Prospects of Arbuscular Mycorrhizal Technology on Growth, Mineral Nutrient Acquisition and Fixed Oil Profile of Selected Sunflow

PROSPECTS OF ARBUSCULAR MYCORRHIZAL TECHNOLOGY ON GROWTH, MINERAL NUTRIENT ACQUISITION AND FIXED OIL PROFILE OF SELECTED SUNFLOWER (Helianthus annuus L.) HYBRIDS AT VARIOUS LEVELS OF ROCK PHOSPHATE IN MARGINAL SOIL

SAYEDA SARAH MUBARAK

DEPARTMENT OF BOTANY

UNIVERSITY OF PESHAWAR

SESSION: 2012-2013 PROSPECTS OF ARBUSCULAR MYCORRHIZAL TECHNOLOGY ON GROWTH, MINERAL NUTRIENT ACQUISITION AND FIXED OIL PROFILE OF SELECTED SUNFLOWER (Helianthus annuus L.) HYBRIDS AT VARIOUS LEVELS OF ROCK PHOSPHATE IN MARGINAL SOIL

The thesis submitted to Department of Botany University of

Peshawar in partial fulfillment of the requirements for the Degree of

Doctor of Philosophy in Botany

SAYEDA SARAH MUBARAK

DEPARTMENT OF BOTANY

UNIVERSITY OF PESHAWAR

SESSION: 2012-2013

DEDICATION

Dedicated to my loving parents, who have been

an intimate source of inspiration throughout my life, in whatever I am and whatever I do.

UNIVERSITY OF PESHAWAR

PESHAWAR

A dissertation submitted in partial satisfaction of the requirement for the degree of Doctor of Philosophy

In BOTANY

By Sayeda Sarah Mubarak

Supervisor: Prof. Dr. Mohammad Ibrar

Graduate Studies Committee 1. Prof. Dr. S. Bashir Ahmad, Member 2. Prof. Dr. Muhammad Ibrar, Member 3. Prof. Dr. Muhammad Nafees, Member 4. Dr. Zahir Muhammad, Member 5. Prof. Dr. Siraj ud Din, Convener

APPROVAL

This thesis, entitled “Prospects of arbuscular mycorrhizal technology on growth, mineral nutrient acquisition and fixed oil profile of selected sunflower (Helianthus annuus L.) hybrids at various levels of rock phosphate in marginal soil” submitted by Ms. Sayeda Sarah Mubarak is hereby approved and recommended as partial fulfillment for the award of Degree of Doctor of Philosophy in BOTANY,

______(External Examiner) Prof.

______(Supervispr) Prof. Dr. Mohammad Ibrar Research supervisor Department of Botany, University of Peshawar.

Dated:______

March 21st , 2016

PUBLICATION OPTION I hereby reserved the rights of publication, including right to reproduce this thesis in any form for a period of 5 years from the date of submission.

Sayeda Sarah Mubarak

VITAE

November 14, 1986, Born, Mardan M.phil: Department of Botany University of Peshawar, 2012 M.Sc: Department of Botany University of Peshawar, 2008 B.Sc: F.G. Degree College for women Peshawar, 2006 F.Sc: F.G. Degree College for women Peshawar, 2004 SSc: F.G. Girls Public High School Peshawar Cantt, 2002 Lecturer in Botany, Govt. Frontier College For women Peshawar, 2014---Present.

Major field Botany (Mycorrhizal Technology). Botany

Courses studied Teacher 1. Agrostology Prof. Dr. Siraj-ud-Din 2. Plant molecular systematic Prof. Dr. Siraj-ud-Din 3. Fresh water algae Prof. Dr. Nadeem 4. Environmental Health and Problems Dr Ghulam dastagir 5. Vegetation ecology Dr. Lal badshah 6. Physiology of plant under Stress Dr. Barkat-ullah

