 plants), carbonic anhydrase

does not have a specific function in photosynthetic process.

Similarly, several functions of Zn were observed for humans (Bell and Dell, 2008). Hence,

normal human growth, protein synthesis, DNA replication and cell division are controlled by Zn in

human body. Immune system functioning, neurological function in human body as well as for

normal human reproductive system functioning is also controlled by Zn. However, Zn deficient diet

can cause damages and infections in human bodies (Walker and Black, 2004). Most important

function of Zn in human body is its resistance for cancer cells production (Ho, 2004). Apart from Zn

vital role in human body, it is of great importance for pregnant women, new born babies and

juveniles. Zinc deficiency also causes a lot of diseases in human body like delayed puberty, diarrhea,

dwarfism and skin lesions etc. (Hambidge, 2000; FAO/WHO, 2004).

2.6. Zinc Deficiency in Plants

Young leaves of crops contain Zn density in the range of 15–20 mg Zn kg–1

(Broadley et al., 2012).

But depending on growth stage and plant type, Zn deficiency could happen on 7–30 mg Zn kg–1

(Reuter and Robinson, 1997). Pronounced incidence of Zn insufficiencies are found in those crops

that are unable to take Zn from the soil under Zn deficient environment like rice, maize and beans

(Alloway, 2009). Optimal Zn concentrations in new leaves for the identification of Zn insufficiency

is 10-22 mg kg–1

for soybean, 11-15 mg kg–1

for maize and 11-14 mg kg–1

for wheat crop. Young

leaves are most prone to Zn deficiency, hence; caused low phloem Zn translocation mobility in

crops. Stunted, little and cup shaped leaves in many crops is the distinctive sign of Zn insufficiency.

Reduction in internodal distance (rosetting) in broad leaves plants is also caused by Zn deficiency.

Additional indications of Zn deficiency have cup shaped leaves and interveinal chlorosis (Alloway,

2009).

In third world countries, Zn deficiency is the 5th

main source of human‟s diseases and also

child deceases (WHO, 2002). About 2.7 billion humans are deficient in Zn around the globe (Muller

and Krawinkel, 2005). World population of approximately 50% is deficient of Zn in their diets.

Least developed continents like Asia and Africa have more occurrences of Zn deficiency as

compared to developed continents (Brown et al., 2001). In a survey about Zn deficiency from health

13

department, 40% mothers and each 3rd

kid in Pakistan are suffered in insufficiency of Zn (Ministry

of Health, 2009).

Sex and development phase require different Zn intake rates. Normally, adult women and

man require 10 and 12 mg Zn d−1

, respectively. But, females in gestation and lactation period want

more than 14 mg Zn d−1

. Due to high dependence on Zn poor cereals in the daily diet, developing

countries have less Zn intake levels as compared to recommended to achieve the daily Zn

requirements of the body (White and Broadley, 2009; Bouis and Welch, 2010). Apart from these

above said reasons, population with more Zn deficiency relies on cereals grown on Zn deficient soils

(Alloway, 2009).

2.7. Factors affecting Zinc Bioavailability

2.7.1. Soil pH

Most significant factor controlling Zn availability to crops is soil reaction (pH). Variations of soil pH

influenced >90% deviation in plant Zn bioavailability (Wear, 1956). In Pakistan, widespread Zn

deficiency is associated with high soil pH (Qadar, 2002). Decrease in Zn solubility in the soil was

observed due to increase in soil pH (Lindsay, 1979). Precipitation of elements occurs at high pH and

Zn adsorption occurs at lower pH that effect solubility of Zn as well as its bioavailability to growing

crops (Gupta et al., 1987). At pH values 6-8, adsorption and precipitation is dominant and pH below

6 can cause Zn adsorption than other mechanisms in the soil (Singh and Abrol, 1985). However,

different pH values control the Zn solubility and its adsorption mechanisms on the soil colloid

(McBride and Blasiak, 1979). In alkaline or neutral soil pH, Zn hydrolyzed form (Zn (OH) +

) might

be adsorbed on clay surface and cause the reduction in plant Zn bioavailability (Jeffery and Uren,

1983). So, this might be possible for the reduction of Zn bioavailability at high pH. Furthermore,

higher pH could cause Zn adsorption on clay colloids and this might be due to change in negative

sites on OM as well as clay colloids (Harter, 1983; McBride, 1989). High pH can bound Zn firmly

on clay colloids (oxides and silicate clay surfaces) which ultimately effect plant Zn bioavailability

from the soil (Jurinak and Bauer, 1956; Lindsay, 1978; Harter, 1983; Jeffery and Uren 1983). For

example, hundred time decrease was found in Zn bioavailability to crop plants as increased in soil

pH by 1 unit from 7 to 8 (Lindsay, 1991).

14

2.7.2. Organic Matter

Zinc bioavailability and solubility to crops is influenced by soil organic matter (Shuman, 1985;

Harter, 1991; Barrow, 1993). Soluble complexes formation was enhanced by the organic materials

that ultimately increase crop Zn uptake from the soil (Ozkutlu et al., 2006). Various organic

materials applications influence the plant Zn uptake and solubility (Sinha and Prasad, 1977;

Tarkalson et al., 1998; Arnesen and Singh 1998). Reaction of organic matter with Zn can cause Zn

immobilization, fatty acid mobilization or insoluble salts complex formation in the soil (Tisdale et

al., 1993).

Zinc can also retain on organic matter surface layer (Kabata-Pendias and Pendias, 1984) as

well as retaining of Zn as weak acids on functional groups (Shuman, 1980). It is observed that small

concentration of metals having comparatively insufficient liginds containing sulfur is the main cause

of metal sorption (McBride, 1989; Barrow 1993). Organic soils are most commonly deficient in Zn

(Verloo, 1980; Stevenson 1991). However, an inverse association was found amid rhizosphere

soluble Zn and organic matter present in the soil (Catlett et al., 2002). Organic matter forms

insoluble Zn complex which cause more Zn adsorption on the soil colloid and hence reduce

environmental pollution. Apart from this, Zn bioavailability and solubility reduction was found in

the soils that have very low organic matter in them (Ozkutlu et al., 2006).

Beside of its importance in soil physical properties improvements, it also forms soluble

complexes with Zn (that acts as a natural chelate) that enhance Zn uptake by plant roots (Amanullah,

2006). Because of micronutrients significance in human nutrition, fertilization of micronutrient

chelates in soils are commonly used in third world countries to cope with soil Zn deficiency

problems. Chelates might either be synthetic or natural in their nature and formation (Bar-Ness and

Chen, 1991; Sadiq and Hussain, 1993; Perez-Sanz et al., 2002). Still, due to high cost of production

and some socio-economic constraints, third world countries cannot use synthetic chelate still now.

There should be the need of synthetic chelates formation that increases the Zn or trace elements

uptake by the plant roots from the soil. On the other hand, synthetic chelate formation, sources and

types are influenced by the use of synthetic sources which enhance the Zn availability in the soil

(Gramss et al., 2003).

15

2.7.3. Calcareousness (CaCO3 Contents)

Pakistani soils are calcareous in nature and suffer from Zn deficiency in soils (Rashid and Rayan,

2004). The reason behind this mechanism should be due to either by Zn can sorbed on the surfaces

of CaCO3 or soluble ZnCO3 formation (Udo et al., 1970), as surfaces of CaCO3 acts as a metal ion

sorption or precipitation on clay surface (McBride, 1979; Papadopoulos and Rowell, 1989; Zachara

et al., 1991). Conversely, CaCO3 presence in calcareous soils influenced the Zn adsorption maxima

(Shukla and Mittal, 1979). Carbonate bound Zn is more in calcareous and high pH soils than low pH

soils (Imtiaz et al., 2006). Apart from it, CaCO3 active fraction was of vital significane as compared

by total CaCO3 present in the soil (Elrashidi and Connor, 1982). Furthermore, Zn coefficient of

diffusion is fifty times less in high pH soils than low pH soils. Reduction in Zn diffusion coefficient

was observed by liming of high pH soils (Moraghan and Mascagni, 1991). Zinc can strongly be

adsorbed on the surfaces of CaCO3 that could be due to its surface area, pH, surface negative charge

as well as the calcium carbonate or magnesium content (Saleh et al., 1998).

2.7.4. Salt Affected Soils

Salt affects soils in arid to semi-arid areas of the world also cause Zn insufficiency problem. Root

interface of plants were influenced by stronger salt cations interaction that cause in the reduction of

plant Zn uptake (Tinker and Lauchli, 1984). Cadmium (Cd) uptake by plants was increased under

saline conditions that ultimately reduced plant Zn uptake. It might be of that reason because Cd and

Zn have an inverse correlation amid them (Jalil et al., 1994). Salt stress in the soil increase Cd

uptake in plants, which finally form CdCl2 complex formation that can easily be up taken by roots of

plants due to its solubility (Khoshgoftar et al., 2004). Toxic metals uptake in the plants can be

reduced by proper Zn fertilization in salt affected soils that increases the plant growth (Shukla and

Prasad, 1974; Shukla and Mukhi, 1980; AbdEl-Hady, 2007). Zinc application under salt stress

improved plant growth that could be by the efflux and influx regulation of Na+ across the cell

membrane and this can occur by Zn association with Na+ (Aktas et al., 2006). As described earlier,

integrity of cell membrane was controlled by Zn (Cakmak, 2000). Furthermore, reactive oxygen

species detoxification (Cakmak and Marschner, 1993) under salt stress condition can also be

controlled by Zn fertilization.

16

Figure 2.3. Release and fixation of zinc in soil–plant system (modified from

Cakmak, 2008)

2.7.5. Clay Minerals

Clay minerals as well as Manganese (Mn) and Fe oxides in the soil also influenced the soil Zn

bioavailability (Barrow, 1993). Zn adsorption is higher in montmorillonite and illite clay minerals

than kaolinite clay minerals (Pickering, 1980). The reason behind this is less permanent and

isomorphic charge on kaolinite clay minerals as compared to montmorillonite and illite clay

minerals.

17

2.8. Zinc Bioavailability Estimation in Human Diet

Zinc bioavailability in human diet is influenced by various nutrient elements. Zinc density in human

nutrition is of prime importance (Lönnerdal, 2000; White and Broadley, 2005) but some anti-

nutrients (which decrease essential elements absorption by the body) and nutrient promoters‟ (which

regulate nutrient absorption by the body) effect Zn uptake and its availability in the human nutrition

(Brown et al., 2001; FAO/WHO, 2004). Main hindrance in the absorption of Zn in human body is

influenced by phytate compound (also called phytic acid or inositol hexakisphosphate; C6H18O24P6)

that is part of many cereals grain and many other fruits of our daily usage. In the intestinal lumen,

phytate permanently forms complex with Zn that hinders in the way of Zn bioavailability in humans.

Apart from phytate, hemaglutinins, lectins, flavonoids, polyphenolics, tannins and fibers are other

anti-nutrients in our daily diet. Zinc competition with other elements also causes a reduced Zn

absorption in the human body. But, relatively low molecular weight molecules having solubility in

organic materials (like methionine, fumarate, histidine, riboflavin, citrate, cysteine, lysine, malate,

palmitic acid and ascorbate etc.) enable the human body to utilize Zn more efficiently (Brown et al.,

2001).

Zinc as well as phytic acid content in human nutrition determine the changes in Zn

bioavailability. Hence, Zn bioavailability was accurately determined by the molar ratio of

[phytate]:[Zn] in human nutrition (Brown et al., 2001; Weaver and Kannan 2002). Classification of

human diet is such as low Zn bioavailability (15%), average Zn bioavailability (30%) as well as high

Zn bioavailability (50%) if nutrition have molar ratio of [phytate]:[Zn] in the range of >15, 5-15 and

<5, correspondingly. Hence, we can say that Zn bioavailability was accurately measured from

human diet by the molar ratio of [phytate]:[Zn] from the specific diet. Net required Zn physiological

absorption is about 3 mg d−1

by mature human body (Institute of Medicine, 2001; Hotz and Brown,

2004). Aimed at the mature people‟s daily Zn nutrition, physiological Zn requirement should met the

criteria of daily Zn requirements. Recently, Zn bioavailability in our daily nutrition is measured by

trivariate model (Hambidge et al., 2010) and it is correlated by Zn homeostasis in human intestine.

In that model, total per day intake of phytate and Zn calculates the estimated Zn bioavailability from

our food. Zinc trivariate absorption model is a quantifiable approximation for Zn bioavailability in

human body alongside from the molar ratio of [phytate]:[Zn] in human diet (Miller et al., 2007).

18

( (

) √( (

))

)

In that equation, AMAX, KR, KP are used for maximum Zinc absorption, zinc-receptor equilibrium

dissociation constant of binding reaction and Zinc-phytate equilibrium dissociation constant of

binding reaction with their values 0.091, 0.680 and 0.033, respectively (Hambidge et al., 2010) and

it is related to Zn homeostasis in human intestine. In the model, TAZ (in mg Zn d−1

; total everyday

Zn absorption) exists as a purpose of TDZ (mmol Zn d−1

; total everyday dietary Zn) as well as TDP

(in mmol phytate d−1

; total everyday dietary phytate).

This model tells the bioavailable Zn from our daily food items through use of this

mathematical model (Bouis and Welch 2010). As a purpose of nutritional phytate and Zn in our

food, this mathematical model (Miller et al. 2007) was positively verified in adult women in labeled

Zn research (Rosado et al. 2009). That model precisely evaluates the human intestine Zn absorption

from cereal grains or other food items if these are used as daily food items. Zinc bioavailability and

its approximation from maize grain or other food items is the requirement for an efficient bio-

fortification program.

2.9. Zinc Fertilization Approaches to Maize

Zinc fertilization in soils could be of prime important approach to ensure freely bioavailable Zn and

its uptake to plants for the maintenance of healthy plant root growth and development. Plants

nutrients used in integration with other nutrients as well as their balance use has the tendency to

improve the grain yield but farmers of least developed countries do not apply balance fertilization to

crop plants. In 20th

century, approximately 50% increase in grain yield was due to Zn fertilization in

soils having deficient in Zn (Fageria, 2008). Non accessibility and unawareness about Zn

fertilization is the main cause for less adoptability to poor growers (Bell and Dell, 2008) and

fertilization approaches affordable to farmers are more quickly accepted by these farmers. For the

approaches to be more farmers friendly and affordable, it must be having less labor cost and time–

efficient to maximize obvious change in yield of crop.

19

Table 2.1. Potential solutions to overcome zinc deficiency in humans

Interventions and strategies Scope Importance and economic

benefits

Supplementations: Clinical

treatments (therapeutic and

preventive) can be done through

mineral drugs.

Recommended for a shorter

period during pregnancy and

severe Zn deficiency.

Active response but costly

Fortification: Enrichment of

nutrients (particularly Zn and Fe)

in foodstuffs during processing

(Juices and flour etc,).

Mostly limited to developed

areas of cities but effective.

Uneconomical for longer

period of times but gives

valuable aspects.

Food

Diversification/modification:

Use of nutritious food (fortified)

should be the part of daily life.

Only developed countries

societies can afford or where

variety of foodstuff is accessible.

Restricted to developed

countries and economically

practical.

Agronomic Biofortification:

Zinc fertilization through

agronomic practices

(broadcasting or foliar nutrients

spray).

Particularly for the people living

in the rural areas and fortified

foodstuff items are out of range.

Cost effective as well as

sustainable than other

approaches. Apart from yield

increase, it is long–lasting

solution to reduce zinc

deficiency problem.

Genetic Biofortification: Use of

nutrient efficient cultivars or

plant breeding techniques to

increase concentration of

bioavailable nutrients.

Widespread scope and

practicable.

Economically it is also

feasible and cost effective

but rural people are unaware

about its use.

Adopted from various sources (White and Broadley, 2005; Stein et al., 2007; Bouis and Welch,

2010; Hussain et al., 2010).

20

Grain yield as well as Zn density in grains is affected by various Zn fertilization approaches. Zinc

fertilization either by surface broadcasting or seed treatment with Zn could improve Zn density in

grain of maize (Harris et al., 2007). Seed treatment with Zn improved shoot growth and

development; however surface broadcasting is the prerequisite for optimum grain yield. As

compared to control, about 4–40% increase in yield was observed by broadcasting fertilization of Zn

(Yerokun and Chirwa, 2014). Likewise by broadcast Zn fertilization, more than 40% increase in

grain Zn density was observed in maize grain (Kanwal et al., 2010). Zinc fertilization in integration

with NPK fertilizers and organic manures produced maximum maize grain yield (3.9 t ha–1

) that was

130% more yield by the fertilization of sole NPK fertilizers (Manzeke et al., 2014). Band placement

of Zn (beside the row) is prominent than soil broadcast fertilization; hence for band placement,

recommended fertilization levels are less than broadcast method (Shapiro et al., 2003). So, band Zn

fertilization was useful to optimize shoot as well as grain Zn density in maize crop.

Zinc fertilizers can also be foliarly sprayed on the vegetative growth stages of crop plants

(Fageria et al., 2009). Zinc foliar fertilization at grain filling stage is related by optimum Zn density

in grain and more appropriate from surface broadcasting. Zinc fertilization by broadcasting+foliar

spray approaches (tasseling stage and flowering initiation stage) gave maximum grain and stover

maize yield as compared to sole Zn broadcast or foliarly spray. There are lot of indications

presenting that foliarly spray or mutual broadcasting+foliarly sprays Zn fertilization were more

prominent and useful to optimize Zn accumulation and uptake in plants as well as in reducing the

disorders of Zn insufficiency. Hence for agronomically biofortification of Zn in maize grain, foliar

spray of Zn or urea coated with Zn stands best approach as compared to Zn surface broadcasting

(Shivay and Prasad, 2014). Hence, it is very convenient and suitable strategy in resolving health

problems effectively that are caused by Zn deficiency (Cakmak, 2008; Yosefi et al., 2011). So,

optimum Zn fertilization should be recommended for human Zn requirement.

Apart from fertilization approaches, each cultivar also has the different response to Zn

application. Zinc efficient maize cultivars respond well to Zn fertilization for maximum yield and

grain Zn density. So, scientists would cautiously define the optimum grain Zn density for a particular

combination of cultivar–environment.

21

Figure 2.4. Approaches for zinc biofortification of major cereal grain (modified

from Hussain et al., 2010)

2.10. Physiological Functions of Zinc in Plants

Most important function of Zn is in bio-chemical or physiological processes in plants is like

photosynthesis, metabolism of carbohydrates, proteins and auxins production, to maintain the

integrity of cell membranes and protection from reactive oxygen species to damage the cells

(Römheld and Marschner, 1990; Marschner, 1995; Cakmak, 2000).