TABLE OF CONTENTS

S. No. Contents Page No.

Acknowledgment i-ii

Abstract iii-iv

CHAPTER 1 INTRODUCTION 1-19

1.1 Mycorrhiza 1 1.2 Classification of mycorrhiza 2 1.3 Arbuscular mycorrhiza 2 1.4 Distribution of AM fungi 3 1.5 Taxonomy of AM fungi 3 1.6 Morphology of AM fungi 4 1.7 Significance of AM fungi 6 1.7.i VAM as bio-Fertilizer 6 1.7.ii Phosphorus (P) uptake 6 1.7.iii Rock phosphate (RP) uptake 7 1.7.iv Nitrogen (N) uptake 8 1.7.v Potassium (K) uptake 9 1.7.vi Carbon (C) 10 1.7. vii Other macronutrients uptake 10 1.7. viii Zinc (Zn) uptake 10 1.7.ix Other micronutrients uptake 10 1.7.x Disease and pathogen 11 1.7.xi Soil aggregation 11 1.7.xii Hormone production 11 1.7.xiii Salinity 12 1.7.xiv Drought resistance 12 1.7.xv Succession 13 1.7.xvi Phytoremediation 13 1.7.xvii Land rehabilitation 13 1.7.xviii Weed control 14 1.8 Mycorrhizal dependency 14 1.9 Test plant sunflower (Helianthus annuus L.) 14 1.9.i Local names 14 1.9.ii Systematic position 14 1.9.iii Cultivation 16 1.9.iv Botanical description 16 1.9.v Chemical constituents 16 1.9.vi Uses 17 1.9.vii Nutritional values of sunflower oil 17 1.10 Objectives of present study 18 1.11 Expected outcomes 19 CHAPTER 2 LITERATURE REVIEW 20-46 2.1 Effect of AM fungal inoculation on plant growth, yield and nutrient 20 acquisition 2.2 Effect of AMF on spores density and root colonization 31 2.3 Effect of AM fungal inoculation on plant growth, yield and nutrient 36 acquisition in relation to fertilizer application 2.4 Effect of AM fungal inoculation on percent root colonization and 41 spores density in relation to fertilizer application 2.5 Mycorrhizal dependency (MD) 44 CHAPTER 3 MATERIALS AND METHODS 47-59 3.1 Field work 47

3.1.i Equipment and reagents 47 3.1.ii Plant material 48 3.1.iii Experimental site 48

3.1.iv Soil 48 3.1.v Application of AMF inoculum 48 3.1.vi Fertilizer application 49 3.1.vii Experimental design, treatments and replications 49 3.1.vii Evaluation 50 3.1.ix Mycorrhizal dependency 51 3.1.x Statistical analysis 51 3.2 Analysis of plant 51 3.2.i Mineral analysis 51 Wet digestion method for nitrogen 51 Wet digestion for other elements 52 Preparation of stock/standard solutions (100 ppm) 52 Preparation of stock/standard solutions (2.5,5,10 ppm) 52 Instrumental condition for different elements in the samples 52 Procedure 52 3.2.ii Proximate analysis 53 Determination of moisture 53 Determination of ash 53 Determination of crude protein 53 Determination of crude fiber 54 Determination of crude fats 55 Determination of carbohydrates 55 3.2.iii Oil fatty acid profile 55 3.3 Laboratory work 56 Equipment and reagents 56 3.3.i Assessment of roots 56 Collection and preservation of roots 56 Preparation of preservative 56 Preservation 56 Preparation of KOH solution (10%) 56 Preparation of acid fuchsin stain 56 Staining procedure 57 Assessment of root colonization 57 3.3.ii Extraction of spores 57 Collection of soil samples 57 Mounting of spores 58 Calculation of density of spores 58 Identification of spores 58 CHAPTER 4 RESULTS AND DISCUSSION 60- 4.1 Growth parameters 61 4.1.i Plant height 61 4.1.ii Root length 65 4.1.iii Number of fresh leaves 68 4.1.iv Number of wilted leaves 72 4.1.v Leaf length 75 4.1.vi Leaf width 78 4.1.vii Dry weight of plant 81

4.1.viii Head (Capitulum) diameter 85 4.1.ix Number of seeds per head 88 4.1.x Weight of seeds per head 92 4. 2 Oil fatty acid profile 95 4. 2.i Oil content (%) 95 4.2. ii Fatty acid profile 99 Unsaturated fatty acids 99 Linoleic acid (C18:2) 99 Oleic acid (C18:1 cis-9) 103