22

2.10.1. Photosynthesis

Reduction in photosynthesis process in plants was observed by Zn deficiency (Shrotri et al., 1981;

Seethambaram and Das, 1985). Various plant species shows a reduction in photosynthetic rate by

50–70 % due to insufficiency of Zn (Pearson and Rengel, 1998; Alloway, 2004). Reduction in

chlorophyll content might to decrease the Hill reactions which ultimately reduce photosynthesis rate

in Zn insufficient crops (Sharma et al., 1992; Brown et al., 1993). Under Zn insufficient soils,

reduction in CO2 supply reduced photosynthesis rate in cauliflower leaves plants by the reduction of

stomatal conductance (Sharma et al., 1994). Furthermore, for appropriate photosynthetic functioning

in plants, Zn is also important for carotenoid and cholorophyll regulation in plants (Aravind and

Prasad, 2004).

2.10.2. Zinc in Enzyme Function

The metabolism of carbohydrate, protein, auxin and reproductive process are generally inhibited

under Zn deficiency (Römheld and Marschner, 1990; Brown et al., 1993). This is because Zn is

constituent of approximately 300 metallo enzymes especially which are required for nucleic acids

and protein metabolism. Meanwhile, tryptophan production is regulated by Zn and important role in

auxin (essential growth hormone) production in plants (Skoog, 1940; Brown et al., 1993; Alloway,

2004; Brennan, 2005). Further, Zn was also involved in protein synthesis (Lin et al., 2005; Hänsch

and Mendel, 2009).

2.10.3. Superoxide Dismutase

Zinc has important effects on the activities of superoxide dismutase (SOD) which occurs in

chloroplasts and is produced by all aerobic organisms (Cakmak et al., 1994). Destructive and

reactive superoxide anion removal is being catalyzed by these enzymes and produces H2O2. Regular

cell metabolism is done by Superoxide radicals as well as its derivatives. Mitochondrial as well

chloroplasts electron transport chain produced their higher concentration than normal (Cakmak,

2000). Under Zn deficiency, the activity of SOD significantly decreased (Marschner, 1993) and Zn

supply to beans insufficient in Zn for almost 72 hours and repaired the enzyme activity and protein

and chlorophyll concentrations to the level of Zn sufficient plants (Cakmak et al., 1994). Rengel

(1999) described in an experiment that Zn deficient lupin caused reduction in the activity of total

23

SOD but Mn-SOD action was unchanged. Moreover, Cakmak and Marschner (1988) suggested that

the decrease of protein synthesis by Zn deficiency was caused by the decrease of antioxidative

enzyme activities.

2.10.4. Zinc in Carbohydrate Metabolism

Marschner, (1993) reported that Zn can affect the carbohydrate metabolism at various levels. The

involvement of Zn in carbohydrate metabolism is co-related by its influence on the transformation of

sugars and rate of net photosynthesis. Dependence of crop and Zn insufficiency, it depressed 50-70%

net photosynthesis rate (Brown et al., 1993). The restoration of phloem loading of sucrose by

resupply of Zn can be attributed by the role of Zn in the structural integrity of bio-membranes

(Welch et al., 1982).

2.10.5. Zinc in Protein Metabolism

Functional and structural integrity of ribosomes is controlled by Zn. Under Zn deficiency, the

amount of protein synthesized is significantly reduced (Marschner, 1993). Meristematic tissue

required high Zn concentration that is vital for the production of nucleic acid, protein as well as

division of cells (Obata and Umebayashi, 1988).

2.10.6. Zinc in Membrane Integrity

Zinc has significant physiological utilization in maintaining integrity and function of the bio-

membranes. Cakmak and Marschner (1988) reported that Zn has a fundamental role in stabilizing

the membrane integrity and acts as a protective membrane from oxidative damage, caused by

reactive oxygen species.

Cakmak and Marschner (1988) reported that in low Zn media, protoplast of yeast cell

membrane stability was lost from its original shape, increased permeability and allowed leakage of

intercellular solutes. Evidence of Zn involvement in membrane integrity of higher plants has also

been reported by Welch et al. (1982). They showed that there was greater leakage of P32

from roots

with Zn deficient than from roots with Zn sufficient of wheat. Apart from this, direct influence of Zn

was observed on cellular function through its effects on the structural integrity of bio-membranes.

Hence, Zn has to be supplied regularly to crop plants for their complete growth period.

24

Reactive oxygen species detoxification is also controlled by Zn application that causes

potentially damaging problems to membrane lipids as well as sulfhydryl groups (Rengel, 1999). Zinc

also plays a key function in trans-membrane fluxes of metal ions and prevents protein oxidation in

sulfhydryl groups. Therefore, it may have vital importance in the alteration of efflux degrees as well

as uptake of metal ions by the plants (Welch and Norvell, 1993; Kochian, 1993).

2.11. Mechanism of Zinc Uptake in Plants

Zinc uptake by plant roots is influenced by the presence of root hairs, Zn crop requirement and its

uptake ability of that element (Fageria et al., 2002). Because our soils are deficient in Zn, root

interception (exchanged by root surfaces) or diffusion (concentration gradient) is the main methods

for plant Zn acquisition (Jungk, 1991); therefore supply by mass flow to the roots is less important

than diffusion and interception. Roots of non-graminaceous species absorb Zn from the soil solution

in the form of Zn2+

ion. But Zn(OH)+ is dominant form of Zn uptake to crop plants in alkaline pH

soils (Marschner, 1995).

Uptake of Zn concentration depends upon Zn transporter genes having 10-100 mmol m-3

KM range

(Fox and Guerinot, 1998). Conversely, Zn efficiency varies in different wheat cultivars due to

different carrier–mediated system for Zn uptake (Hart et al., 1998). In the beginning, there is the

quick uptake of Zn because of its binding by plant root cells. After that, it slows down in the cell

membrane to linearity level (Veltrup, 1978). Hence, movement of Zn ion to the cell wall from

outside solution is of compelled by diffusion, passive or non-metabolic in nature (Kochian, 1993;

Marschner1995). Nutrients can travel through xylem vessel by root epidermis after arriving at the

root surface, such as epidermis, cortex and endodermis, and from xylem. Then, these are supplied

into the shoot where they are required (Marschner, 1995).

Zinc can also be absorbed as Zn2+

ion to the graminaceous plants (Halverson and Lindsay,

1977) as well as forms phytosiderphores (amino acids having non protein in nature form complex

with Zn; Taghavi et al., 1984; Welch, 1993). In gramineae especially rice, early work showed that

Fe deficiency stimulated the release of phytosiderophores in the rhizosphere that are non-protein

amino acids in nature. Phytosiderophores can effectively chelate Fe2+

and facilitate its uptake across

the root cell plasma membrane (Takagi et al., 1984). A Study by Zhang et al. (1989) indicated that

25

phytosiderophores released into the rhizosphere were not only specific for Fe deficiency, but also

responded to Zn deficiency. It was reported that phytosiderophores were also effective in

solubilizing Zn. They also demonstrated in a similar experiment using 65Zn2+

with wheat plants, that

these compounds have the ability to chelate Zn2+

, Cu2+

and Mn2+

(Zhang et al., 1991a). In addition,

Treeby et al. (1989) reported that under Fe deficiency, root exudates of barley can effectively

mobilized Zn, Mn, Cu and Fe from a calcareous soil. Therefore a more accurate general term for

phytosiderophore is phytochelate or phytometallophore.

2.12. Zinc Translocation in Plants

Amount of Zn supply is dependent on translocation of Zn in plants as well as plant N concentration

and possibly upon the Zn requirement in crop plants. In subterranean clover, Zn concentration was

more in above ground parts as compared to below ground parts. Presumably, reduction in the

concentration of Zn from the roots might be due to its uptake and translocation to the newly

developed plant organs to meet the demand of the grain (Riceman and Jones, 1958). However, Zn

mobility varies within the phloem in different plant parts. So, Zn supply and its distribution to other

plan parts is a complex mechanism (Longnecker and Robson, 1993). Plant parts, growth stage and

supply of Zn to crops determine the Zn concentration in crops. Zinc concentration is about 10-15 mg

kg-1

in Zn insufficient plants and about 15-100 mg kg-1

in Zn sufficient plants (Longnecker and

Robson, 1993). In low or average Zn application, its concentration is maximum on its reproductive

stage as compared to its vegetative stage. Plants with sufficient Zn application have normally the

same leaf Zn concentration in specific leaf blades at different plant ages. However, developing plant

parts contains more Zn concentrations as compared to matured plant parts by average Zn application

(Longnecker and Robson, 1993).

The distribution of Zn between roots and shoots is dependent in the Zn supply. It was

observed that high Zn fertilization to below ground clover roots uptake more Zn as compared to low

soil Zn fertilization (Reuter, 1980). With high Zn supply, Zn concentration was higher within the

root cortical cell walls, but this high Zn concentration in roots was not matched by increased Zn

accumulation in shoots (Longnecker and Robson, 1993).

26

2.13. Secretions of Chelating Agents and Organic Acids

In nutrient deficiency especially Zn, organic acids are exudates by plant roots (Grinsted et al., 1982).

More organic acid exudates in white lupin crops were reported in soils with P insufficiency (Gardner

et al., 1983). Apart from this, decrease in pH of rhizosphere in lupin roots caused production of citric

acid. Then it mobilizes the soil Zn bioavailability that plant can uptake more efficiently as compared

to Zn insufficient soil (Dinkelaker et al., 1989).

Under nutrient deficient environment, chelating compounds are also excreted by plant roots.

Iron uptake by grasses can also be increased if there is Fe deficient environment in the soil by

producing phytosiderophores (Römheld and Marschner, 1990). Copper (Cu) and Zn formed complex

chelates with Fe, remobilize them in the soil particularly of Zn as well as Fe in alkaline calcareous

soils to increase their uptake by crop plants (Marschner, 1993). Zinc deficiency caused roots induced

changes in the field of wheat and barley and this response might be an effective mechanism to elicit

the mobilization of Zn in the rhizosphere (Zhang et al., 1989).

2.14. Genotypic Variation for Zinc in Plants

Zinc efficiency may be defined as cultivars growth and yield capability in soils having insufficient

Zn (Graham, 1984). Tolerance of Zn insufficiency is different in many plant cultivars as well as in

their species (Hasisalihoglu and Kochian, 2003). Zinc concentration variation among maize crop

cultivars as well as its utilization efficiencies were described by many scientists (Maziya-Dixone et

al., 2000; Bänziger and Long, 2000; Oikeh et al., 2003).

Zinc utilization efficiency are controlled by some plant mechanism that are a) increased in root to

shoot Zn uptake, b) roots are of that type that enhance plant Zn bioavailability, c) shoot cell can

uptake Zn more efficiently as compared to other plant parts, and d) variations in shoot Zn

compartment (Hasisalihoglu and Kochian, 2003).

2.15. Zinc Uptake Efficiency in Plants

Zinc uptake efficiency was changed by Zn fertilization rates as well as every cultivar has

different efficiency to applied Zn. Different rates of Zn fertilization in the maize field

significantly increased maize crop harvest. Zinc uptake as well as its content increased by

27

increasing the Zn fertilization rates (Tariq et al., 2002). Another study was performed to find

out the optimal Zn fertilization rates in the cropping system of mungbean-maize-rice. Zinc

fertilization respond positively for all crops during these years. 4–0–2 kg Zn fertilization ha-1

was best Zn rate for these crops during the first year of experiment and 2–0–2 kg Zn

fertilization ha-1

was most suitable for the second year for these crops. Zinc fertilization is

positively related with grain N concentration as well as Zn fertilization increased the grain Zn

content. Maize grain Zn density was also increased by 27 mg kg-1

by applying 2 kg Zn per

hectare. Screening experiments were conducted on eight maize cultivars for the consecutive

three years on the same soil. They found that only three cultivars were Zn efficient and uptake

Zn more efficiently than all others (Hossain et al., 2008).

In another trail of higher pH soil, Zn fertilization efficiency was checked for wheat crop by

using NPK related Zn lignosulfonate source (ZnLS). NPK+ZnLS were the best Zn source for

maize dry matter yield and shoot Zn concentration in parallel to NPK+ZnSO4. Efficient Zn

source on all plant growth stages was NPK+ZnLS as compared to NPK+ZnSO4. At harvest, no

significant dissimilarities were found in all these treatments and sources. However at high pH

conditions, NPK+ZnLS Zn source was most beneficial and prominent for wheat crop growth

and production (Ortiz et al., 2010).

2.16. Extent of Soil Deficient in Phosphorous

In the world, soil deficient in Phosphorus (P) is of major nutritive limitation and is the problems of

all soils. Four forms of P are found in our soils. Soil colloid firmly adsorbed by organic as well as

inorganic form of P and these forms are difficult to uptake by the crop plants. Soil P of about 80% to

90% is present either in the form of comparatively insoluble or slow bioavailability forms. Soil P

present in available form is approximately 10-20% and is lightly adsorbed on the soil colloid. While,

the residual ≤1% is found in the freely bioavailable form and is most important for crop nutrient

uptake (Memon, 2005). Phosphorous bioavailability is decreased by many reasons in the soils which

might be due to soil pH higher than 8, fine textured soils with maximum clay contents (as higher

amounts of amorphous oxide and allophones in andisols soil order), and deficient in organic matter

as well as maximum P adsorption capacity soils.

28

Man-made activities, soil ploughing, imbalance animals dung application, imbalance grazing

of fields by livestock and deforestation are the main reasons of P losses from the soil. Soil ploughing

by different methods disturbs the soil properties and increase P losses from the soils if all are done

without proper farming management (Brady and Weil, 2008). Deforestation, wild fire as well as

cutting of trees for fire use are also the main reasons of P losses. Phosphorous deficiency is not

common in all soils. High P fertilization is not required to P deficient soils if these soils are found

deficient in P (Fox and searle, 1978). Phosphorous fertilization by higher rates to P deficient soils

caused economic and environmental constrains. Apart from it, low P fertilization can be tolerated by

paddy crop as associated with other crop plants. It might be because of that the P bioavailability

increased under reduced condition as in normal soil condition (Tadano and Tanaka, 1980).

2.17. Phosphorus Utilization efficiency in Plants

Phosphorous efficient cultivars ensure their capability to utilize suitable P rates from soil as well as

its translocation to different plant parts. Different crops have different response to P uptake and

its efficiency. By fertilizing the soil with 60-120 kg P ha-1

, wheat crop growth and yield was

significantly and positively increased. Various plant growth and yield attributes were

significantly changed by P fertilization. Maximum increase in all parameters as well as yield

was found by the application of 120 kg P ha-1

(Hussain et al., 2008). Cultivars also differ in

their P utilization efficiencies, cation density and plant growth attributes. Zinc was applied by

two levels. Under P deficient environment, Krichauff variety (lower uptake of P) had maximum

cation composition as compared to Brookton cultivar (high uptake of P). Zn fertilization by

different levels was not involved in variation of cation density in wheat crop. But, P

fertilization is involved in the reduction of cations density (Zhu et al., 2001a).

2.18. Zinc Interaction with Phosphorus

Phosphorous-zinc interaction in main cereal crop plants is still unknown, particularly in

exhaustive farming methods by higher P fertilization as well as minimum Zn fertilization

bioavailability. Phosphorous (P) and zinc (Zn) interrelate in soil plant systems. Interaction of

29

that type caused either one nutrient insufficiency interrelating by other, if there was an

antagonistic interaction (Imran et al., 2016). If root environment increased P bioavailability then it

could enhance plant Zn insufficiency by changing shoot Zn utilization and its re-translocation to

other plant parts (Robson and Pitman, 1983).

However, the reason behind this is not justified. It was observed in many experiments that

soils with marginally insufficient in Zn, if received higher P application; significantly improved

uptake of P. That should might also be the reason of P toxicity and have the same indications as of

insufficiency of Zn (Webb and Loneragan, 1990). Yet, data on the influence of P bioavailability on

Zn utilization and re-translocation in maize plant is not enough to understand its mechanism.

However, Optimal P fertilization should be managed for intensive agricultural system to

maximize high crop yield and Zn in grain required for the nutrition of human.

Phosphorous application by different rates influenced on yield, biomass accumulation

and Zn remobilization in all crop stages of wheat. Increase in P fertilization positively

improved production of wheat grain, straw weight and P concentration but reduced Zn

concentration in shoots. Higher P fertilization (>50 kg P ha−1

) significantly decrease Zn

accumulation and remobilization of wheat (Zhang et al., 2015). In maize crop, sole Zn

fertilization responds positively. However, combined application of Zn+P improved the maize

yield and its nutritional attributes (Verma and Minhas, 1987). That might be due to that plants

insufficient in Zn improved P density in grains and it was due to Zn influence in straw and not

in the rhizosphere (Webb and Loneragan, 1988).

In another experiment, reduction in the growth of wheat crop was observed by P or Zn

imbalance fertilization. Phosphorous insufficiency causes more reduction in plant growth that

surplus P fertilization or by surplus Zn fertilization than sufficient Zn. Surplus Zn or

insufficient P influence appears on plants on their vegetative growth stages. Under surplus P

fertilization, a clear antagonism relationship was found in wheat plant roots which cause a

considerable reduction of root Zn concentration in wheat plants. But, recommended P

fertilization rate enhanced plant Zn uptake from root to shoot (XiWen et al., 2009).

Oseni (2009) conclude the influence of various Zn and P fertilization rates influence on

many crops. As incremental P application > 60 kg P ha−1

, cowpea and sorghum crops reduced

their ability to uptake Zn efficiently. For both crops P and Zn utilization, a significant negative

30

correlation was obtained. Other elements in the potato crops were also influenced by P and Zn

interaction. Phosphorous density in potato tubers were reduced as by increased Zn fertilization.

Phosphorous mobilization to upper plant parts is reduced by high root Zn concentration. Hence,

cause the P to remain in the root rhizosphere (Barben et al., 2007).

Zinc-phosphorus interactions in various wheat cultivars have different P utilization

efficiencies. It was observed that soils low/deficient in Zn or P, increased in wheat Zn density

was observed due to low P utilization efficiencies (Zhu et al. 2001b). Increase in P application

in a calcareous soil increase growth and nutritional attributes of maize. Then fertilization of P

caused the reduction in shoot dry weight (23%), Zn density in shoots (82%) and improved P

density in shoots (3.0 fold) in maize. So, heavy P fertilization can induce P toxicity and Zn

deficiency (Kizilgoz and Sakin, 2010).