Saturated fatty acids 106 Palmitic acid (C16:0) 106 Stearic acid (C18:0) 109 4.3 Proximate analysis 118 4.3.i Crude protein (%) 118 4.3.ii Ash content (%) 122 4.3.iii Moisture content (%) 125 4.3.iv Crude fats (%) 128 4.3.v Crude fibers 131 4.3.vi Carbohydrate contents 134 4.4 Mineral composition 137 4.4.i Macronutrients 137 Phosphorus (P) 137 Nitrogen (N) 141 Potassium (K) 144 4.4.ii Micronutrient 147 Zinc (Zn) 147 4.5 Mycorrhizal dependency 151 4.6 Effect of indigenous AMF and rockphosphate levels on spore 153 density and root colonization 4.6.i AMF spore density 153 4.6.ii AMF species 154

4.6.iii AMF colonization in roots 160 CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 165-167 5.1 Conclusion 165 5.2 Recommendations 167 CHAPTER 6 REFERENCES 168-225

LIST OF TABLES

Table Title Page No. No.

1. Biological materials, time schedule and cultural practice 59 2. Plant Height (cm) in different hybrids of Helianthus annuus L. at various levels of 63 rock phosphate (RP). 3. Root Length (cm) in different hybrids of Helianthus annuus L. at various levels of 66 rock phosphate (RP). 4. Number of Fresh Leaves/plant in different hybrids of Helianthus annuus L. at 70 various levels of rock phosphate (RP). 5. Number of Wilted Leaves/plant in different hybrids of Helianthus annuus L. at 73 various levels of rock phosphate (RP). 6. Leaf Length (cm) in different hybrids of Helianthus annuus L. at various levels of 76 rock phosphate (RP). 7. Leaves Width (cm) in different hybrids of Helianthus annuus L. at various levels of 79 rock phosphate (RP). 8. Dry Weight (gm) in different hybrids of Helianthus annuus L. at various levels of 83 rock phosphate (RP). 9. Head (capitulum) diameter (cm) in different hybrids of Helianthus annuus L. at 86 various levels of rock phosphate (RP). 10. Number of seeds/ Head in different hybrids of Helianthus annuus L. at various levels 90 of rock phosphate (RP). 11. Seeds weight/ Head in different hybrids of Helianthus annuus L. at various levels of 93 rock phosphate (RP). 12. Oil content (%) in different hybrids of Helianthus annuus L. at various levels of rock 97 phosphate (RP). 13. Linoleic acid (%) in different hybrids of Helianthus annuus L. at various levels of 101 rock phosphate (RP). 14. Oleic acid (%) in different hybrids of Helianthus annuus L. at various levels of rock 104 phosphate (RP) 15. Palmatic acid (%) in different hybrids of Helianthus annuus L. at various levels of 107 rock phosphate (RP). 16. Stearic acid (%) in different hybrids of Helianthus annuus L. at various levels of rock 110 phosphate (RP). 17. Crude Protein (%) in different hybrids of Helianthus annuus L. at various levels of 120 rock phosphate (RP). 18. Ash content (%) in different hybrids of Helianthus annuus L. at various levels of 123 rock phosphate (RP). 19. Moisture content (%) in different hybrids of Helianthus annuus L. at various levels 126 of rock phosphate (RP). 20. Crude Fats (%) in different hybrids of Helianthus annuus L. at various levels of rock 129 phosphate (RP). 21. Crude fibers content (%) in different hybrids of Helianthus annuus L. at various 132 levels of rock phosphate (RP). 22. Carbohydrates content (%) in different hybrids of Helianthus annuus L. at various 135 levels of rock phosphate (RP). 23. Phosphorus concentration in different hybrids of Helianthus annuus L. at various 139 levels of rock phosphate (RP). 24. Nitrogen concentration (%) in different hybrids of Helianthus annuus L. at various 142 levels of rock phosphate (RP). 25. Potassium concentration (%) in different hybrids of Helianthus annuus L. at various 145 levels of rock phosphate (RP). 26. Zinc concentration (%) in different hybrids of Helianthus annuus L. at various levels 149 of rock phosphate (RP). 27. Effects of rock phosphate (RP) fertilizers on mycorrhizal dependency (MD) 152 % of four hybrids of sunflower 28. Effect of mycorrhiza on AMF spores in the roots of sunflower hybrids at 154 various levels of rock phosphate (RP) 29. Effect of mycorrhiza on AMF spores species in sunflower hybrids. 156 30. Effects of various levels of RP fertilizers on REC index in the roots of 162 sunflower hybrids. Effects of various levels of RP fertilizers on AM infection morphologies, 163 external hyphae, internal hyphae, arbuscules, vesicles, (%) in roots of mycorrhizal sunflower hybrids.

LIST OF FIGURES

Fig. Title Page No. No.

1. Mean Plant height (cm) in different hybrids of Helianthus annuas L. 64 2. Effect of RP treatment on plant height (cm) in four hybrids of 64 Helianthus annuas L. 3. Effect of mycorrhiza on plant height (cm) under different RP levels in 64 four hybrids of Helianthus annuas L. 4. Mean root length (cm) in different hybrids of Helianthus annuas L. 67 5. Effect of RP treatment on root length (cm) in four hybrids of 67 Helianthus annuas L. 6. Effect of mycorrhiza on root length (cm) under different RP levels in 68 four hybrids of Helianthus annuas L. 7. Number of fresh leaves/plant in different hybrids of Helianthus annuas 71 L. 8. Effect of RP treatment on number of fresh leaves in hybrids of 71 Helianthus annuas L. 9. Effect of mycorrhiza on number of fresh leaves/plant under different 71 RP levels in Helianthus annuas L 10. Number of wilted leaves/plant in different hybrids of Helianthus 74 annuas L. 11. Effect of RP treatment on number of wilted leaves/ plant in hybrids of 74 Helianthus annuas L. 12. Effect of mycorrhiza on number of wilted leaves under different RP 74 levels in Helianthus annuas L. 13. Leaf length in different hybrids of Helianthus annuas L. 77 14. Effect of RP treatment on leaf length (cm) in four hybrids of Helianthus 77 annuas L. 15. Effect of mycorrhiza on leaf length (cm) under different RP levels in 77 four hybrids of Helianthus annuas L. 16. Leaf width in different hybrids of Helianthus annuas L. 80 17. Effect of RP treatment on leaf width (cm) in four hybrids of Helianthus 80 annuas L. 18. Effect of mycorrhiza on leaf width(cm) under different RP levels in 80 four hybrids of Helianthus annuas L. 19. Dry weight of plant (gm) in different hybrids of Helianthus annuas L. 84 20. Effect of RP treatment on dry weight of plant (gm) in four hybrids of 84 Helianthus annuas L. 21. Effect of mycorrhiza on dry weight of plant (gm) under different RP 84 levels in four hybrids of Helianthus annuas L. 22. Head diamter in different hybrids of Helianthus annuas L. 87 23. Effect of RP treatment on head diameter (cm) in four hybrids of 87 Helianthus annuas L. 24. Effect of mycorrhiza on head diameter (cm) under different RP levels in 88 four hybrids of Helianthus annuas L. 25. Number of seeds/ head in different hybrids of Helianthus annuas L. 91 26. Effect of RP treatment on number of seeds/ head in four hybrids of 91 Helianthus annuas L. 27. Effect of mycorrhiza on number of seeds/ head under different RP 91 levels in four hybrids of Helianthus annuas L. 28. Seeds weight/ head in different hybrids of Helianthus annuas L. 94 29. Effect of RP treatment on seeds weight/ head in four hybrids of 94 Helianthus annuas L. 30. Effect of mycorrhiza on seeds weight/ head under different RP levels in 94 four hybrids of Helianthus annuas L. 31. Oil content (%) in different hybrids of Helianthus annuas L. 98 32. Effect of RP treatment on oil content (%) in four hybrids of Helianthus 98 annuas L. 33. Effect of mycorrhiza on oil content (%) under different RP levels in 99 four hybrids of Helianthus annuas L. 34. Linoleic acid (%) in different hybrids of Helianthus annuas L. 102 35. Effect of RP treatment on linoleic acid in four hybrids of Helianthus 102 annuas L. 36. Effect of mycorrhiza on linoleic acid (%) under different RP levels in 102 four hybrids of Helianthus annuas L. 37. Oleic acid (%) in different hybrids of Helianthus annuas L. 105 38. Effect of RP treatment on oleic acid (%) in four hybrids of Helianthus 105 annuas L. 39. Effect of mycorrhiza on oleic acid (%) under different RP levels in four 105 hybrids of Helianthus annuas L. 40. Palmitic acid (%) in different hybrids of Helianthus annuas L. 108 41. Effect of RP treatment on palmitic acid (%) in four hybrids of 108 Helianthus annuas L. 42. Effect of mycorrhiza on palmitic acid (%) under different RP levels in 108 four hybrids of Helianthus annuas L. 43. Stearic acid content (%) in different hybrids of Helianthus annuas L. 111 44. Effect of RP treatment on Stearic acid (%) in four hybrids of Helianthus 111 annuas L. 45. Effect of mycorrhiza on stearic acid (%) under different RP levels in 111 four hybrids of Helianthus annuas L. 46. Crude protein (%) in different hybrids of Helianthus annuas L. 121 47. Effect of RP treatment on crude protein (%) in four hybrids of 121 Helianthus annuas L. 48. Effect of mycorrhiza on crude protein (%) under different RP levels in 121 four hybrids of Helianthus annuas L. 49. Ash content (%) in different hybrids of Helianthus annuas L. 124 50. Effect of RP treatment on ash content (%) in four hybrids of Helianthus 124 annuas L. 51. Effect of mycorrhiza on ash content (%) under different RP levels in 124 four hybrids of Helianthus annuas L. 52. Moisture content (%) in different hybrids of Helianthus annuas L. 127 53. Effect of RP treatment on moisture content (%) in four hybrids of 127 Helianthus annuas L. 54. Effect of mycorrhiza on moisture content (%) under different RP levels 127 in four hybrids of Helianthus annuas L. 55. Crude fat content (%) in different hybrids of Helianthus annuas L. 130 56. Effect of RP treatment on crude fat content (%) in four hybrids of 130 Helianthus annuas L. 57. Effect of mycorrhiza on crude fat content (%) under different RP levels 130 in four hybrids of Helianthus annuas L. 58. Crude fiber content (%) in different hybrids of Helianthus annuas L. 133 59. Effect of RP treatment on crude fiber content (%) in four hybrids of 133 Helianthus annuas L. 60. Effect of mycorrhiza on crude fiber content (%) under different RP 133 levels in four hybrids of Helianthus annuas L. 61. Carbohydrate content (%) in different hybrids of Helianthus annuas L. 136 62. Effect of RP treatment on carbohydrate content (%) in four hybrids of 136 Helianthus annuas L. 63. Effect of mycorrhiza on carbohydrate content (%) under different RP 137 levels in four hybrids of Helianthus annuas L. 64. Phosphorus concentration (%) in different hybrids of Helianthus 140 annuas L. 65. Effect of RP treatments on phosphorus concentration (%) in four 140 hybrids of Helianthus annuas L. 66. Effect of mycorrhiza on phosphorus concentration (%) under different 141 rock phosphate levels in four hybrids of Helianthus annuas L. 67. Nitrogen concentration (%) in different hybrids of Helianthus annuas 143 L. 68. Effect of RP treatments on nitrogen concentration (%) in four hybrids 143 of Helianthus annuas L. 69. Effect of mycorrhiza on nitrogen concentration (%) under different rock 144 phosphate levels in four hybrids of Helianthus annuas L. 70. Potassium concentration (%) in different hybrids of Helianthus annuas 146 L. 71. Effect of RP treatments on potassium concentration (%) in four hybrids 146 of Helianthus annuas L. 72. Effect of mycorrhiza on potassium concentration (%) under different 147 rock phosphate levels in four hybrids of Helianthus annuas L. 73. Zinc concentration (%) in different hybrids of Helianthus annuas L. 150 74. Effect of RP treatments on zinc concentration (%) in four hybrids of 150 Helianthus annuas L. 75. Effect of mycorrhiza on zinc concentration (%) under different rock 150 phosphate levels in four hybrids of Helianthus annuas L. 76. Effects of rock phosphate (RP) fertilizers on mycorrhizal dependency 152 (MD) % in four hybrids of Helianthus annuas L. 77. Effects of rock phosphate (RP) fertilizers on spore density/100 gm. soil 154 of four hybrids of Helianthus annuas L. 78. Effects of rock phosphate (RP) fertilizers on % infection of four hybrids 162 of sunflower