31

Chapter 3

MODELING TEMPORAL VARIATIONS IN ADSORPTION

OF APPLIED ZINC AND PHOSPHORUS IN

ALKALINE-CALCAREOUS SOILS

3.1. Abstract

Zinc (Zn) and Phosphorus (P) deficiencies are widespread in most alkaline-calcareous soils.

Most of the applied Zn and P are adsorbed on soil colloidal surfaces short after their

applications. The objective of current investigation was to determine interactive influence of

Zn and P on adsorption and bioavailability of applied nutrients. Levels of Zn (0, 4, 12 mg kg−1

soil) and P (0, 40, 120 mg kg−1

soil) were applied in all possible combinations to three

differently textured soils (clay, loam and loamy sand) in pots. The soils were incubated for 20,

40, 60, 90 and 120 days at 25+5 °C and adsorption of Zn and P in these soils were measured by

AB-DTPA method. Adsorption of Zn and P depend on the rate of application and inherent soil

properties. Percent adsorption of added Zn and P increased with increasing clay content as

described by Michaelis-Menten equation. Most of the fixation of Zn and P took place within

55−60 days after their addition in soils. Zinc availability was reduced by P application but P

availability was not as much affected at a very high Zn rate. In conclusion, Zn and P adsorption

largely depend on soil physico-chemical properties, rate of application and time. Michaelis-

Menten model worked best about 120 days that was good to determine the fertilizer

requirement to complete the growth of crops.

3.2. Introduction

Zinc (Zn) deficiency is considered one of the major micronutrient problems for crop yield and

quality. It appears to be more in calcareous soils than in neutral or acidic soils (Cakmak et al., 1999;

Alloway, 2008). Even though Zn deficiency is common in calcareous soils, there are not large

differences in the total Zn content of calcareous and non-calcareous soils (Zahedifar et al., 2010).

McBride (1989) reported that aluminosilicate clays affect Zn sorption due to clay mineral impacts on

cation exchange capacity (CEC). Alloway (2008) suggested four major Zn fractions in soils; the

32

water soluble pool (which exists in the soil solution), an organically bound pool (that is adsorbed,

chelated or complexed (with the organic matter), and an exchangeable pool (loosely bound to soils

due to electrical charges to soil particles).

Most important factors affecting Zn availability are solubility of Zn minerals, type and

quantity of organic matter, and the amount of Zn which could be adsorbed on clay surfaces

(Vodyanitskii, 2010; Hettiarachchi et al. 2010). Obrador et al. (2003) found that during a 15 to 60

day incubation trial, residual form of Zn is dominant in these soils and it was related with soil

minerals.

For proper plant growth, P is vital nutrient and its deficiency is common in calcareous soils.

Therefore, plant available soil P is increased by addition of P fertilizer (Jalali and Ranjbar, 2010).

Because limiting resources of rock phosphate, proper management of P fertilizer is necessary. It

should be done by optimizing yield of crops as well as reducing its losses in the environment.

Farming soils have three fractions of P: organic P (25-30%), insoluble inorganic P (about 75%), and

a small soluble P fraction and 90% soils are deficient in P in Pakistan (Ozanne, 1980; Ahmad &

Rashid, 2004). Nearly 80% of applied P becomes sorbed on soil colloids. Hence after that, soil

equilibrium contains very minute concentration of P that is important for plant use (Leytem &

Mikkelsen, 2005). Furthermore, more P fixation/adsorption in soil restricts it for plant use reduction

(Delgado et al., 2002). Hence with time, applied P caused a significance reduction of P efficiency.

Phosphorous has the capability for enhancing Zn adsorptions as well as reducing Zn

desorption in soils (Agbenin, 1998; Rupa and Tomar, 1999). Phosphorous adsorption enhanced

cation exchange capacity of soils and caused maximum Zn adsorption/fixation in soil (Xie and

Mackenzie 1989). In presence of applied P, organic matter and Fe/Al oxide caused to the increase of

Zn fractions associated in the soil (Pardo, 1999; Sarret, 2002).

Phosphorus and Zn interrelate with each other in plants or soils; hence cause a significance

reduction in their bioavailability and uptake by the plants (Nayak and Gupta, 1995). It was

experienced that higher or normal P fertilization in the soils caused a significance Zn insufficiency in

soils. Numerous scientists stated as positive interaction, but challenging and negative interaction also

exists for this (Boawn and Leggett 1964; Paulsen and Rotimi, 1968).

33

Surprisingly, exact mechanisms of Zn-P interaction in soil are still unclear. The best evidence

for more Zn adsorption resulting from P treatments is found not oly in alkaline or calcareous soils

where Zn deficiencies are most common, but also in acid soils rich in variable-charge colloids

(Adams et al., 1982).

All the earlier adsorption models work best for very short period of time. Freundlich and

Langmuir models work best for 1−2 days. Apart from these models, multireactional model (MRM)

performs best from 16-32 days (Zhao and Selim, 2010). But these models are not enough to

complete the nutritional requirement of crop growth.

For this, Michaelis-Menten model was used to check temporal variations in adsorption of applied Zn

and P in alkaline-calcareous soils.

3.3. Materials and Methods

3.3.1. Soils

A soil incubation study was carried out in pots to check Zn and P adsorption in different textured

soils. Bulk samples of the soils were collected from [30.259° N 71.515° E Loam from 0-15 cm and

clay about 2 feet depth); 30.153° N 71.253° E (Loamy sand)] Agricultural Research Farm,

Bahauddin Zakariya University, Multan. Air-dried soil samples were crushed to pass through a 2

mm sieve. Various physico-chemical features of these soil samples were determined. Hydrometer

process was used to measure soil textural class (Gee and Bauder, 1982). Clay, loam and loamy sand

soil‟s pH of saturated soil paste (pHs) and electrical conductivity of extract (ECe) were measured in

1:1 soil to water suspension. Walkley-Black method was used to measure organic matter content in

these soil samples (Nelson and Sommer, 1982). Acid dissolution process (Allison and Moodie,

1965) was used to measure free lime (CaCO3) content (Table 3.1).

34

3.3.2. Incubation

Collected bulk samples of the soils were thoroughly mixed and 2.5 kg of soil samples were filled in

each of 54 polyethylene lined plastic pots. In all possible combinations, three Zn (0, 4, 12 mg Zn

kg−1

soil) and three P (0, 40, 120 mg P kg−1

soil) rates were uniformly applied to all pots in solution

form. The sources of Zn and P used were (ZnSO4.7H2O) and potassium dihydrogen phosphate

(KH2PO4), respectively. The pots were arranged according to a completely randomized design in the

glasshouse in duplicates.

Table 3.1. Physico-chemical properties of soils used in the incubation trial

Soil Characteristic Unit Clay Loam

Loamy Sand

Sand % 42 48 82

Silt 12 35 13

Clay 46 17 5

CaCO3 % 4.5 5.0 5.2

Organic Matter 0.3 0.4 0.2

ECe dS m−1

2.3 2.4 2.5

pHs --- 8.5 8.6 8.2

AB-DTPA-extractable Zn mg kg−1

0.8 1.4 0.9

Olsen P 6.4 10.6 8.6

Throughout the experimental period, the mean temperatures in the glasshouse were 30 ± 5 °C at

different times of the day and 20 ± 3 °C during the night. In all pots, moisture content at field

capacity was maintained by distilled water throughout the investigational period.

3.3.3. Availability and adsorption of applied nutrients

Subsamples of incubated soils were collected at 20, 40, 60, 90 and 120 days after incubation.

From each subsamples, AB−DTPA extractable P was determined on a spectrophotometer

(Biotechnology Medical Services, UV-1602, BMS, Canada Inc.) by developing blue colour

(Soltanpour, 1985).

35

Table 3.2. Analysis of variance for P and Zn at varying Zn and P levels

Source DF P Zn

F value P F value P

P applied 2 149428 0.00 2587 0.00

Time 4 10392 0.00 2752 0.00

Zn applied 2 345 0.00 709432 0.00

Soil 2 13472 0.00 126186 0.00

P applied × Time 8 885 0.00 14.0 0.00

P applied × Zn applied 4 9.36 0.00 54.6 0.00

P applied × Soil 4 165 0.00 24.4 0.00

Zn applied × Time 8 2.17 0.03 4055 0.00

Time × Soil 8 83.4 0.00 774 0.00

Zn applied × Soil 4 6.75 0.00 7437 0.00

P applied × Time × Zn applied 16 0.07 1.00 0.36 0.99

P applied ×Time × Soil 16 0.94 0.52 0.27 0.99

P applied × Zn applied × Soil 8 3.13 0.00 10.2 0.00

Time × Zn applied × Soil 16 0.04 1.00 46.9 0.00

P applied × Time × Zn applied × Soil 32 0.03 1.00 0.11 1.00

Error 135

Total 269

CV (%) 1.24 0.75

Zinc was also measured in the same extraction on an atomic absorption spectrophotometer (Thermo

Scientific 3000 Series, Waltham, MA, USA).

The data were fitted to linear and Michaelis-Menten models of nutrient adsorption. The linear

equation used to describe the data was;

Y = a x + b

Where Y is Zn or P adsorption (mg kg−1

) in a day, a is slope that is change in nutrient adsorption (mg

kg−1

) per unit change in time (days), x is extraction time (days) and b is intercept.

Michaelis-Menten equation was used to describe Zn and P adsorption as a function of time (Zhu et

al., 2002):

Q(t)= (Qmax×t)/(k+t)

36

where, t is incubation time (days); Q(t) is the amount of Zn or P (mg kg−1

of soil) adsorbed at time t

(days); Qmax is the maximum Zn or P adsorption (mg kg−1

of soil) at a given time t that is obtained

from linear equation (1/b); k is constant at >50% adsorption and it is obtained by k = a Qmax. When

the sorption activity follows Michaelis-Menten model, a reciprocal graph of 1/Q vs 1/t is made.

3.3.4. Statistical analysis

Analysis of variance was based on four way factorial design at P≤0.05 (Steel et al., 1997). Various

statistical computations were run on Statistix 9®

for Windows (Analytical Software, Tallahassee,

USA). The computer package Excel Graphics was used for the preparation of graphs.

3.4. Results

3.4.1. Availability and adsorption of phosphorus

Influence of applied Zn and P rates on the extractable P in differently textured soils at different time

intervals can be seen from Table 3.2. At all Zn levels and in all the extractions, the concentration of

extractable P significantly increased with increasing P application. On average, greater available P

was found in loam soil followed by clay and loamy sand.

Time (days) also had a significant effect on the availability of applied P in the soils (Figure

3.1). With time, more P was un-extractable from the soils and soil solution P was found to become

constant after 55 to 60 days of incubation. At both soil Zn levels, increasing the P level from 40 to

120 mg kg−1

significantly increased extractable P. Increase in the level of P applied from 40 to 120

mg P kg−1

of soil, 35.93−107.17 mg P kg−1

of soil was fixed in clay soil, 35.4-106 in loam and

34.77−106.26 in loamy sand soil respectively after 20 days of incubation.

Linear model of nutrient adsorption clearly indicated that P adsorption (Y) increased with

increase in Zn levels from 0 to 4 mg kg−1

soil (Table 3.3). Twenty days after the incubation,

maximum P adsorption (Qmax) of 36.9 mg P kg−1

in clay soil followed by loam (36.23 mg P kg−1

)

and loamy sand (35.02 mg P kg−1

) soils were observed at nutrient application level of 40 mg kg−1

P

and 4 mg Zn kg−1

soil. Adsorption of P level increased as there was increased in P applied level from

37

40 to 120 mg kg−1

soil. However, higher application of P level (120 mg P kg−1

soil) also had positive

influence on P adsorption at all Zn application levels (Table 3.3).

The coefficient of linear regression, R2, was always >93 (Table 3.3). Therefore, Michaelis-

Menten model is suitable to present changes in P adsorption over crop growth period (Fig. 3 and

Table 3). Parameters of Michaelis-Menten model varied with applied level of nutrient and soil type

(Table 3). Zinc application levels had only a non significance effect on P maximum adsorption

(Qmax) in different soils. Maximum Qmax of 125 mg P kg−1 of

soil was found in clayey soils whereas

loam and loamy sand had Qmax of 111 mg P kg−1

. Value of rate constant, k varied with soil. The

results showed that P adsorption decrease as the k value increase. Highest k value was found in

loamy sand soil (1.15−1.14) followed by loam (1.23−1.35) and clay (0.85-0.92) respectively (Table

3.3).

3.4.2. Availability and adsorption of zinc

Zinc adsorption significantly changed over time in soil types at varying P and Zn application levels.

Under controlled environmental conditions, Zn concentration in all soils decreased with time, but

attained equilibrium at about 55 to 60 days after incubation (Figure 3.2). Increased P application

levels (40 to 120 mg P kg−1

of soil), significantly decreased Zn concentration in all soil types. For

example, increase in applied P @ 40 to 120 mg kg−1

of soil, a decrease of Zn concentration about 25

and 32% in clay soil, 32 and 31% in loam soil and 32 and 31.5% in loamy sand soil was found

respectively (Figure 3.2b and c). At all P application rates (0, 40 and 120 mg P kg−1

of soil), there

was a significant decrease in Zn concentration at 4 mg Zn kg−1

of soil Zn application (Figure 3.2). At

higher Zn rate (12 mg Zn kg−1

of soil) to varying P application levels (40 to 120 mg kg−1

of soil),

there was a substantial increase in Zn density as compared to all other applied P and Zn

combinations.

However, a decrease in soil Zn concentration was observed with increase in applied P level

(120 mg P kg−1

of soil). It was also noticed that when only Zn levels (4 and 12 mg Zn kg−1

of soil)

were applied, adsorbed P content was increased at higher rates than Zn and P combinations. It was

also inferred that Zn adsorption rate were changed with change in soil texture (Figure 3.2).

38

Table 3.3. Parameters of Lineweaver-Bulk and Michaelis-Menten models for adsorption of Zn and P

on differently textured soils Soil Treatment Lineweaver-Bulk Linear Expression Michaelis-Menten Model

A B R2 Qmax (1/b) K (a Qmax)

Model parameters for P adsorption

Clay P40Zn0 0.026 0.026 0.95 38.5 1.00

P40Zn4 0.022 0.026 0.95 38.5 0.85

P40Zn12 0.024 0.026 0.96 38.5 0.92

P120Zn0 0.008 0.008 0.95 125.0 1.00

P120Zn4 0.008 0.008 0.95 125.0 1.00

P120Zn12 0.008 0.008 0.96 125.0 1.00

Loam P40Zn0 0.027 0.027 0.93 37.0 1.00

P40Zn4 0.032 0.026 0.98 38.5 1.23

P40Zn12 0.035 0.026 0.96 38.5 1.35

P120Zn0 0.009 0.009 0.94 111.1 1.00

P120Zn4 0.009 0.009 0.95 111.1 1.00

P120Zn12 0.009 0.009 0.94 111.1 1.00

Loamy Sand P40Zn0 0.023 0.027 0.92 37.0 0.85

P40Zn4 0.031 0.027 0.95 37.0 1.15

P40Zn12 0.043 0.028 0.93 35.7 1.54

P120Zn0 0.009 0.009 0.95 111.1 1.00

P120Zn4 0.009 0.009 0.94 111.1 1.00

P120Zn12 0.009 0.009 0.93 111.1 1.00

Model parameters for Zn adsorption

Clay P0Zn4 0.362 0.271 0.94 3.7 1.34

P40Zn4 0.431 0.272 0.97 3.7 1.58

P120Zn4 0.417 0.269 0.98 3.7 1.13

P0Zn12 0.229 0.090 0.95 10.5 2.41

P40Zn12 0.228 0.096 0.95 10.4 2.38

P120Zn12 0.226 0.095 0.95 10.5 2.38

Loam P0Zn4 1.386 0.312 0.95 3.2 4.44

P40Zn4 1.411 0.309 0.97 3.2 4.17

P120Zn4 1.403 0.311 0.96 3.2 4.51

P0Zn12 0.310 0.098 0.96 10.2 3.16

P40Zn12 0.293 0.097 0.96 10.3 3.02

P120Zn12 0.269 0.097 0.96 10.3 2.77

Loamy Sand P0Zn4 0.384 0.270 0.97 3.7 1.42

P40Zn4 0.438 0.270 0.98 3.7 1.61

P120Zn4 0.415 0.271 0.98 3.7 1.53

P0Zn12 0.201 0.098 0.95 10.6 2.14

P40Zn12 0.206 0.094 0.97 10.6 2.19

P120Zn12 0.196 0.094 0.96 10.6 2.09

39

Figure 3.1. Temporal changes on P adsorption under different Zn and P rates. Under script shows P

and Zn rates in mg kg−1

of soil.

40

Figure 3.2. Temporal changes on Zn adsorption under different Zn and P rates. Under script shows

P and Zn rates in mg kg−1

of soil.

41

Time (days) also has also a great effect on the Zn adsorption in all textured soils (Figure 3.2).

As time passed, more Zn was found to be adsorbed in all soils and it was found to become constant

after 55 to 60 days of incubation. At 120 mg P kg−1

of soil, increasing the Zn levels from 4 to 12 mg

Zn kg−1

of soil significantly increased extractable Zn from 3.33−4.13 in clay, 4.30-2.99 in loam and

3.11−2.99 in loamy sand soil respectively after 20 days of incubation. More the level of Zn (4 to 12

mg Zn kg−1

of soil) applied, more adsorption of Zn was found in all soils (Figure 3.2).

As from linear equation, Zn adsorption (y) depended upon slope (change in nutrient

adsorption with time) and Zn application levels rates. Slope value was maximum for loam soil

followed by clay and loamy sand for Zn adsorption. Linear model of Zn adsorption noticeably

showed that Zn adsorption (Y) increased with increase in P levels from 40 to 120 mg P kg−1

soil

(Table 3.3). Twenty days after the incubation, maximum Zn adsorption (Qmax) of 3.42, 3.41 and 2.63

mg Zn kg−1

was in clay, loamy sand and loam soil respectively at nutrient application level of 4 mg

Zn kg−1

and 40 mg P kg−1

soil respectively. However, Zn adsorption increased as there was

increased in P to120 mg P kg−1

soil to 3.53, 3.42 in clay and loamy sand soil respectively. More Zn

adsorption was found in clay soil followed by loamy sand and loam soil respectively (Table 3.3).