LIST OF APPENDICES Appendix Title Page No. No. 1. Composition of hoagland's solution used in this experiment 226 2. ANOVA for plant height of Helianthus annuas L. 227 3. ANOVA for root length of Helianthus annuas L. 227 4. ANOVA for number of fresh leaves of Helianthus annuas L. 228 5. ANOVA for number of wilted leaves of Helianthus annuas L. 228 6. ANOVA for leaf length of Helianthus annuas L. 229 7. ANOVA for leaf width of Helianthus annuas L. 229 8. ANOVA for dry weight of plant of Helianthus annuasL 230 9. ANOVA for head diamter of Helianthus annuas L. 230 10. ANOVA for number of seeds/head of Helianthus annuas L. 231 11. ANOVA for weight of seeds/head of Helianthus annuas L. 231 12. ANOVA for oil of Helianthus annuas L. 232 13. ANOVA for linoleic acid of Helianthus annuasL 232 14. ANOVA for oleic acid of Helianthus annuas L. 233 15. ANOVA for palmatic acid of Helianthus annuas L. 233 16. ANOVA for steric acid of Helianthus annuas L. 234 17. ANOVA for protein of Helianthus annuas L. 234 18. ANOVA for ash content of Helianthus annuas L. 235 19. ANOVA for moisture of Helianthus annuas L. 235 20. ANOVA for fats of Helianthus annuas L. 236 21. ANOVA for fibers of Helianthus annuas L. 236 22. ANOVA for carbohydrates of Helianthus annuas L. 237 23. ANOVA for phosphorus of Helianthus annuas L. 237 24. ANOVA for nitrogen of Helianthus annuas L. 238 25. ANOVA for potassium of Helianthus annuas L 238 26. . ANOVA for zinc of Helianthus annuas L. 239