Zinc linear correlation regression was maximum of R2

>94 (Table 3) and from the data,

Michaelis-Menten model is suitability for all soil types on the growth of crops (Fig. 3.4 and Table

3.3). Variation in applied nutrients and soil types change the parameters of Michaelis-Menten model.

Phosphorous applications levels have significantly decrease Zn maximum adsorption (Qmax) in

different soils. Maximum Zn adsorption of 10.64 mg Zn kg−1

soil was found in loamy sand, 10.53

mg Zn kg−1

soil in clay and 10.31 mg Zn kg−1

soil in loam soil respectively. Value of k is dependent

on soil types and Zn adsorption decrease as increase in k value. Maximum k value was observed in

loam soil (4.51-2.77) followed by clay (2.41-1.13) and loamy sand (2.19-1.42) respectively (Table

3.3).

42

Figure 3.3. Lineweaver-Bulk expression of P adsorption data in three different textured soils

43

3.5. Discussion

As for most of the Pakistani soils (Maqsood et al., 2015; Alloway, 2009), the soil types under this

incubation experiment were alkaline calcareous and deficient in Zn and P; having <1.5 mg of AB-

DTPA extractable Zn kg−1

soil and <10 mg kg−1

soil Olsen-P (Table 3.1). On average, over 90%

soils of Pakistan are low in plant bioavailable P, resulting in adequate to high P insufficiency in the

soils (Iqbal et al., 2003). Moreover, most of the P is fixed in alkaline high pH (pH>7) and (Sharif et

al., 2000) calcareous (CaCO3>3%) soils. Calcareous soils are the main cause of Zn insufficiency in

soils (Table 3.1) with pH of 7.4 or more. Hence, increase in soil pH caused the reduction of soluble

Zn in the soils (Hussain et al., 2011; Alloway 2008). In calcareous soils, carbonates sorption with Zn

might be the other cause of low Zn phyto-availability (Lindsay, 1972b).

In present study, most of the fixations took place within 55 to 60 days after addition of Zn

and P but thereafter the solution attained equilibrium in the soil (Figure 3.1 and 3.2). Thus it was

evident that when Zn alone was added particularly of 12 mg Zn kg−1

soil, bioavailable P content of

soils decreased. More adsorption was found in loam soil followed by clay and loamy sand.

High application of P fertilizer induced Zn deficiency when soils are low in phyto-available

Zn. The resulting disorders are usually called "P-induced Zn deficiencies", and may require Zn

application (Loneragan et al., 1982). Additionally, Zn sorption by P fertilization might be due to the

possible reason of co-precipitation of Zn resulted in the formation of intermediary product (ZnHPO4)

of solid-solution (Agbenin, 1998).

Application of P decreased the content of AB-DTPA extractable Zn, the rate of Zn

adsorption gradually increased with increasing P level (Figure 3.2). There was depressive effect of P

on the extractable Zn that was found to be more apparent in respect of the initial Zn status of soil

than of the applied one (Megalah et al., 1993). Phosphorous fertilizations in soil may also enhanced

Zn sorption by increasing the net negative charges on Fe/Al oxides (Zhao et al., 2010). Therefore,

Zn adsorption is influenced by P fertilizations, consequential of increase in definite Zn sorption

locates

44

Figure 3.4. Lineweaver-Bulk expression of Zn adsorption data in three different textured

soils

45

Hence, it caused the reduction of bioavailable Zn in the soil. Phosphorous stimulated Zn sorption

and it may be related to increased surface negative charges and/or creation of specific sorption sites

resulting in increased surface Zn retention (Bolland et al., 1977; Barrow, 1987; Xie and Mackenzie,

1989). There was found the percentage adsorption decrease in added Zn and P as the clay content

decrease in soil textural classes and CaCO3 content as compared to other textural classes (Megalah et

al., 1993; Kalyanasundaram et al., 1970). More adsorption in different textured soils (Figure 3.1,

3.2) may be due to high clay and silt content in the soil (Armour et al., 1989).

Different variations in different soils were observed on the degree of Zn adsorption. More

adsorption was found in neutral soil, CaCO3 and pH incremental (Alloway, 2008; Harter and Naidu,

2001). The minimum Zn adsorption occurred in soils that received no P application (Figure 3.2d, g)

but the adsorption increase as the P application rates increased (Figure 3.2b, c and e-i). Rupa et al.

(2000) found that Zn availability was significantly decreased with the addition of P. Zinc

bioavailability is also associated with its distribution among various soil fractions that is helpful to

understand its chemistry and mobilization for plant uptake (Behera et al., 2009).

Higher P fertilization to the soils is also the main reason of soil Zn deficiency. Decrease in

plant Zn uptake was observed as increase in CaCO3 content and P fertilization (Spencer, 1960). Zinc

bioavailability in the soil was also reduced by higher P concentration in the soils (Mandal and

Mandal, 1990). Correspondingly, P fertilization to average Zn insufficient soils also increases the

problem of Zn deficiency in soil (Salimpour et al., 2010).

As P fertilization caused more sorption of Zn that might be due to the formation of Zn–P

complex in the soil environment. Lindsay (1972b) told that more soluble source of Zn in soil is Zn

phosphate [Zn(PO4)3·H2O]. If properly applied, this complex could be more efficient source of Zn

and P. However, adsorption of Zn increased simultaneously with its application rate, irrespective of

the textural type of soils (Diatta et al., 1998). Bingham and Garber, (1960) reported that P

fertilization enhanced the Zn solubility. However, contradiction findings are also present (Spencer,

1960) in which CaCO3 and P content present in the soil decreased the Zn bioavailability to crop

plants.

46

3.6. Conclusion

In conclusion, it can be inferred that Zn and P applied at different levels influenced the sorption

ability of different textured soils. Zinc and P availability was observed maximum at 12 and 40 mg

kg−1

respectively in all soil types. Soil Zn and P adsorption largely depend on incubation time and

soil types as after specific time period equilibrium stage reached and no further adsorption takes

place after that. Similarly, more Zn and P adsorption was observed in loam, followed by clay and

loamy sand soils. Model bioavailability adsorption is the logical approach to the complexity of this

approach. Using Michaelis-Menten equation, one can estimate adsorption of a specific nutrient in

different textured soils at a specific time that may be helpful for soil and crop specific

recommendations for Zn and P application to the farming community to attain maximum economic

benefits. Further research is also necessary to access the Zn-P interaction and their adsorption in

soils for yield and Zn-P uptake of the crops grown on the calcareous soils.

47

Chapter 4

EFFICIENCY OF ZINC AND PHOSPHORUS APPLIED

TO OPEN-POLLINATED AND HYBRID CULTIVARS

OF MAIZE

4.1. Abstract

Improving efficiency of applied nutrients is important to produce optimum crop yields with reduced

fertilizer inputs. Phosphorous (P) has antagonistic effect on zinc (Zn) uptake by plants and

information on the efficiency of these each nutrients in maize cultivars are limited. This study

evaluated the response of different levels of Zn (0, 9 mg kg–1

soil) and P (0, 40 mg kg–1

soil) on

growth, nutrient uptake and their utilization efficiency in four maize cultivars differing in their

growth behavior (DK–6142, P1543, Neelam and Afghoi) when grown under natural greenhouse

conditions. Maize cultivars significantly differed for above given traits and among treatments,

combined Zn+P application increased dry matter, nutrient uptake and their efficiency as compared

with control. Agronomic, physiological and recovery efficiency of P increased in Neelam, Afghoi

and DK–6142 cultivars with Zn applied and vice versa. Afghoi and DK–6142 cultivars were more

responsive for agronomic, physiological and apparent Zn and P recovery efficiency than other ones.

For P1543 cultivar, Zn and P physiological efficiency decreased while recovery efficiency increased,

respectively, with combined application of both nutrients. However, for each of the nutrients

utilization efficiency, none of these were related to open pollinated or hybrid maize cultivars and

rather dependent on genetic makeup for internal higher utilization efficiency. Overall, nutrient

efficiency of applied Zn or P are interdependent on each other and maize cultivars had a differential

response to their applications.

4.2. Introduction

Zinc (Zn) and phosphorous (P) are important nutrients for growth of plants and often deficient in

calcareous and high pH soils (Gianquinto et al., 2000; Imran et al., 2015, 2016).

________________________________________________________________________________

The study has been published in International Journal of Agriculture and Biology (2016) 18:1249-

1255; DOI: 10.17957/IJAB/15.0239) and entitled „Efficiency of zinc and phosphorus applied to

open-pollinated and hybrid cultivars of maize ‟. The authors are M. Imran, A. Rehim, S. Hussain,

M.Z. Hye and H. Rehman

48

Soil deficiency of the one nutrient can regulate plant status of the other, as excessive Zn application

to soil has been shown to decrease P concentration in plants (Soltangheisi et al., 2013). Under soil

Zn deficiency, uptake of P by roots and its accumulation in leaves increases (Cakmak et al., 1986;

Huang et al., 2000; Khan et al., 2014). On other hand, excessive application of P fertilizers to soil

decreases plant available Zn (Lambert et al., 2007; Zorrig et al., 2010). Zinc and P fertilization has

negative interaction for each other with respect to their concentration and uptake by maize plants and

individual application of each to soil improves their uptake and concentration. However, P

application when combined with Zn increases Zn concentration in plants that might be due to

dilution effect of increased shoot growth rather than reduced Zn uptake by roots (Singh et al., 1988).

On other hand, some evidences support that interaction of P and Zn occurs within plants and not in

soils (Cakmak et al., 1986; Khan et al., 2014).

Maize is an exhaustive crop which requires high P and also sensitive to Zn deficiency (Sattar

et al., 2001; Imran and Rehim, 2016). Breeding for nutrient efficient cultivars of maize can be a best

approach to manage deficiencies of Zn and P. However, this will require avoiding speculated

antagonistic interaction between P and Zn especially for calcareous soils where both P and Zn are

widely deficient and recommended for optimum crop growth (Maqsood et al., 2015).

Maize genotypes differ in uptake and efficiency of applied nutrients (Hussain et al., 2012).

Nutrient efficiency of crop plants depends on their performance in soils of low nutrient status. The

efficiency of a certain nutrient for genotype depends on its higher uptake from a deficient soil, better

translocation to aerial parts and better utilization of absorbed nutrients within plant body (Clark,

1990). For example, P–efficient crop cultivars are designed to grow in soils deficient with P and Zn

in many cases; hence, interaction between P and Zn uptake efficiency become important when P

supply may affect the expression of uptake efficiency of Zn in crop plants (Zhu et al., 2001). Hence,

it is essential to measure the influence of soil P bioavailability on Zn utilization efficiency in

different maize cultivars. Further, improving fertilizer use efficiency by maintaining an appropriate

level of each nutrient is of primary importance from economic perspective and for sustainable

agriculture (Welch and Graham, 2005). Therefore, P–Zn interaction in maize for improving nutrient

utilization efficiency needs particular attention. The present study has been therefore planned with

the objectives to study the dependence of nutrient utilization efficiency of applied P and Zn on each

other in maize cultivars contrasting in their growth behavior.

49

4.3. Materials and methods

4.3.1. Soil Physico-Chemical Analysis

Bulk soil samples (0–15 cm depth) were collected from Agricultural Research Farm of Bahauddin

Zakariya University, Multan. Collected soil was sun dried, thoroughly mixed and crushed to pass

through a 2 mm sieve. A subsample of the soil was analyzed for physico-chemical characteristics by

following standard methods. Hydrometer method was used for the determination of soil textural

class (Gee and Bauder, 1986). Soil EC and pH was measured in 1:1 soil to water suspension.

Organic matter (OM) and free lime (CaCO3) was determined by Walkley–Black (Nelson and

Sommer, 1982) and acid dissolution methods (Allison and Moodie, 1965). Zinc was extracted with

AB-DTPA (Soltanpour, 1985) and its concentration in the extract was estimated on an atomic

absorption spectrophotometer (Perkin Elmer, AAnalyst 100, Waltham, USA). Soil was extracted

with sodium bicarbonate (Olsen and Sommers, 1982) and plant available P in soil was

determined on spectrophotometer (Biotechnology Medical Services, UV-1602, BMS, Canada).

Table 4.1. Physical and chemical properties of soil used in the experiment

Soil Property Unit Value

Textural class --- Loam

Sand % 42.8

Silt % 39.3

Clay % 17.9

pH (1:1 ratio) --- 8.04

EC (1:1 ratio) dS m−1

0.55

CaCO3 % 4.63

Organic matter % 0.47

AB-DTPA extractable Zn mg kg−1

0.93

Olsen P mg kg−1

9.57

50

4.3.2. Experimental Details

Five (5) kg of thoroughly mixed soil was filled each in 48 polyethylene lined plastic pots. Two Zn

levels (0 or 9 mg Zn kg–1

soil as ZnSO4.7H2O) and two P rates (0 or 40 mg P kg–1

soil as KH2PO4)

were applied in all possible combinations to four cultivars of maize (open pollinated cultivars

Neelam and Afghoi, and hybrid cultivars DK–6142 and P1543). Basal rates of 60 mg N kg–1

and 60

mg K kg–1

to soil were added respectively as urea and potassium sulphate. Before sowing, soil in all

the pots was moistened with distilled water, dried and thoroughly mixed for equilibration. The pots

were arranged according to a two factorial completely randomized design in a glasshouse (Steel et

al., 1997).

Five seeds of each maize cultivar were sown per pot. Ten days after germination, three plants

were maintained in each pot. Distilled water was used to maintain moisture contents at field capacity

in all the pots during the experimental period. Two weeks after sowing, a second dose of 30 mg N

kg-1

soil as urea was applied uniformly to each pot. For plant roots and shoots, seedlings were

harvested 30 days after sowing, washed with distilled water and blotted dry with tissue papers. The

plant samples were oven dried at 70 °C to a constant weight for dry matter yield. Finely ground plant

samples were digested in di-acid mixture (2:1 ratio of HNO3:HClO4). An atomic absorption

spectrophotometer was used to measure Zn concentration in the digest. Phosphorous in the digested

material was analyzed by metavanadate yellow color method (Chapman and Pratt, 1961a). Shoot P

and Zn contents were calculated as: Nutrient content (mg pot–1

) = shoot dry matter (SDM, g pot–1

) ×

shoot nutrient concentration (mg g–1

).

4.3.3. Phosphorus and Zn Utilization Efficiency

Phosphorous and Zn utilization efficiency was calculated as (Siddique and Glass, 1981):

( )

( ) ( )

(Equation i)

51

( )

( ) ( )

(Equation ii)

Agronomic, physiological and recovery efficiencies were calculated as (Gerloff and Gabelman,

1983):

( )

(Equation iii)

( )

(Equation iv)

( )

(Equation v)

4.3.4. Statistical Analysis

Analysis of variance (ANOVA) was based on two factorial completely randomized design. Least

Significant Difference (LSD) test was used to compare various significantly treatments mean

(Steel et al., 1997). Computer based software; Statistix 9®

was used for statistical analysis.

4.4. Results

4.4.1. Effect on Plant Growth

There was significant effect of Zn or P treatments, cultivars and their interaction on shoot fresh and

dry matter yield of maize (Table 4.2). Shoot fresh and dry matter of three cultivars (Afghoi, P1543

and Neelam) was highest for combined Zn and P treatment followed by individual application.

However, maximum shoot fresh and dry matter in DK–6142 was recorded for individual P treatment

followed by Zn+P treatment (Table 4.2). Root fresh weight of Afghoi and Neelam was highest for

combined Zn+P treatment followed by individual applied P. However, P1543 produced maximum

shoot fresh and dry weights by individual applied Zn. Apart from this, root dry weight was highest in

all the four cultivars with combined Zn+P followed by individual P treatment.

52

4.4.2. Nutrients Concentration in Plant Tissues

Zn and P concentrations in shoot and root tissues varied with genotype, nutrient and their

interactions (Table 4.3). In interactive effects, Zn concentration in shoots and roots ranged from 29.8

to 16.9 μg g−1

and 30.4 to 17.4 μg g−1

in all maize cultivars respectively. As compared to control,

shoot and root Zn concentrations were significantly improved by Zn+P fertilization in DK–6142

followed by P1543, Neelam and Afghoi cultivars.

Table 4.2. Effect of Zn and P applications on growth attributes of four maize cultivars

Cultivars

Shoot Fresh

Weight

(g pot-1

)

Shoot Dry

Matter

(g pot-1

)

Root Fresh

Weight

(g pot-1

)

Root Dry

Matter

(g pot-1

)

P0Zn0

Neelam 56.1 ± 0.6 e–g 9.1 ± 0.1 g 25.0 ± 1.4 h 4.5 ± 0.1 k

Afghoi 59.3 ± 0.8 de 9.8 ± 0.1 f 29.7 ± 2.4 ef 6.0 ± 0.3 h

DK–6142 55.3 ± 3.1 fg 8.0 ± 0.3 i 27.1 ± 0.8 g 5.3 ± 0.2 j

P1543 54.8 ± 3.7 g 9.3 ± 0.1 g 38.4 ± 0.5 b 7.4 ± 0.1 f

P0Zn9

Neelam 60.8 ± 2.1 d 9.3 ± 0.1 g 30.6 ± 0.4 d–f 6.5 ± 0.2 g

Afghoi 66.5 ± 2.8 c 10.1 ± 0.4 e 31.4 ± 1.7 c–e 7.3 ± 0.1 f

DK–6142 59.1 ± 0.9 d–f 8.6 ± 0.2 h 29.0 ± 0.9 f 5.7 ± 0.1 i

P1543 58.5 ± 1.6 d–g 10.3 ± 0.1 e 38.6 ± 0.1 b 7.5 ± 0.1 ef

P40Zn0

Neelam 61.7 ± 0.9 d 10.7 ± 0.1 d 30.8 ± 0.7 c-f 7.4 ± 0.3 ef

Afghoi 75.4 ± 1.8 b 10.7 ± 0.3 d 32.7 ± 0.1 c 7.7 ± 0.1 e

DK–6142 59.8 ± 3.2 de 8.6 ± 0.1 h 30.4 ± 1.5 ef 7.2 ± 0.1 f

P1543 60.4 ± 0.7 d 10.9 ± 0.1 d 38.5 ± 0.4 b 7.5 ± 0.2 ef

P40Zn9

Neelam 69.0 ± 0.4 c 11.4 ± 0.1 c 32.4 ± 0.2 cd 8.7 ± 0.1 c

Afghoi 84.8 ± 0.5 a 13.4 ± 0.5 a 38.4 ± 1.2 b 9.2 ± 0.4 b

DK–6142 67.3 ± 1.7 c 10.3 ± 0.1 e 37.2 ± 0.9 b 8.2 ± 0.1 d

P1543 75.1 ± 1.4 b 11.9 ± 0.1 b 51.3 ± 2.0 a 9.9 ± 0.2 a

Means not sharing the same letter within a column differ significantly at 5% level of probability by

LSD test and ± indicate standard deviation of three replications. The subscript numbers after P and

Zn shows their respective application rates in mg kg-1

of soil.

53

Phosphorous concentration ranged from 1.61 to 2.85 g kg−1

in shoots and 1.56 to 2.80 g kg−1

in roots (Table 4.3) and highest was recorded for combined Zn+P treatment followed by individual P

and Zn. Among cultivars, P concentration in plant tissues was greater for DK–6142 and P1543 as

compared to Neelam and Afghoi.

Highest shoot Zn uptake in all the cultivars was found for combined Zn+P application

followed by individual Zn and P treatments (Fig. 4.1). Similarly, shoot P uptake also increased with

combined Zn+P application; however, it was greater for individual P applied following combined

Zn+P.

Table 4.3. Effect of Zn and P applications on Zn and P concentration in four maize cultivars

Cultivars

Shoot Zn

(μg g−1

)

Root Zn

(μg g−1

)

Shoot

Phosphorous

(g kg−1

)

Root

Phosphorous

(g kg−1

)

P0Zn0

Neelam 18.79 ± 0.01 j 19.14 ± 0.03 j 1.61 ± 0.04 m 1.58 ± 0.03 m

Afghoi 18.25 ± 0.05 k 18.61 ± 0.05 k 1.77 ± 0.01 l 1.73 ± 0.01 l

DK–6142 19.53 ± 0.17 i 19.93 ± 0.07 i 1.86 ± 0.01 jk 1.81 ± 0.02 jk

P1543 18.98 ± 0.03 j 19.36 ± 0.26 j 1.90 ± 0.01 ij 1.85 ± 0.2 ij

P0Zn9

Neelam 25.70 ± 0.07 g 26.20 ± 0.02 g 1.82 ± 0.03 k 1.78 ± 0.03 k

Afghoi 25.01 ± 0.04 h 25.51 ± 0.04 h 1.84 ± 0.03 k 1.80 ± 0.03 k

DK–6142 26.71 ± 0.03 e 27.25 ± 0.12 e 1.93 ± 0.03 hi 1.89 ± 0.03 hi

P1543 26.01 ± 0.04 f 26.53 ±0.04 f 1.97 ± 0.03 h 1.93 ± 0.03 h

P40Zn0

Neelam 17.38 ± 0.41 m 17.72 ± 0.07 m 2.05 ± 0.05 g 2.02 ± 0.06 g

Afghoi 16.94 ± 0.04 n 17.29 ± 0.07 n 2.13 ± 0.02 f 2.08 ± 0.03 f

DK–6142 18.05 ± 0.09 k 18.43 ± 0.12 k 2.23 ± 0.03 e 2.19 ± 0.03 e

P1543 17.62 ± 0.07 l 18.01 ± 0.03 l 2.28 ± 0.03 e 2.23 ± 0.03 e

P40Zn9

Neelam 28.61 ± 0.02 c 29.19 ± 0.27 c 2.44 ± 0.03 d 2.38 ± 0.02 d

Afghoi 28.15 ± 0.25 d 28.72 ± 0.25 d 2.53 ± 0.02 c 2.47 ± 0.02 c

DK–6142 29.75 ± 0.05 a 30.35 ± 0.18 a 2.85 ± 0.02 a 2.79 ± 0.03 a

P1543 29.27 ± 0.09 b 29.91 ± 0.07 b 2.71 ± 0.02 b 2.65 ± 0.03 b

Means not sharing the same letter within a column differ significantly at 5% level of probability by

LSD test and ± indicate standard deviation of three replications. The subscript numbers after P and

Zn shows their respective application rates in mg kg-1

of soil.

54

Fig. 4.1. Effect of Zn and P applications on uptake of Zn and P by maize cultivars. Means not

sharing the same letter within a box differ significantly at 5% level of probability by LSD test and

error bars indicate ± standard deviation of three replications.

55

4.4.3. Phosphorus and Zinc Utilization Efficiency

Zinc and P utilization efficiency varied significantly with treatments and their interactions (Fig. 4.2).

Among interactions, Zn utilization efficiency ranged from 399 to 627 g−2

of shoot dry weight in all

maize cultivars. As compared to control, Zn utilization efficiency increased by individual applied P

in Afghoi followed by P1543, Neelam and DK–6142 cultivars for combined Zn+P and individual

applied Zn treatments, respectively. Nonetheless, zinc utilization efficiency increased with shoot dry

weight and decreased with shoot Zn concentration (Fig. 4.2A).

Phosphorus utilization efficiency was dependent on applied Zn. All the cultivars had

maximum P utilization efficiency for Zn application only (5.61 to 4.79 g−2

shoot dry matter mg−1

of

shoot P) followed by combined Zn+P and individual Zn application (Fig. 4.2B).

Agronomy efficiency for Zn or P signficantly increased either with or without of each

nutrient applied and highest of it was observed in Afghoi except for P1543 when recommnded level

of both nutrients was applied (Fig. 4.3A).

Physiological Zn efficeincy was significantly highest for Agfhoi and DK–6142 cultivars at

recommended level of P as compared to control (Fig. 4.4A). Similarly, physiological efficiency for P

increased significantly in all maize cultivars from indiviual P to combined Zn+P application except

P1543 cultivar with highest efficiency when no Zn applied (Fig. 4.4B).

Among maize cultivars, highest apparent Zn recovery efficiency was observed in Afghoi at

recommended level of P as compared to control (Fig. 4.5A and 4.5B). Likely, highest apparent P

recovery efficeincy was observed for Afghoi followed by DK–6142, Neelam and P1543 when

recommended Zn applied.

56

Fig. 4.2. Utilization efficiency of Zn and P in maize cultivars affected by applied P and Zn levels.

Means not sharing the same letter within a box differ significantly at 5% level of probability by LSD

test and error bars indicate ± standard deviation of three replications. ZnUE and PUE indicate Zn

utilization efficiency and P utilization efficiency respectively.

57

Fig. 4.3. Agronomic Zn and P efficiency of maize cultivars at control (without) and recommended

(with) rates of P and Zn. Means not sharing the same letter within a box differ significantly at 5%

level of probability by LSD test. Error bars indicate ± standard deviation of three replications.

58

4.5. Discussion

Due to Zn and P deficiency in calcareous soils (Table 4.1), combined application of Zn+P improved

plant growth attributes in all maize cultivars (Table 4.2). Shoot concentration of micronutrients may

play critical role in accumulation of it in grain (Cakmak et al., 2010).

Concentration of Zn in plant tissues was improved by Zn fertilization and decreased with application

of P (Table 4.3). It might be due to possibility of individual applied P to induce Zn deficiency due to

higher than adequate shoot P concentration in maize plants or yield dilution due to increased dry

matter with P application (Das, 2005b; Rehim et al., 2014).

However, extent of variation in Zn concentration with P application was genotype specific

(Table 4.3). Hence, it is essential to measure the influence of soil P bioavailability on Zn uptake and

utilization efficiency in different maize species. Zinc efficient genotypes can increase its

translocation to the shoot and regulate P transport in order to maintain balanced nutrient of Zn

(Cayton et al., 1985). The higher Zn uptake in maize plant was associated with a higher grain yield

(Fageria et al., 2008).

Combined application of both Zn+P caused increase in shoot P concentration (Table 4.3) that

relates with increased soil P supply probably to intensification of root system (Taiz and Zeiger,

2008). Application of P tends to decrease the root Zn concentration and this might be due to

formation of insoluble complex with Zn where deficient in soil (Li et al., 2003; Sarwar et al., 2010,

2015).

As compared with control without P or Zn, Zn uptake in the shoots was improved by the Zn

and decreased with P application (Fig. 4.1). Maximum P and Zn uptake in maize shoot was found by

Zn+P application (Fig. 4.1). Excessive P application without Zn fertilization reduces Zn uptake in

maize (Nichols et al., 2011).

Zinc utilization efficiency decreased in maize cultivars with combined Zn+P application to

plants grown with only P application (Fig. 4.2). Differential P and Zn utilization efficiency in maize

cultivars was influenced by P–Zn interactions (Gill et al., 2004). Maximum nutrient use efficiency

(NUE) at low nutrient rate would possibly be due to intense root structure in the soil causing

59

efficient utilization of applied nutrient. At higher nutrient fertilization, plants used smaller fraction

of fertilizer nutrient which causes decrease in low NUE (Rehim et al., 2012).

Different cultivars also have different P and Zn use efficiencies (Irshad et al., 2004).

However, degree of variation in Zn concentration under P applications may be genotype specific.

Genotypic variations in response to P and Zn deficiencies could be exploited to increase crop

production in soils low in available Zn and P and can be a better strategy in low input sustainable

agriculture systems especially in developing countries. Individual applied P also reduces grain Zn

that ultimately decreases the nutritional quality of cereal grain which is a major concern (Cakmak,

2002). Hence, combined fertilization of Zn+P may also be good to improve better nutritional quality

of maize crop (Gill et al., 2004).

In fact, plant physiological and genetic components affect uptake and utilization of nutrients

under many ecological and environmental surroundings (Baligar et al., 2001). The nutrient

utilization efficiency for the shoot dry matter decreased with increasing nutrient supply to roots

(Furlani et al., 2005). In present study, Zn utilization efficiency of all the tested maize cultivars was

highest for individual applied nutrients (Fig. 4.2). Among cultivars, DK–6142 had highest Zn as well

as P utilization efficiency as compared to all other cultivars. According to Furlani et al. (2005),

genetic make up for different varieties have their different nutrient utilization efficiency. Fageria et

al. (2008) demonstrated that higher Zn utilization efficiencies of cereals were found as compared to

legumes and these are associated with higher grain yields of maize. Differences in nutrient

utilization efficiency may also be attributed to difference in internal utilization efficiency of cultivars

contrasting in their growth rates.

Recovery efficiency of Zn and P was significantly higher in the presence of other nutrient

(Fig. 4.5). However, recoveries of applied Zn and P by four cultivars of maize were low from the

soil and never more than 50%. Low efficiencies might be due to nutrients fixation on soil colloids

(Baligar et al., 2001). These losses could possibly add towards the soil degradation and high

fertilizer demand. Higher nutrient efficiency by crops not only reduces the fertilizer input costs but it

decreases the nutrient losses and protect the environment. Modern genotypes of different crops are

more efficient in absorption and utilization of nutrients from the soil as compared to obsolete

cultivars (Clark and Duncan, 1991).

60

Fig. 4.4. Physiological Zn and P efficiency of maize cultivars at control (without) and recommended

(with) rates of P and Zn. Means not sharing the same letter within a box differ significantly at 5%

level of probability by LSD test. Error bars indicate ± standard deviation of three replications.

61

Fig. 4.5. Apparent Zn and P recovery efficiency of maize cultivars at control (without) and

recommended (with) rates of P and Zn. Means not sharing the same letter within a box differ

significantly at 5% level of probability by LSD test. Error bars indicate ± standard deviation of three

replications.

62

Highest agronomic, physiological and apparent recovery efficiency for Zn or P is directly

correlated with grain yield and fertilizer use efficiency (Fageria et al., 2005). Agronomic and

physiological efficiency of applied Zn and P also significantly increased with either of nutrients

applied (Fig. 4.3 and 4.4). If appropriate Zn and P quantities are selected for crop production, the

relationship between these two nutrients could turn to be positive or stabilizer and more affective

(Srivastava et al., 2014). Efficient cultivars may absorb more quantities of nutrients from deficient

soils by maintaining required physiological processes and higher enzyme activities. By this, efficient

cultivars may produce greater yields with low fertilizer rates.

63

4.6. Conclusion

This study revealed significant effects of Zn and P application on growth, nutrient concentration and

efficiency in four maize cultivars. The study also demonstrated that optimum Zn supply may not

have antagonist effect on P uptake by maize plants. Further, NUE was highly genotype dependent. It

was observed that both P and Zn applied to calcareous soils, having deficiency of Zn and P,

increased efficiency of each other. This is possibly due to the improved metabolism which may

enhance uptake and assimilation of plant nutrients. In conclusion, balanced nutrition of P and Zn in

nutrient–deficient calcareous soils is imperative for optimum plant growth and NUE.

64

Chapter 5

COMPARISON OF ZINC AND PHOSPHOROUS RATES

IN MAIZE HYBRID AND NON-HYBRID FOR ZINC

BIOFORTIFICATION UNDER FIELD CONDITIONS

5.1. Abstract

Phosphorous (P) and zinc (Zn) are found to interact widely in soil-plant systems. This interaction

may leads to deficiency of one or both nutrients interacting with each other in case of antagonistic

interactions. In the current investigation, a field trial was conducted to study the interactive effect of

P (0 and 60 kg ha–1

) and Zn (0, 16 kg ha–1

) on growth, yield and grain Zn content of two maize (Zea

mays L.) cultivars i.e. Neelam and DK–6142. Both the cultivars showed increased growth and yield

with Zn and P treatments as compared to unfertilized control, however, combined Zn and P

application was more productive than individual effects. Application of Zn+P improved grain Zn and

P concentrations by 52% and 32%, respectively as compared to control, averaged across genotypes.

Sole P application decreased grain Zn 10% over control. Combined P and Zn application also

decreased the grain [phytate] : [Zn] ratio significantly resulting in increased estimated human Zn

bioavailability in both maize genotypes. In conclusion, combined Zn and P fertilization appeared to

be more appropriate to enhance grain yield and grain Zn concentration in maize grain. However,

fertilization of Zn should be aimed to increase yield and grain Zn density should consider the

genotypic variations and P rate.

5.2. Introduction

Phosphorus and Zn insufficiencies are well-known dietary limitations for growth and yield in

agriculture worldwide and observed extensively (Marschner, 1995).

___________________________________________________________________________

The study has been published in Journal of Plant Nutrition and Soil Science (2016) 179:60−66, and

entitled „Zinc bioavailability in maize grains in response of phosphorous–zinc interaction‟. The

authors are M. Imran, A. Rehim, N. Sarwar and S. Hussain.

65

Higher P availability in the rhizosphere may induce Zn deficiency in plants by decreasing Zn uptake

and Zn translocation into the shoots (Robson and Pitman, 1983) but the mechanisms are still not

clear. Many studies have revealed that high P fertilization with low Zn supply remarkably increased

P absorption, which in turn caused P toxicity and symptoms are as in Zn deficiency (Cakmak and

Marschner, 1986; Webb and Loneragan, 1990). However, little information on the effect of P

availability on uptake and translocation of Zn in maize is available.

After wheat and rice crop, maize crop is of vital importance in cereal crops worldwide.

Maize grain is consumed as staple food worldwide comprising of Asia, Africa, America (Ortiz-

Monasterio et al., 2007; Menkir et al., 2008) and for animals forage (Harris et al., 2007). It provides

an expected 15% of the protein and 20% of the calories for the worldwide as well as a nutritional

staple crop for > 200 million individuals around the globe. Unluckily, maize grain fulfill a number of

macro and micronutrients for human dietary needs, but the amount of Zn is insufficient for persons

who depend on maize as a major staple food (Chomba et al., 2015; Nuss, et al., 2010). Particularly,

Zn may be limiting for the growth and development of young children (Kreb, 2013).

Genetic variation of Zn concentrations in maize grains has been reported (Maziya et al.,

2000; Banziger et al., 2000; Fahad et al., 2015). On an average, maize contains about 20 mg kg-1

Zn

concentration in grain (Ortiz et al., 2007). However, concentration and bioavailability are lower in

food. Many national and international ventures are focusing on Zn biofortification of cereal crops

(White and Broadley, 2009). The HarvestPlus Program set targets of Zn concentrations is 28 mg kg-1

in polished rice, 38 mg kg−1

in maize and wheat, 56 mg kg−1

in beans, 66 mg kg−1

in pearl millet, 70

mg kg−1

in roots of sweet potatoes and 34 mg kg−1

in cassava roots (Bouis and Welch, 2010). Plant

species differ widely in their Zn requirements as well as high Zn tolerance in their tissues (Broadley

et al., 2007; Fageria, 2009). Maximum crop plant requires Zn density in leaves of 15-30 mg kg−1

for

optimum yield; however their growth might be inhibited by leaf Zn density of >100–700 mg kg−1

(Reeves and Baker, 2000; Broadley et al., 2007).

Maize consumption to meet energy and protein needs is not sufficient to avoid the risk of Zn

deficiency, especially in young children (Kreb, 2013). The bioavailability of minerals is reduced by

phytic acid (PA) in food crops (Lesteinne et al., 2005). Phytic acid is considered an anti-nutrient

because of its ability to form strong metal-ion complexes, mainly Zn2+

, Ca2+

, and Fe3+

(Harland and

66

Oberkas, 1987). As a strong chelating agent, PA reduces the bioavailability of Zn+2

by the

development of insoluble compounds (Weaver and Kannan, 2002). As a result, PA produces deficit

absorption of some dietary minerals e.g. Zn and Fe (Reddy and Pierson, 1994; Saharan et al., 2001).

Grain products have high concentrations of PA (Alabaster et al., 1996) and about 1-2% seed weight

is PA and it can reach up to 3-6%. 90% of PA are found in maize germ, while in rice and wheat it is

found to a larger extent in the external covers of husk and pericarp layer (Cheryan, 1980).

Imbalanced use of fertilizers is very critical among many other factors which may cause a

decline in maize yield. Maize requires rich nutrient supply which also requires appropriate supply of

micronutrients, especially Zn (Armani et al., 1999; Obrador et al., 2003) with macronutrients.

Moreover, enhanced yield with high yielding genotypes with the advent of Green Revolution has

further aggravated the issue (Dar, 2004). Due to intensive use of high-purity fertilizers, soils have

become deficient in micronutrients. Moreover, calcareousness of soils also intensified this

problematic. In developing countries particularly Pakistan, farmers generally apply nitrogen (N) and

P while no micronutrients, leading to micronutrients deficiency. One of the strategies to alleviate Zn

deficiency in maize-consuming populations in developing countries is Zn biofortification of maize

grains (Saltzman et al., 2013).

The present trial was conducted to explore the influence of Zn as well as P fertilization on

maize growth, yield, tissue concentrations of Zn and P, and estimated Zn bioavailability in two

contrasting maize genotypes.