ABBREVIATIONS AMF Arbuscular mycorrhizal fungi H Hybrid Gl. Glomus spp. EH. External hyphae IH. Internal hyphae Kg. Kilogram M Mycorrhizal NM Non mycorrhizal Mycorrhizal dependency (MD) RP Rock phosphate T Treatment P Phosphorus N Nitrogrn K Potassium Zn Zinc

ACKNOWLEDGEMENTS

In the name of Allah, the Most Beneficent and the Most Merciful, all praise is due to Almighty Allah, who bestowed me with strength and His blessing to finalize this thesis. Then, I owe my deepest gratitude to my honorable and respectable supervisor Prof. Dr. Muhammad Ibrar, Department of Botany, University of Peshawar for his invaluable help, constructive comments, kind suggestions and inspiring guidance throughout the experimental and thesis work, which contributed to the success of this research. Without that it would not have been possible for me to complete this work. I would like to express my deep appreciation and thanks to Prof. Dr. Siraj-ud-Din, Chairman, Department of Botany, University of Peshawar, for their guidance, encouragement and co-operation at every stage of my research work. My sincere thanks also go to Dr. Tanvir burni, Prof. Dr. Abdur Rashid, Dr. Nadeem, Dr. Zahir Muhammad, Dr. Gulam Dastagir, Dr. Lalbadshah and Mr. Rehmanullah, Department of Botany, University of Peshawar for providing every possible help in the completion of this research work. I am also indebted to Dr. Farukh Hussain and Dr. Barkatullah for their useful comments and suggestions. My deep regards are forwarded to Dr. Ihsan-ul-Allah, Pakistan Council of Scientific & Industrial Research, Peshawar, Dr. Ihsanullah (Director/ Chief scientist), Dr. Iftikhar Ali (Deputy Chief Scientist), and Dr. Taufiq Ahmad (Deputy Chief Scientist) of Nuclear Institute for Food & Agriculture, Peshawar for providing professional guidance and help during my research analysis.. I am also grateful to Centralized Resource Laboratory, Dept. of Physics, University of Peshawar and Department of Water Management, Khyber Pakhtunkhwa, University of Agriculture Peshawar for their help in my research work. I am thankful to Pakistan Agricultural Research Council, Islamabad for providing authentic sunflower seeds and Higher Education Commission, Islamabad for providing funds for my research samples analysis. I feel heartiest gratitude to my friend and colleague Maryam Ehsan for her sincere help and encouragement at every step of my research.

With profound sense of devotion I am much obliged to my loving family, specially my father Prof. Syed Mubarak Ali, for providing guidance and moral support, throughout my research work. Lastly, I would like to extend my thanks to all my class fellows, other staff members including, Clerical staff and Gardner’s of botany department who supported me in every respect of work during the completion of my research work.

Sayeda Sarah Mubarak

ABSTRACT The experiment was carried out in a net house in the University of Peshawar Pakistan, to find out the effects of arbuscular mycorrhizal fungi (AMF) inoculation along with application of various levels (0%, 25%, 50% and 100%) of rock phosphate (RP) fertilizer on growth and yield of selected sunflower hybrids (Helianthus annuus L.) in P- deficient soil. The results revealed that AM fungi effectively promoted the productivity of sunflower hybrids and proved their role as bio-fertilizer. It was noted that dual use of AMF and low-moderate dose of rock phosphate has profound effect regarding plant height, root length, number of leaves/plant, leaf size, head diameter, seed number/ head, seeds weight/head, dry weight of plant, oil content, fatty acid content, mycorrhizal dependency, proximate and mineral composition of the sunflower as compared to control non-inoculated plants. While, at high RP level (RP3) the non mycorrhizal plants outperformed. However the response varied in different hybrids. As far as hybrid response is concerned Hysun-33 performed better in most of the measured aspects as compared to other hybrids. Proximate analysis showed significant (p < 0.05) increase in crude protein, crude fats, moisture, ash and crude fiber content in mycorrhizal plant, however carbohydrates content was reduced. Rock phosphate fertilization showed no significant effect on nitrogen (N), potassium (K) and Zinc (Zn) uptake however phosphorus (P) uptake increases with increasing RP levels in both AMF inoculated and non-inoculated plants. The findings also provided useful information on oil yield and its fatty acid profiles as affected by AMF inoculation. The present study strongly suggests that the AMF-rock phosphate combination produces better results in the enhancement of the oil content of sunflower hybrids even in P-deficient soils. It was noted that the use of AMF along with low doses of rock phosphate promote mono unsaturated (oleic acid) and polyunsaturated fatty acids (linoleic acid), while the use of AMF along with high doses of rock phosphate bring increase in the production of saturated fatty acids like Palmitic acid and stearic acid in sunflower seeds. It has been observed that spore density and AMF root colonization was higher in the soil of control (RP0) plants, which decreases progressively with increasing fertility level, less number of spores and percent root colonization was found at high (RP3)

iii level in all hybrids. Higher P doses declined the sporulation and colonization. Seven AMF species were recorded. The dominant genus was Acaulospora followed by Glomus, Sclerocystis and Gigaspora. The average AMF spore densities ranged from 56-260 spores/ 100gm soil while root colonization ranged from 32-100 %. Mycorrhizal enhancement regarding AMF spore density and root colonization followed RP0>RP1>RP2>RP3 trend in all hybrids. All selected sunflower hybrids were more responsive to mycorrhizal association but degree of dependency also varies according to rock phosphate levels. This study clearly indicates the potential of using indigenous biofertilizer such as AMF for oil seed crops in low fertility soils, to achieve adequate production level with least utilization of synthetic fertilizers for sustainable agriculture practice. The use of biofertilizer is not only eco-friendly but also economical as it reduces our dependence on expensive chemical fertilizers.

CHAPTER 1

INTRODUCTION

1.1 MYCORRHIZA

The German Botanist Frank (1885) introduced the Greek word 'Mycorrhiza' which literally means fungus and roots (Myco-fungus; Rhiza-Root). The Paleontological studies reveal that the evolution of fungi (including ancestral mycorrhizal fungi) took place about 460 million years ago (Simon et al., 1993). The Rhynie chert of the Lower Devonian (400 m.yrs ago) shows fossils of the earliest land plants like Rhynia and Aglaophyton major, in which the roots were colonized with Arbuscular mycorrhizal fungi (AMF). Structure resembling vesicles and spores of present Glomus species were also found (Remy et al., 1994). The AMF helped primitive plants in extracting nutrients from primitive soil (Bellgard & Williams, 2011). Thus fungal root symbiosis has played major role in the plant succession on primitive land (Allen, 1987). Mycorrhizal fungi refer to the symbiotic relationship between Arbuscular mycorrhizal fungi (AMF) and roots of higher plants in which both the organisms are benefited. The higher plant (macrosymbiont) gains better exploration of the soil with the complex network of hyphae of AMF that increases water and nutrient uptake from the soil and the fungus (microsymbiont) is benefited by getting carbon from the plant for its growth, development and physiological functions (Davies, 2011). In this symbiotic relationship, roots of the host plant provide food to the fungus in the form of simple carbohydrates and also serve as a suitable substrate for the fungus (Wilkinson, 2008). Mycorrhizas are universal mutualistic associations between soil fungi and vascular plants and are essential in improving plant growth and soil quality. It improves the resilience of plant communities against environmental, nutritional and drought stresses (Barea et al., 2011). About 95% of the vascular plants and some of pteridophytes and mosses (especially liverworts) in the world are capable of forming symbiotic mycorrhizal association (Quilambo, 2000), with the exception of few plant families which are without these symbiont like Cactaceae (Neeraj et al., 1991), Amaranthaceae, Brassicaceae, Caryophyllaceae, Chenopodiaceae, Commelinaceae, Cyperaceae, 2