5.3. Material and Methods

5.3.1. Growth Conditions

A field trial was carried out in the research area of the Soil Science Department of Faculty of

Agricultural Sciences and Technology (FAS&T), Bahauddin Zakariya University, Multan. Afore

crop planting, samples from soil surface (0–15 cm depth) were randomly collected. Randomly

collected surface samples were air-dried and these were to pass from 2 mm sieve. Then, these

surface soil samples were examined aimed at different physico−hemical properties (Table. 5.1). The

nature of the examined soil was calcareous and alkaline having low plant available Zn (AB–DTPA:

0.77 mg kg−1

) and P (Olsen–P: 7.49 mg kg−1

).

67

Table 5.1. Physical and chemical soil properties.

Character Unit Value Method

Textural class - Loam USDA classification

Sand % 47.5 Hydrometer method (Gee and Bauder, 1986)

Silt % 34.7

Clay % 17.8

pHs - 7.85 pH of saturated soil paste

ECe dS m–1

0.643 Electric conductivity of saturated soil paste extract

CaCO3 % 0.63 Acid dissolution (Allison and Moodie, 1965)

Organic Matter % 0.59 Walkley-Black method (Jackson, 1962)

AB-DTPA

extractable Zn

mg kg–1

0.77 Extracted with AB-DTPA

(Soltanpour, 1985)

Olsen P mg kg–1

7.49 NaHCO3 method (Olsen and Sommers, 1982)

One maize hybrid (Zea mays L. cv. Monsanto DK–6142) and one indigenous (cv. Neelam)

genotype were used in this experiment. Two levels of Zn (0, 16 kg ha-1

) and two levels of P (0, 60

kg ha-1

) were used in this trial. Zinc was applied as hydrated zinc sulfate (ZnSO4 × 7 H2O) and P as

di-ammonium phosphate. All Zn and P were applied at the time of sowing. Zinc was applied into the

field by mixing it in the soil. Randomized complete block design (RCBD) was used to arrange the

experimental units. The investigational plot was separated into three blocks having twenty four (24)

total plots. Each plot dimension was 3 m × 5 m as well as eighty nine (89) uniform size and healthy

maize seeds were sown in each plot. Plant−to−plant and row−to−row distance was maintained 22.5

and 75 cm, respectively. At sowing, plots were uniformly supplied with 140 N and 60 K (in kg ha−1

)

by applying urea and potassium sulfate, respectively. Zinc was applied 15 d after sowing. Thirty

days after the crop sowing date, second fertilization of N (60 kg N ha−1

) was applied. During the

entire investigational time span, soil moisture was maintained by canal water near to field capacity in

entire plots.

5.3.2. Plant Analysis

At maturity of the crop, entire plots were harvested as well as threshed manually check stover and

grain yield. Sub−samples of cobs and stover were taken representing each plot. Distill water was

used for the washing and cleaning of stover samples. Then these samples were sun−dried afore

68

oven−drying at 65°C for 72 h. after that, a mill with stainless blades was used to finely ground these

samples (IKA WERKE, MF 10 Basic, Staufen, Germany). Di-acid (HNO3:HClO4, ratio 2:1) mixture

was used for finely powdered samples by wet digestion procedure (Jones and Case, 1990). Atomic

absorption spectrophotometry (PerkinElmer, Analyst 100, Waltham, USA) instrument was used to

determine Zn density from the digested sample.

Vanadate-molybdate yellow color procedure was used for the determination of P density in

digested grain and shoot samples by spectrophotometrically (Chapman and Pratt, 1961a). For the

determination of PA in maize grain, To calculate grain phytate by indirect method, 0.05 g finely

ground grain aliquots were extracted in 10 mL of 0.2 N HCl at room temperature for 2 h constant

shaking. This method of phytate determination use pink color absorption developed by unreacted Fe

(III) with 2,2‟-bi-pyridine. In short, 0.5 mL extract was mixed with 1 mL 0.4 mM ammonium ferric

sulphate solution in a caped glass tube and heated in a boiling water bath for 30 min. Then, these

samples were allowed to adjust at room temperature. After adjusting, 2 mL of 2,2‟-bi-pyridine

solution (5 g dissolved in 500 mL water with 1% v/v thioglycollic acid) was added to the mixture

and the absorbance was measured by using a spectrophotometer (Shimadzu, UV–1201, Kyoto,

Japan) at 519 nm wavelength.. Phytate in the extract was calculated from a calibration curve

established using standard phytate solutions made with sodium phytate (Haug and Lantzsch, 1983;

Sigma-Aldrich P-8810, St. Louis, USA).

5.3.3. Human Zinc Bioavailability in Grain

Zinc as well as phytate density in food affects food Zn bioavailability. Hence in maize grain, molar

ratio of [phytate]:[Zn] was used for estimation (Brown et al., 2001; Weaver and Kannan, 2002) of

human Zn bioavailability.

For quantifiable estimation of bioavailable Zn, it was measured by using trivariate (Miller et al.,

2007) Zn absorption model.

( (

) √( (

))

)

69

From the above equation, AMAX, KR and KP are for maximum zinc absorption, equilibrium

dissociation constant of zinc-receptor binding reaction and equilibrium dissociation constant of zinc-

phytate binding reaction and their values are 0.091, 0.680 and 0.033, respectively and it is correlated

with human intestinal Zn homeostasis (Hambidge et al., 2010). In the model, total daily absorbed Zn

(TAZ; mg Zn d−1

) is a function of total daily dietary phytate (TDP; mmol phytate d−1

) and total daily

dietary Zn (TDZ; mmol Zn d−1

).

5.3.4. Statistical Analysis

Two factorial arrangements was the base of Analysis of variance (ANOVA) with RCBD.

Experimental units were differentiated significantly (P≤0.05) By Least Significant Difference test

(LSD; Steel et al., 1997). Statistical software (Statistix 9®

) was used for statistical analysis

(Analytical Software, Tallahassee, USA).

5.4. Results

5.4.1. Growth and Yield Attributes

A significant influence of various fertilization treatments and genotypes was observed for the cob

length of maize (Fig. 5.1A). Cob length ranged from 12.8–23.5 cm in both maize genotypes at

different P and Zn rates. Maximum cob length (23.5 cm) was found in Zn+P combination followed

by sole Zn application (19.8 cm), in DK–6142. Averaged across different fertilization treatments,

maize genotype DK-6142 recorded a higher cob length compared to Neelam.

Grain and straw yield (t ha-1

) of maize varied significantly under the influence of various

fertilization treatments and genotypes (Fig. 5.1B and 5.2A). Grain and straw yield ranged from 4.23

to 7.4 and from 7.8 to 13.3 t ha–1

respectively, under different fertilization treatments. The maximum

grain and straw yields were found with combined of Zn+P. In Neelam, the grain yield was increased

by 12, 28, and 42% by application of P, Zn, and Zn+P, respectively, as compared with control. The

same trend was found for DK–6142 but the increase was more than for Neelam. However, increase

in straw yield was more in Neelam as compared with DK-6142. The Zn+P application resulted in 57

and 53% increase in straw yield of Neelam and DK–6142, respectively (Fig. 5.2A).

70

Figure 5.1. Cob length (A) and grain yield (B) of maize genotypes Neelam and DK–6142 after

different Zn and P application. Different letters indicate significant differences by LSD at P ≤

5%. Error bars indicate ± standard deviation of three replications.

71

Figure 5.2. Straw yield (A) and 1000-grain weight (B) of maize genotypes Neelam and DK–

6142 after different Zn and P application. Different letters indicate significant differences by

LSD at P ≤ 5%. Error bars indicate ± standard deviation of three replications.

72

Figure 5.3. Phosphorus concentrations in grains (A) and stover (B) of maize genotypes Neelam

and DK–6142 after different Zn and P application. Different letters indicate significant

differences by LSD at P ≤ 5%. Error bars indicate ± standard deviation of three replications.

73

The 1000-grain weight showed highly significant effects of genotypes and different

fertilization treatments (Fig. 5.2B). All the fertilization treatments increased the 1000-grain weight

as compared to control. The 1000-grain weight was increased by 6.2, 12.76, 20.56% in Neelam and

6.67, 13.95, 26.52% in DK-6142 by application of P, Zn and Zn+P, respectively. Overall, maize

genotype DK-6142 had a higher 1000-grain weight in all the treatments than Neelam.

5.4.2. Zinc and Phosphorous Accumulation in Plant Parts

Significant main and interactive effects of genotypes and different fertilization treatments were

observed for P concentration (μg g−1

) in grains and stover (Fig. 5.3 A and B). The P concentration in

grains ranged between 1616 –2231 μg g−1

for Neelam and 1876–2371 μg g−1

for DK–6142.

A stronger increase was found for combined application of Zn+P (2232 and 2371 μg g−1

)

followed by sole P application (1972 and 2140 μg g−1

) in Neelam and DK-6142, respectively. A

similar trend was found for P concentration (μg g−1

) in stover of both maize genotypes.

Significant main and interactive effects of genotypes and different fertilization treatments

were observed for Zn concentration (μg g−1

) of grains and stover of maize. Density of Zn in maize

grain was from 22.48 to 34.56 μg g−1

in Neelam and 25.16-37.26 μg g−1

for DK–6142 (Fig. 5.4). It

was observed that Zn concentrations of maize grains and stover were decreased (about 10%) with

increasing level of P rate but combined application of Zn+P increased the Zn concentration (about

49%) as compared with control (Fig. 5.4 A and B). The same trend was found for the Zn

concentration (μg g−1

) of stover for both maize genotypes. Addition of sole P fertilization reduced

the Zn contents in tissues (4–5%). However, addition of combined Zn+P fertilizer increased the Zn

contents by 78 and 87% in Neelam and DK–6142 grains, respectively (Figure 5.4 C).

5.4.3. Phytate in Grains

Significant main and interactive effects of genotypes and different fertilization treatments were

observed for phytate concentration (mg g−1

) and content (mg seed−1

) of maize (Fig. 5.5 A and B).

Phytate concentration was in the range of about 11.8 to 14.8 mg g−1

in Neelam and 13.2-16.2 mg g−1

in DK–6142, respectively under different fertilization treatments.

74

Figure 5.4. Zn concentrations of grain (A) and stover (B), and Zn content per seed (C) of

maize genotypes (Neelam and DK–6142) at different Zn and P rates. Different letters indicate

significant differences by LSD at P ≤ 5%. Error bars indicate ± standard deviation of three

replications.

75

The maximum increase in phytate concentration was found by sole P application. In Neelam, the

phytate concentration was increased by 22 and 2.5% for sole P and Zn+P, respectively. The same

trend was found for DK–6142 but the increase was more than for Neelam. Reduction (2.5%) in

phytate concentration was found by single Zn application in Neelam. Phytate content (mg seed−1

)

also followed a similar trend as phytate concentration in maize grains (Fig. 5.5 B).

5.4.4. Estimated Human Zn Bioavailability in Grains

Significant main and interactive effects of genotypes and different fertilization treatments were

recorded for the [phytate] : [Zn] ratio and a non-significant interactive effect for the Zn

bioavailability over control (Fig. 5.6A and B). The minimum [phytate] : [Zn] ratio of 35.6 and 39.4

(33 and 26% less than unfertilized control) in maize grain was recorded by combined Zn+P followed

by sole Zn fertilization in the genotypes Neelam and DK–6142, respectively.

However, in DK-6142, the maximum reduction in the [phytate] : [Zn] ratio was found by

sole application of Zn (29%). The maximum increase in [phytate] : [Zn] ratio was found for single P

application in Neelam (38%) and DK–6142 (34%), respectively.

Contrary to the [phytate] : [Zn] ratio, the estimated Zn bioavailability was the minimum

(1.08 and 1.18 mg Zn for 300 g maize grains) for single P application and maximum (1.66 and 1.77

mg Zn for 300 g maize grains) for combined Zn+P application over control in the genotypes Neelam

and DK–6142, respectively. As compared to control, single Zn application also increased the

estimated Zn bioavailability by 34 and 24% in Neelam and DK–6142, respectively.

5.5. Discussion

Low availability of Zn (< 1.0 mg of AB-DTPA extractable Zn kg−1

soil) and P (< 10 mg of Olsen-P

kg-1

soil) to plants occurs in calcareous soils because of their high fixation and low organic matter

contents (Okalebo et al., 2002; Maqsood et al., 2011; Ahmad et al., 2012; Imran et al., 2015; Tab.

5.1). Therefore, Zn and P fertilization increased plant growth and yield parameters (Sattar et al.,

2011; Hussain et al., 2012, 2013; Rehim et al., 2014), in agreement with the present study (Fig. 5.1

and 2). Generally, Zn fertilization was found more effective in increasing yield at higher P levels.

76

Figure 5.5. Phytate concentrations of grains (A) and phytate content per seed (B) of maize

genotypes Neelam and DK–6142 after different Zn and P application. Different letters

indicate significant differences by LSD at P ≤ 5%. Error bars indicate ± standard deviation of

three replications.

77

Response of nutrient fertilization was obvious in grain yield as compared to straw yield. Sole

fertilization of either Zn or P significantly decreased the stress factor of the other, indicating more

requirement of one of the nutrients at a higher rate of the other nutrient among two. Farmers in

developing countries particularly Pakistan are only using macronutrient (e.g. N, P, and K) while

ignoring micronutrient fertilization (Hussain et al., 2008; Aziz et al., 2011). The current

investigation obviously indicates that Zn deficiency in maize may cause considerable reduction in

grain yield.

Applications of Zn and P to the soil increased their concentrations of grains and stover (Fig.

5.3 and 4). At crop harvest, the grain concentrations of Zn as well as P were increased by combined

application of Zn+P. Reduction in Zn and P concentration in maize grains and stover was observed

after the single application of P and Zn to maize genotypes, respectively (Fig. 5.3 and 4).

Comparatively higher Zn content in grain compared to stover (Fig. 5.4) is important for

better human nourishment (Kanwal et al., 2010; Graham et al., 1992). Decreased Zn concentration

due to excessive P fertilization might decrease the food nutritional quality that is of major limiting

factor (Cakmak, 2002; Verma et al., 1987) because of known Zn insufficiency in human nutrition

(Buerkert et al., 1998).

Single fertilization of Zn caused a significant improvement in Zn concentration of maize

(Fig. 5.4). This is in general agreement with Kaya and Higgs (2001) and Montilla et al. (2003). The

current results reveal that single P application markedly reduced shoot Zn concentration of both

maize genotypes (Fig. 5.4). Large applications of P fertilizers to soils without Zn fertilization (low in

bioavailable Zn) can limit tissue Zn concentration particularly in grain (Robson and Pitman, 1983;

Gianquinto et al., 2000; Gill et al., 2004).

The treatments receiving no P application had the highest Zn concentration in both genotypes

whilst single P application tended to reduce the Zn concentration. These results corroborate earlier

findings of Farah and Solimon (1986), Olsen (1972), and Prabhakarannair and Babu (1975). The

decrease in Zn concentration due to P application at 60 kg ha-1

may result from the formation of

soluble Zn-P compounds in the soil as reported by Krishnasamy (1993). Similarly, Harrel (2005)

reported that P-Zn interactions in corn decreased P concentration and increased Zn bioavailability;

they suggested that Zn and P fertilizers should be applied separately to overcome this problem.

78

A higher Zn content in maize was found after combined application of Zn+P followed by

single Zn application in both genotypes (Fig. 5.4 C). High Zn concentration of the grain was due to

the genotypes and not tightly linked to agronomic Zn-efficiency traits, and varieties may have to be

selected independently to increase the nutritional value of the grain for humans (Graham et al., 1992;

Fageria, 2007). Phytate concentration was also decreased by single Zn application (Fig. 5.5). As

most of the P in cereals grains is bound in phytate (an anti-nutritional factor) P concentration is

strongly related with grain phytate concentration that reduces the Zn availability to human beings

(Erdal et al., 2002; Stangoulis et al., 2007).

Single Zn application reduced the [phytate] : [Zn] ratio in both maize genotypes (Fig 5.6 A).

A low [phytate] : [Zn] ratio might have caused an increase in Zn bioavailability (Fig. 5.6B). Hence,

Zn combined with P fertilization would be considered for improved nutritional quality of the

products (Gill et al., 2004). The [phytate] : [Zn] ratio has recently been reported as a bioavailability

trait in cereal grains and it should be in the range of 15-20 (Joy et al., 2014; Šimić et al., 2012).

Single P application results in greater [phytate] : [Zn] ratio (73–71) indicating a Zn-deficiency risk

for plant growth as well as for human health. The [phytate] : [Zn] ratio in grains was decreased by

25.8–33.2% with single Zn and combined Zn+P application in both maize genotypes.

Similarly, combined Zn+P fertilization followed by single Zn fertilization improved Zn

bioavailability from 1.19 to 1.66 and 1.34 to 1.77 mg Zn for 300 g in grain of Neelam and DK-6142,

respectively (Fig. 5.6 B). The Zn-absorption requirement of an adult human is 3 mg d-1

(Institute of

Medicine, 2001) while consumption of 300 g maize grain can only ensure about 55-59% of the daily

dietary Zn requirement (Fig. 5.6 B).

79

Figure 5.6. Grain [phytate] : [Zn] ratio (A) and estimated Zn bioavailability (B) in grains of

maize genotypes Neelam and DK–6142 after different Zn and P application. Different letters

indicate significant differences by LSD at P ≤ 5%. Error bars indicate ± standard deviation of

three replications.

80

5.6. Conclusions

The current investigation inferred that the combined Zn+P fertilization improved growth and yield

attributes in both maize genotypes compared to sole fertilization of either. Zinc and P concentration

in grain were also increased with combined Zn+P application. Single Zn application decreased grain

P concentration, phytate content, and [phytate] : [Zn] ratio in both genotypes. However, single P

fertilization significantly reduced grain Zn density and bioavailable Zn in maize grain. The Zn

density, [phytate] : [Zn] ratio and bioavailable Zn was much dependent on Zn fertilization in both

maize genotypes, especially when combined with P application.

81

Chapter 6

EFFECTIVENESS OF DIFFERENT ZN APPLICATION

METHODS IN MAIZE FOR GRAIN

ZINC BIOFORTIFICATION

6.1. Abstract

Maize (Zea mays L.) is generally low in bioavailable zinc (Zn); however, agronomic biofortification

can cure human Zn deficiency. In the present experiment, Zn was applied in pots as ZnSO4·7H2O to

maize cultivar DK–6142 as foliar spray (0.5% w/v Zn sprayed 25 days after sowing and 0.25% w/v

at tasseling), surface broadcasting (16 kg Zn ha–1

), subsurface banding (16 kg Zn ha–1

at the depth of

15 cm), surface broadcasting + foliar and subsurface banding + foliar in comparison to an

unfertilized control. As compared to control, all treatments significantly (P≤0.05) increased growth,

yield and nutritional attributes in maize. Grain Zn and protein concentrations were correlated and

ranged from 22.3 to 41.9 mg kg−1

and 9 to 12 %, respectively. Zinc fertilization also significantly

reduced grain phytate and increased grain Zn concentration. Zinc fertilization, especially

broadcasting and subsurface banding combined with foliar spray decreased grain [phytate]:[Zn] ratio

to 28 and 21 and increased Zn bioavailability by trivariate model of Zn absorption to 2.04 to 2.40,

respectively. Conclusively, broadcasting and subsurface banding combined with foliar spray is

suitable for optimal maize yield and agronomic Zn biofortification of maize grain. This would also

be helpful to optimize Zn and protein concentration in maize grain.

6.2. Introduction

Zinc (Zn) is vital micronutrient for plants, humans and animals. In plants, Zn is of vital importance

for healthy root structure, free radical de-toxification, enzymatic precursor, as well as tolerance to

plant stress (Cakmak, 2008; Peck and McDonald, 2010).

The study has been published in Archives of Agronomy and Soil Science (2016; DOI:

10.1080/03650340.2016.1185660) and entitled „Zinc fertilization approaches for agronomic

biofortification and estimated human bioavailability of zinc in maize grain‟. The authors are M.

Imran and A. Rehim.

82

In third world countries, where growth and mental development of offspring can be impaired due to

Zn deficiency, achievement of sufficient grain Zn concentration is of great interest (Brown et al.,

2004).

In the world, about 50% cereal growing area has low phyto–available Zn (Graham and

Welch, 1996; Cakmak, 2002) and prevalent deficiencies of Zn have also been identified in 50−70%

soils of Pakistan (Hamid and Ahmad, 2001), which might be due to low Zn phytoavailability caused

by calcareousness, high pH and high fixation. However, maximum grain Zn concentration of maize

grain was also obtained when grown on calcareous soils but the exact mechanism of this situation is

still unknown (Joy et al., 2015). Cereals are identified as naturally low in bioavailable Zn, especially

in the areas low in phyto–available Zn (Shivay and Prasad, 2012), resulting in low Zn concentration

of 13–23 mg Zn kg–1

in maize grain (Manzeke et al., 2012) compared to a pre–dietary prerequisite of

about 40–60 mg Zn kg–1

in maize gain (Pfeiffer and McClafferty, 2007). Several projects in the

world are in in process with the aim to improve Zn density in edible crops. The main objective of

HarvestPlus program is to maintain Zn concentrations of 38 mg Zn kg−1

(Bouis and Welch, 2010) in

maize grain.

Maize (Zea mays L.) is the third most important cereal crop. It adds 2.1% to the value added

in agriculture sector and 0.4% to gross domestic product in Pakistan. Area under maize crop in

Pakistan has decreased from 1.168 million hectares in 2013-2014 to 1.130 million hectares in 2014–

2015. Maize grain production was 4.94 million tonnes during 2013–2014, which decreased to 4.70

million tonnes in 2014–2015 (Government of Pakistan, 2014–2015). Maize provides about 20%

calories and 15% protein of the world and is dietary staple food crop for >0.2 billion people around

the world. Unfortunately, the amount of essential nutrient like Zn is inadequate for people who are

depending on maize grain as staple food (Nuss et al., 2010). For the production of cost-effective

crop yields and maximum Zn concentration in grain, Zn fertilization is recommended for soils low in

bioavailable Zn (Imran et al., 2015).

Diffusion of Zn in soils is limited; hence soluble Zn sources are readily bioavailable to plants

(Amrani and Westfall, 1999). More soluble sources of Zn are ZnSO4 as well as Zn chelates (Zn-

EDTA), but chelated sources are more expensive (and are less concentrated) than other sources of

Zn (Mortvedt and Gilkes, 1993). Zinc fertilization approaches can be done in the soils either before

83

crop sowing or by foliarly sprays to crops (Mortvedt and Gilkes, 1993). For Zn fertilization in the

soil, Zn can be soil broadcast or banding beneath the seed (Martens and Westermann, 1991); hence

most appropriate amount, approach and fertilization time is very important topics in Zn nutrition of

maize grain.

Phytic acid (PA; A phosphorous storing compound) is considered as an anti-nutrient in

agriculture, food and nutritional sciences. In maize, rice and wheat, efforts have been made to reduce

PA content (0.52–4.56 mg g–1

of PA) by plant breeding and genetic engineering (Guttieri et al.,

2006). Higher phosphorous (P) fertilization cause reduction in Zn concentration and uptake; hence

requires higher Zn fertilization (Rehim et al., 2014). Numerous studies investigating P and Zn

interactions in cereals have shown the value of agronomic biofortification in reducing PA:Zn molar

ratio within the known critical levels of between 15–20 (Joy et al., 2014). Considerable decrease in

PA levels in staple food leads to improvements in calcium (Ca) and Zn consumption (Li et al.,

2000). On the other side, PA may also have important benefits on human health (Fox et al., 2002).

Dietary PA beneficial effects included decreased heart attack danger, colon cancer and formation of

renal stone (Fox et al., 2002). Phytic acid is also important for plant growth, reduction in PA

influence plant health and seed germination. Therefore, substantial removal of PA might not be a

good approach, as it may have a dominant role in plant seed function, cellular functioning as well as

positive human‟s health benefits. Appropriate (~10 mg g–1

) PA levels in food can be attained by

processing, cooking and plant breeding (Thavarajah and Thavarajah, 2014).

Agronomic biofortification of maize grain by Zn fertilization is an easy way to resolve Zn

deficiency problems in Pakistan. For this, Zn absorption trivariate model measures the quantifiable

approximation of bioavailable Zn in human nutrition (Bouis and Welch, 2010). By using PA and Zn

as a function in our food, a Zn absorption model (Miller et al., 2007) was effectively verified in adult

women with labeled Zn absorption experiments (Rosado et al., 2009). The trivariate model based on

estimation of Zn absorption in the intestine of humans correlated with actual Zn absorption from

maize grain if maize grain is a major food in the daily diet. Zinc estimation in maize grain in

addition to its availability is essential positive approach for agronomic biofortification program.

84

This study therefore aims to investigate and quantify the added benefits of Zn fertilization as a

pathway to agronomic biofortification of staple maize crop under different Zn fertilization

approaches.

6.3. Materials and Methods

6.3.1. Soil

The pot trial was performed at Research Area of Soil Science Department, Bahauddin Zakariya

University, Multan (30.258° N, 71.515° E, 125.27 m elevation above sea level) to assess the effects

of various Zn fertilization approaches on the productivity and grain Zn concentration of maize crop.

Before crop sowing, multiple soil samples were collected from the field and these were examined for

various soil physico-chemical characteristics (Table 1). The hydrometer protocol (Gee and Bauder,

1986) was used for soil texture analysis. EC and pH meters were used to measure electrical

conductivity of soil extract (ECe) and pH of saturated soil paste (pHs). Walkley-Black and acid

dissolution procedures were used for organic matter determination in soil (Nelson and Sommer,

1982) as well as for soil free lime (CaCO3; Allison and Moodie, 1965) determination. The AB–

DTPA method was used to determine the soil bioavailable Zn for plants with an atomic absorption

spectrophotometer (Thermo Scientific 3000 Series, Waltham, MA, USA; Soltanpour, 1985). The soil

was calcareous with low Zn phyto-availability (AB–DTPA: 0.43 mg Zn kg–1

soil; Table 6.1).

Table 6.1. Physical and chemical properties of soil used in the experiment

Soil Property Unit Value

Textural class --- Loam

Sand % 47.2 ±0.3

Silt % 34.7 ±0.7

Clay % 18.1 ±0.2

pHs --- 8.1 ±0.1

ECe dS m–1

0.64 ±0.07

Calcium Carbonate % 4.70 ±0.08

Organic Matter % 0.49 ±0.05

AB-DTPA extractable Zn mg kg–1

0.43 ±0.02

Three samples were analysed after random composite sampling

85

6.3.2. Growth Conditions

Twenty five kilogram of carefully mixed soil was filled in 18 pots lined with poly-ethylene. In this

trial, different Zn application methods like control (without Zn), foliar spray (0.5% w/v Zn sprayed

25 days after sowing and 0.25% w/v at tasseling stage), surface broadcasting (16 kg Zn ha–1

before

sowing of crop), subsurface banding (16 kg Zn ha–1

at the depth of 15 cm), surface broadcasting +

foliar and subsurface banding + foliar were applied as ZnSO4·7H2O. Recommended doses of NPK

fertilizers were added respectively at 140–100–60 kg ha–1

(1.37–6.22–1.5 g pot–1

) in soil as urea, di–

ammonium phosphate and potassium sulphate respectively. Before sowing of crop, distill water was

used to moisten the soil of all pots and carefully mixed to attain equilibrium. Completely randomized

design (CRD) was used to organize all pots in the green house (Steel et al., 1997). Throughout the

investigational span, the mean greenhouse temperature was 35 ± 6 °C at different day times as well

as 23 ± 3 °C for the period of the night. Six seeds of maize hybrid cultivar DK–6142 were sown in

triplicates per pot. About ten days after germination, three healthy plants were allowed to grow in

each pot. During the experimental period, distilled water was used to maintain moisture contents at

field capacity in all the pots. Three weeks after sowing, second dose of 50 kg N ha–1

(0.75 mg N pot–

1) as urea was applied uniformly to all pots in solution form.

6.3.3. Crop Harvest and Chemical Analysis

Plant shoots and cobs were harvested at maturity (110 days after germination). Air dried plant

samples were oven–dried at 70 °C to attain a constant weight for dry matter yield in a forced air

oven. These dried plant samples were finely ground with a Wiley mill (IKA Werke, MF 10 Basic,

Staufen, Germany) fitted with a stainless steel chamber and blades. Mixture of di-acid (2:1 ratio of

HNO3:HClO4) was used to digest the portion of finely ground plant samples. Atomic absorption

spectrophotometer (Thermo Scientific 3000 Series, Waltham, MA, USA) was used for the

determination of plant Zn concentration. Indirect method (Haug and Lantzsch, 1983) was used on a

spectrophotometer for grain phytate determination (Shimadzu, UV–1201, Kyoto, Japan; see protocol

from previous experiment).

86

6.3.4. Human Zinc Bioavailability in Grain

Phytate as well as Zn density influenced the food Zn bioavailability; hence, qualitative method was

used to estimate Zn bioavailability in humans as molar ratio of [phytate]:[Zn] (Brown et al., 2001;

Weaver and Kannan, 2002) in maize grain. By knowing a quantitative value of bioavailable Zn, Zn

bioavailability was also calculated by using the model of trivariate Zn absorption (Miller et al.,

2007).

( (

) √( (

))

)

Where, AMAX (maximum Zn absorption) = 0.091, KR (equilibrium dissociation constant of

zinc-receptor binding reaction) = 0.680 and KP (equilibrium dissociation constant of Zn-phytate

binding reaction) = 0.033 relate to Zn homeostasis in human intestine (Hambidge et al., 2010). In the

model, total daily absorbed Zn (TAZ) (mg Zn d−1

) is a function of total daily dietary phytate (TDP)

(mmol phytate d−1

) and total daily dietary Zn (TDZ) (mmol Zn d−1

).

Average per capita consumption of maize flour in major maize consuming countries (such as

Mexico, Zambia and Zimbabwe) is about 300 g d–1

(Food and Agriculture Organization, 2013).

Then, TAZ was estimated for 300 g of maize flour and was termed as „estimated bioavailable Zn‟.

On the other hand, estimated bioavailable Zn results are based on adults consuming 300 g maize

flour as a sole daily diet.

6.3.5. Total protein

Total protein in grain was determined by Chapman and Parker method (1961b). Concentrated

sulphuric acid (H2SO4) was used to digest the grain samples by digestion mixture (K2SO4:CuSO4:

FeSO4 in 90:10:1 ratio). The resulting mixture was again diluted and distilled with NaOH using

condensation in distillation apparatus (micro Kjeldahl). The produced ammonia gas was collected in

2% boric acid solution and nitrogen concentration was determined by titrating against 0.1 N

sulphuric acid. Protein concentration (%) was tabulated by multiplying nitrogen with a value of 6.25.

87

6.3.6. Statistical analysis

Completely randomized design was the base of analysis of variance (ANOVA). Treatments were

distinguished significantly (P≤0.05) from each other‟s by Least Significant Difference (LSD; Steel

et al., 1997) test. Statistical software was used (Statistix 9®) on windows for statistical analysis

(Analytical Software, Tallahassee, FL, USA).

6.4. Results

6.4.1. Growth attributes

Plant height was significantly (P≤0.05) increased in all Zn treatments as compared to control

(without Zn; Table 6.2). As compared to control (without Zn), maximum increase of 13% was by

banding + foliar followed by 10% with broadcasting + foliar Zn fertilization respectively. After

combined applications, increase in plant height was 9% by banding, 6.2% by broadcasting and 3.8%

by foliar Zn fertilization respectively as compared to control.

Table 6.2. Effect of various Zn treatments on the growth attributes of maize plant

Zn Treatments Plant Height

(cm)

Shoot Fresh Weight

(g pot−1

)

Shoot Dry Weight

(g pot−1

)

Control 177±1.53 E 232±1.88 F 110±0.91 F

Surface Broadcasting 189±1.79 C 267±1.45 D 141±0.88 D

Foliar 184±2.08 D 249±1.83 E 123±1.15 E

Subsurface Banding 195±1.20 B 302±1.45 C 174±1.53 C

Surface Broadcasting+Foliar 196±1.33 B 313±1.18 B 191±1.09 B

Subsurface Banding+Foliar 204±1.73 A 322±1.79 A 198±1.76 A

Zinc treatments included: control (Without Zn), Zn foliar (0.5% w/v Zn sprayed at 25 days after sowing and

0.25% w/v at tasseling stage), surface broadcasting (16 kg Zn ha–1

before sowing of crop), subsurface banding

(16 kg Zn ha–1

at the depth of 15cm), broadcasting+foliar and banding+foliar. Different letters in the same

column indicate significant differences by LSD at P≤0.05 and ± indicate standard error (n=3).

88

Various Zn treatments significantly (P≤0.05) influenced shoot fresh weight of maize plants (Table

6.2). Shoot fresh weight was maximum (322 g pot−1

) with banding + foliar followed by broadcasting

+ foliar (313 g pot−1

) and banding (302 g pot−1

), respectively. Apart from these treatments,

broadcasting and foliar spray significantly influence shoot fresh weight compared to control.

Shoot dry weight was also significantly (P<0.05) influenced by Zn fertilization (Table 6.2). It

was maximum in banding + foliar (198 g pot−1

) followed by broadcasting + foliar (191 g pot−1

),

banding (174 g pot−1

), broadcasting (141 g pot−1

) and foliar (123 g pot−1

) Zn fertilization

respectively. Minimum shoot dry weight (110 g pot−1

) was found without Zn fertilization.

6.4.2. Yield attributes

All the treatment significantly (P<0.05) increased the cob length (Table 6.3) of maize. Maximum

increase in cob length was by combined application of banding + foliar (20.9 cm) followed by

broadcasting + foliar (20.4 cm), banding (19.6 cm), broadcasting (18.5 cm) and foliar (17.6 cm) Zn

applications respectively.

Grain yield (g pot–1

) was significantly different for each Zn application methods (Table 6.3).

Foliar spray combined with banding and broadcasting has maximum grain yield of 442 g and 427 g

pot-1

respectively. After combinations, banding (420 g pot–1

), broadcasting (411 g pot–1

) and foliar

(397 g pot–1

) have maximum grain yield as compared to control (377 g pot–1

).

Significant difference in 1000 grain weight was observed among various Zn treatments

(Table 6.3). As compared to control, maximum increase was of 20% by banding + foliar Zn

application, 15.8% by broadcasting + foliar, 11.3% by banding, 8.9% by broadcasting and 7.1% by

foliar Zn application.

6.4.3. Zinc Concentration in Grain

Different Zn treatments significantly (P≤0.05) increased Zn concentration in grain (Table 6.4). Grain

Zn concentration ranged from 22−42 mg kg−1

in various Zn treatments. As compared to control,

89

maximum increase was of 46.8% with banding + foliar, 40.5% with broadcasting + foliar, 34.5% by

banding, 26% by foliar and 17.5% by broadcasting Zn fertilization respectively.

Table 6.3. Effect of various Zn treatments on the yield attributes of maize plant

Zn Treatments Cob Length

(cm)

Grain Yield

(g pot−1

)

1000 Grain weight

(g)

Control 14±0.30 F 377±1.45 F 234±2.08 E

Surface Broadcasting 19±0.13 D 411±2.03 D 257±1.15 D

Foliar 18±0.11 E 397±1.15 E 252±1.73 D

Subsurface Banding 20±0.16 C 420±1.89 C 264±2.03 C

Surface Broadcasting+Foliar 20±0.17 B 427±1.60 B 278±1.02 B

Subsurface Banding+Foliar 21±0.14 A 442±1.43 A 293±2.33 A

Zinc treatments included: control (Without Zn), Zn foliar (0.5% w/v Zn sprayed at 25 days after sowing and

0.25% w/v at tasseling stage), surface broadcasting (16 kg Zn ha–1

before sowing of crop), subsurface banding

(16 kg Zn ha–1

at the depth of 15cm), broadcasting+foliar and banding+foliar. Different letters in the same

column indicate significant differences by LSD at P≤0.05 and ± indicate standard error (n=3).

As compared to control, maximum increase (136%) in grain Zn content (µg seed−1

) was

achieved with banding + foliar Zn fertilization, 100% by broadcasting + foliar, 72% by banding,

45% by foliar and 33% by broadcasting Zn application (Table 6.4). However, Zn concentration in

stover was also statistically significant in all Zn treatments. Zinc concentration in stover was found

less as compared to Zn concentration in grain.

6.4.4. Phytate, Protein and Estimated human bioavailability of zinc in grain

Phytate concentration in grain differed significantly between Zn treatments (Fig. 6.1a). Grain phytate

concentration ranged from 8.9 to 14.23 mg g−1

under different Zn treatments. All treatments

significantly (P≤0.05) decreased grain phytate concentration except broadcasting and foliar methods

that were at par with each other. Phytate concentration in grain decreased 60% by banding + foliar,

33% by broadcasting + foliar and 16.54% by banding as compared to control.

90

Various Zn treatments significantly (P≤0.05) decreased grain [phytate]:[Zn] molar ratio (Figure

6.1b). Minimum [phytate]:[Zn] molar ratio of 21 (two-fold less than control) in maize grain was

attained by banding + foliar, 28 by broadcasting + foliar, 36 by banding, 43 by foliar, 48 by soil and

63 by control.

Table 6.4. Effect of various Zn treatments on the nutritional attributes in maize grain

Zn Treatments Zinc

Concentration in

grain (mg kg−1

)

Zinc content in

grain

(µg seed−1

)

Zinc

Concentration

in stover (mg

kg−1

)

Control 22.3±0.37 F 5.2±0.12 F 13.9±0.08 F

Surface Broadcasting 26.7±0.15 E 6.9±0.09 E 22.9±0.05 D

Foliar 30.1±0.14 D 7.6±0.05 D 19.0±0.07 E

Subsurface Banding 34.0±0.11 C 9.0±0.08 C 24.6±0.10 C

Surface Broadcasting+Foliar 37.4±0.17 B 10.4±0.04 B 28.0±0.15 B

Subsurface Banding+Foliar 41.9±0.15 A 12.3±0.11 A 30.3± 0.09 A

Zinc treatments included: control (Without Zn), Zn foliar (0.5% w/v Zn sprayed at 25 days after sowing and

0.25% w/v at tasseling stage), surface broadcasting (16 kg Zn ha-1

before sowing of crop), subsurface banding

(16 kg Zn ha-1

at the depth of 15cm), broadcasting+foliar and banding+foliar. Different letters in the same

column indicate significant differences by LSD at P≤0.05 and ± indicate standard error (n=3).

Different Zn treatments significantly (P≤0.05) increased grain protein concentration in maize grain

(Figure 6.2a). Grain protein concentration ranged from 9% to 12% in different Zn treatments.

However, increase was 33% by banding + foliar, 21% by broadcasting + foliar, 15.6% by banding,

11.6% by broadcasting and 6% by foliar Zn application as compared to control.

91

Figure 6.1. Phytate concentration (6.1a) and Grain [phytate]:[Zn] ratio (6.1b) in grain of maize crop

grown in pots and differentially treated with Zn. Zinc treatments included: control (Without Zn), Zn

foliar (0.5% w/v Zn sprayed at 25 days after sowing and 0.25% w/v at tasseling stage), surface

broadcasting (16 kg Zn ha-1

before sowing of crop), subsurface banding (16 kg Zn ha-1

at the depth

of 15cm), broadcasting+foliar and banding+foliar. Different letters indicate significant differences

by LSD at P≤0.05 and error bars indicate ±standard error (n=3).

AB B

C

D

E

0

3

6

9

12

15

Ph

ytat

e C

oncc

entr

atio

n (

mg

g−1 )

1a

A

BC

D

E

F

0

13

26

39

52

65

Cont

rol

Surf

ace

Broa

dcas

ting

Folia

r

Subs

urfa

ceBa

ndin

g

Surf

ace

Broa

dcas

ting

+Fo

liar

Subs

urfa

ceBa

ndin

g +

Folia

r

Gra

in [

phy

tate

]:[Z

n]

rati

o

1b

92

Trivariate model based on estimated Zn bioavailability in maize grain was significantly (P≤0.05)

influenced by different Zn fertilization approaches (Figure 6.2b). In all Zn treatments, estimated Zn

bioavailability was minimum of 1.16 mg Zn for 300 g maize grain at control (without Zn) and

maximum of 2.40 mg Zn for 300 g maize grain (52% than control) by banding + foliar Zn

fertilization. After banding + foliar Zn application, increase in estimated Zn bioavailability was of

43% broadcasting + foliar, 34.5% by banding, 25% by foliar and 18.6% by broadcasting Zn

fertilization respectively as compared to control.

6.5. Discussion

As for most of the Pakistani soils, the soil in this trial was alkaline calcareous (Table 6.1) and

deficient in Zn (<1.0 mg kg−1

of AB-DTPA extractable Zn in soil; Maqsood et al., 2015; Imran et

al., 2016). Therefore, Zn fertilization to the soil deficient in phytoavailable Zn (0.43 mg Zn kg−1

soil,

Table 6.1) significantly (P≤0.05) increased the growth and yield of maize crop (Table 2 and 3). The

beneficial effect of Zn fertilization on growth has been reported for maize and other crops (Manzeke

et al., 2014; Ehsanullah et al., 2015). However, combined Zn fertilization approaches (broadcasting

+ foliar and banding + foliar) provided better results than a single fertilization approach (Table 6.2

and 6.3).

Combined fertilizations of Zn by broadcasting + foliar approaches are required for optimum

rice yield from the soil of low Zn bioavailability (Imran et al., 2015). Our results show that for

maize, banded applications of Zn are more effective than broadcast application. Banding usually

involves placement of the fertilizer 5 cm to one side and 5 cm below the row-planted seed

(Mortvedt, 2007).

Maize crop receiving combined banding + foliar Zn fertilization had more growth and yield

than all other treatments. It might be due to more auxin formation [Zn is essential for tryptophan

production in plants that is precursor of indole–3–acetic acid] plus it has a vital function in

internodal elongation and cell division in plants (Khan et al., 2007). The increased Zn uptake by

plants in case of banding, compared to surface broadcasting of Zn and foliar Zn spray, might

enhance Zn pool in leaves and increase IAA levels. So the combined effect of banding and foliar Zn

spray might be more promising in improving plant growth as compared to other fertilization

approaches.

93

Figure 6.2. Grain protein concentration (6.2a) and Estimated Zn bioavailability (6.2b) in grain of

maize crop grown in pots and differentially treated with Zn. Zinc treatments included: control

(Without Zn), Zn foliar (0.5% w/v Zn sprayed 25 days after sowing and 0.25% w/v at tasseling

stage), surface broadcasting (16 kg Zn ha-1

before sowing of crop), subsurface banding (16 kg Zn

ha-1

at the depth of 15cm), broadcasting+foliar and banding+foliar. Different letters indicate

significant differences by LSD at P≤0.05 and error bars indicate ±standard error (n=3).

F

ED

C

B

A

0.00

0.50

1.00

1.50

2.00

2.50

Cont

rol

Surf

ace

Broa

dcas

ting

Folia

r

Subs

urfa

ceBa

ndin

g

Surf

ace

Broa

dcas

ting

+Fo

liar

Subs

urfa

ceBa

ndin

g +

Folia

r

Est

imat

ed Z

n B

ioav

aila

bil

ity

(mg/

300

g) 2b

F

DE

CB

A

0.0

2.5

5.0

7.5

10.0

12.5

Pro

tein

Con

cen

trat

ion

(%

)

2a

94

Compared with Zn control, a significant (P≤0.05) increase was found in grain as well as stover Zn

concentration of maize crop. However, maximum Zn concentration in grain (41.87 mg Zn kg−1

of

grain) was achieved by banding + foliar fertilization (Table 6.4) and is adequate for desired level of

Zn in maize grain. Increase in Zn concentration in maize grain might also be due to its

remobilization from leaves to grain (Harris et al., 2007; Xue et al., 2012). Grain Zn content (µg

seed−1

) by Zn application can be related to increased maize yield, grain weight and grain Zn

concentration (Table 6.4).

Roots of maize crop are usually distributed in the topsoil, with about half of the maize roots

in the 0–15 cm layer (Peng et al., 2010). Therefore, Zn fertilization in 0–15 cm layer is expected to

increase Zn uptake by roots and its translocation into the shoot (Zhang et al., 2013). There was

greater efficiency of banded Zn fertilization as compared to broadcast, but higher Zn rates are

required for broadcasting on severely Zn deficient soils.

Phytate is a main P storage compound in cereal grain and it performs as a metal chelator in

the intestine of human; hence, obstructs the bioavailability of dietary nutritional Zn in humans (Bohn

et al., 2008). In the present trial (Figure 6.1a), it was noted that combined Zn fertilization

significantly (P≤0.05) decreased phytate concentration in maize grain, in agreement with findings in

rice (Mabesa et al., 2013; Imran et al., 2015). One possible cause of this might be yield dilution

effect as phytate concentration was primarily decreased with Zn fertilization alongside by increase in

grain yield (Figure 6.1a; Table 6.3).

Desired [phytate]:[Zn] molar ratio for optimal Zn nutrition in human food still exceeded the critical

value of 15–20, implying that maize grain has poor Zn bioavailability (Hambidge et al., 2008; Joy et

al., 2014). Treatments receiving combined Zn application especially banding + foliar increased Zn

concentration and decreased phytate concentration in maize grain that resulted in the lowest

[phytate]:[Zn] molar ratio about to 21 (Figure 6.1b). So, foliar spray combined with banding had a

significant effect on grain [phytate]:[Zn] ratio, and decreased the ratio to near the desired level for

optimum Zn bioavailability.

Grain Zn concentration has a direct positive effect on the protein concentration in maize

grain (Figure 6.2a). During senescence, cereals are known to re-translocate Zn to grain and N is

known to be the main factor stimulating the translocation of Zn (Barunawati et al., 2013). A

95

synergetic N-Zn interaction was described for rice, wheat and maize (Kutman et al., 2012; Pooniya

and Shivay, 2013; Sarwar et al., 2015). This increase in protein concentration is also associated with

the reduction of phytate concentration in cereal crops (Liu et al., 2013; Li et al., 2015; Figure 6.2a).

Similarly, Zn bioavailability is higher by the use of trivariate model in treatments receiving

combined broadcasting + foliar and banding + foliar Zn fertilization by 2.04 and 2.40 mg Zn for 300

g maize grain. The physiological net Zn requirement to be absorbed by a mature human is 3 mg d-1

(Institute of Medicine, 2001), while 300 g maize biofortified grain could guarantee approximately

80% of the day-to-day Zn requirement (Figure 6.2b). So, a suitable Zn fertilization approach through

which Zn could easily move from stover to grain will remain the best way for agronomic

biofortification of maize grain.

96

6.6. Conclusion

All Zn fertilization approaches increased maize growth and yield. However, foliar Zn spray

combined with broadcasting or banding Zn fertilization was the prerequisite for optimal maize yield

from the soil low in phyto-available Zn. Grain Zn concentration, estimated human bioavailability of

Zn and protein concentration in maize grain was optimum with the combined Zn fertilization

approaches. Fertilization with Zn by broadcasting or banding at 15 cm depth, combined with foliar

Zn sprays are required for maximum redistribution of grain Zn concentration, increased Zn

bioavailability and protein concentration in maize grain. This will not only reduce dietary Zn

deficiencies in humans but also improve the grain nutritional quality. However, desired Zn

concentration in grain by Zn fertilization recommended that genetic and molecular approaches must

be the focused for Zn biofortification programs and deserves further research under field conditions.

97

Chapter 7

SUMMARY

In cereal crops, maize grain is of vital importance throughout the globe (Nuss et al., 2010). Because

of a staple food crop in the various continents of the world, maize grains are of prime importance

(Ortiz-Monasterio et al., 2007; Menkir et al., 2008). It delivers almost 15% of the protein and 20%

of the calories as well as > 200 million people nutritive staple crop around the globe. Apart from

this, some important mineral like zinc (Zn) is inadequate in maize grain especially for those people

who rely on its grain as staple food crop (Nuss et al., 2010). Beside the recommended dose of Zn for

human consumption in maize grain of about 40–60 mg Zn kg–1

(Pfeiffer and McClafferty, 2007); it

has as low as13–23 mg Zn kg–1

(Manzeke et al., 2012) in Zn-deficient soils. Hence, Zn fertilization

to such soils not only increased the grain yield but also grain Zn density in Zn deficient soils (Imran

et al., 2015). So, to decrease the problems of human Zn deficiency; the best way is the agronomic

biofortification of cereal grains with Zn fertilization (Cakmak, 2008).

Studies on soil phosphorous (P) and Zn are comparatively very rare (Xue et al., 2016).

Phosphorus and Zn both are of vital importance for plant growth and developmental stages; however

their bioavailability was often limited in the soil. But, a lot of P–Zn interaction contradictory reports

are available and these might be possibly associated with growth conditions. Higher P soil

fertilization improved P uptake but decreases the Zn uptake on the other side that caused deficiency

of Zn by the crop plants (Zhao et al., 2007; Mousavi et al., 2011). This interaction in the crop plants

is likewise referred to as P-induced Zn deficiency (Marschner, 2012). In maize plants, P toxicity and

Zn deficiency was found by higher P and lower Zn concentrations that decreased the shoot growth

(Shah et al., 2015). This shows that higher P fertilization could cause not only the P toxicity in crop

plants but the reason of Zn deficiency in them. Therefore, P fertilization rate in combination with Zn

should be selected wisely. Apart from this, judicious use of these both nutrients not only improved

their uptake in plants but also improved the yield and grain nutritional quality which ultimately

affect the human health.

Zinc bioavailability in maize crop is reduced by phytic acid (PA) contents (Lesteinne et al.,

2005). In cereal food crops, Zn bioavailability is usually measured by the qualitative [phytate]:[Zn]

98

ratio (Brown et al., 2001). However in recent advances of human nutrition, daily diet Zn

bioavailability is measured by quantitative estimation of trivariate (Miller et al., 2007; Rosado et al.,

2009) Zn absorption model.

Even though widespread investigation on the interaction of Zn–P in cereal, it was not been

explained that whose element among them could predominantly be the cause of the adverse effects

of high P fertilization on plant dry matter Zn concentration. Complete understanding of this

mechanism may also have of vital importance for Zn biofortification efforts in cereals grain.

However, Zn and P adsorption in different textured soils especially at different time intervals were

unknown. Therefore, three different textured soils (clay, loam and loamy sand) were incubated at

different time intervals to check their adsorption/bioavailability by different Zn and P fertilization

(Chapter 3). Adsorption of Zn and P depend on the soil physic-chemical properties, time, rate of

application and inherent soil properties. As described by Michaelis–Menten model, added Zn and P

percent adsorption increased by increased clay contents. Correspondingly, maximum adsorption of

Zn and P was found in loam soil, clay and loamy sand soils, respectively. Maximum adsorption of

Zn and P was found within 65−80 days after their addition in these soils. On the other hand, high P

application caused to reduce the Zn bioavailability but higher Zn application does not influence as

much the P bioavailability. By using Michaelis-Menten model, one could estimate adsorption in

different textured soils of a specific nutrient at a specific time which might be useful for Zn and P

application in the fields to the farming community for soils and crop specific recommendations to

attain maximum cost-effective benefits.

After wheat and rice, maize is major cereal crop in Pakistan having more prone to soil Zn

deficiency because of heavier P fertilization in these soils. Different cultivars required different

nutrients levels for their optimum yield. Four maize cultivars were grown in Zn deficient soils

having different Zn and P rates (Chapter 4). This experiment investigates the significant variances on

the growth, nutrient concentration and efficiencies in four maize cultivars for applied Zn and P

fertilization. As compared to unfertilized control, significant increase was observed in Zn and P

concentration in all cultivars by single and combined fertilization of Zn and P, respectively.

However, P fertilization alone to all the maize cultivars caused the reduction of root and shoot Zn

concentration. Overall, Zn and P various efficiencies were improved if the plants were fertilized with

other element. Applied Zn and P efficiencies were also depending on the other nutrient and varied

99

with different cultivars of maize. Consequently, appropriate genotypes selection and plant nutrients

management are of prime importance to improve applied nutrients efficiencies.

Two cultivars were selected for the next experiment (For chapter 5) on the basis of their higher

Zn concentration in leaves (From chapter 4) because shoot concentration of a micronutrient plays an

important function for grain buildup of that micronutrient (Zubaidi et al., 1999; Cakmak et al.,

2010). Different Zn and P rates were given to selected maize cultivars in the field for maize grain Zn

biofortification (Chapter 5). As compared with single nutrient fertilization, all maize genotypes

growth and yield attributes continued to improve by Zn+P fertilization in combination. Average of

two genotypes, combined Zn+P fertilization also improved grain P and Zn concentration by 32% and

52%, correspondingly as compared to unfertilized control. Phosphorous fertilization alone not only

reduced the grain Zn concentration but also human Zn bioavailability. On the other hand, Zn

fertilization alone reduced the grain P density, phytate concentration and [phytate]:[Zn] molar ratio

in both maize cultivars. In both maize cultivars maize grain, Zn+P fertilization in combination also

reduced the grain [phytate]:[Zn] molar ratio that ultimately improved the bioavailability of Zn in

humans based on Zn trivariate model. In both maize crop cultivars, Zn fertilization was more

dominantly effected Zn concentration, [phytate]:[Zn] molar ratio and human Zn bioavailability

particularly when combined with P fertilization.

Different Zn fertilization approaches were the prerequisite to improve Zn density in maize grain

and its quantitative availability in humans to optimal levels (Chapter 6) by recommended P

fertilization (From chapter 5). All Zn fertilization approaches improved maize growth, yield and

dietary aspects in maize grain. In various Zn fertilization approaches, a positive association was

observed amid grain Zn and protein concentration. Significant increase in grain Zn concentration and

reduction in grain phytate concentration was found by various Zn fertilization approaches. Zinc

fertilization approaches, especially foliar spray in combination with broadcasting and subsurface

banding reduced grain [phytate]:[Zn] molar ratio (<20) and improved human bioavailability of Zn

(>2 mg Zn per 300 g maize grain) by trivariate model and these fertilization approaches were the

prerequisite for optimum maize grain yield and agronomic Zn biofortification from Zn deficient

soils. Apart from the grain nutritional and yield attributes, it could also decrease dietary Zn

malnutrition problems in humans.

100

General Recommendation and Conclusion

Zinc and phosphorous should be applied on the basis of soil test.

Michaelis-Menten equation may be used to access Zn and P bioavailability in different

textured soils.

Michaelis-Menten equation works better about 120 days that might be helpful for soil and

crop specific recommendations for Zn and P application to the farming community to

attain maximum benefits.

Genotypic difference exist in different maize cultivars having different Zn and P

concentration, uptake as well as their utilization efficiencies in them.

Banding or broadcast Zn fertilizer application combined with foliar sprays improved

grain Zn and protein content in maize grain.

If high protein content grain is required then Zn should be added as a fertilizer by

Pakistani farmers in their fertilizer list.

Studies in plant breeding and genetics are required to ensure maximum grain Zn

concentration in maize genotypes.

101

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