Juncaceae, Polygonaceae, Proteaceae (Brundrett et al., 1996; Sawers et al., 2007), Betulaceae, Dipterocaceae, Fagacaeae, Myrtaceae (Nicholson,1967) and Zygophyllaceae (Varma, 1998 ). Their root exudates are unfavourable to AM due to antifungal products like isothiocyanates, moreover, they also lack hyphal branching signal molecules, the strigolactones (Schreiner & Koide, 1993; Yoneyama et al., 2008)

1.2 CLASSIFICATION OF MYCORRHIZA

Mycorrhiza was first classified by Frank (1887) into two type‟s i.e ectotrophic and endotrophic. This classification was based on location of fungal hyphae in relation to the root tissues of plant (ecto-outside; endo- inside the root). These terms have now been replaced by ectomycorrhiza and endomycorrhiza respectively. There had been repeated mycorrhizal classifications made in recent years by many researchers (Harley & Smith, 1983; Bhandari & Mukerji, 1993). But most recent and commonly followed classification is that proposed by Smith and Read, (2008), who classified mycorrhiza on the basis of plant host fungal species involved and fungal morphological characteristics into seven categories i.e

1. Arbuscular mycorrhiza (AM)

2. Ectomycorrhiza

3. Ectendomycorrhiza

4. Arbutoidmycorrhiza

5. Ericoidmycorrhiza

6. Monotropoidmycorrhiza

7. Orchidmycorrhiza

1.3 ARBUSCULAR MYCORRHIZA

The arbuscular mycorrhiza fungi (AMF) are a biological mutualistic association between fungus of the phylum Glomeromycota and roots of higher plants (Willis et al., 2013).They enhance plant nutrition, particularly the uptake of phosphorus (P) resulting in 3 better plant growth (Colard et al., 2011). This association is the result of long evolutionary process of most of the terrestrial plants to increase efficiency for uptake of P (Brundrett, 2009). This is a typical association in which both the partners are equally benefited (Hodge et al., 2010). The Fungal hyphae develop arbuscules in the root cortex which are highly branched intracellular structures; simultaneously they also form dense mycelial network in the soil. Within the roots the fungus is supplied with hexoses with the expenditure of 20% of photosynthetic products which are produced by carbon fixation (Smith & Read, 2008). Arbuscular mycorrhizal (AM) fungi are of great significance and have ecological importance to the plants as they form symbiotic relationship improving their growth and productivity by enhancing water and nutrient absorption in marginal soils (Fedderman et al., 2010; Khalafallah & Abo-Ghalia, 2008).

1.4 DISTRIBUTION OF AM FUNGI Arbuscular mycorrhizal fungi are found in more than 95% of the higher plant roots in diverse ecological habitats all over the world (Jaya-Kumari, 2011), except Antarctica (Kivlin et al., 2011). They are ubiquitous in geographic distribution ranging from tropical rainforest to arctic tundra. They have been recovered from different habitats in the form of chlamydospores, zygospores, and azygospores. These habitats includes; polluted sites, nutrient deficient soils, eroded sites, sand dunes and desserts, sodic soils, industrial wastes, sewage, and others like different forests, savanna, open wood lands and grasslands etc (Chandra, 1992).

1.5 TAXONOMY OF AM FUNGI

The classification and identification of AMF are based on, their spore morphology and spore wall structure (Gerdemann & Trappe, 1974; Morton & Benny, 1990; Schenck & Pérez 1990). Schubler et al. (2001) classified AM fungi on the basis of SSU rRNA gene sequencing into monophyletic phylum, the Glomeromycota. Recently, Oehl et al. (2011) classified phylum Glomeromycota into three classes (Glomeromycetes, Archaeosporomycetes, and Paraglomeromycetes), five orders (Glomerales, Gigasporales, Paraglomerales, Archaeosporales and Diversisporales), fourteen families, 4 twenty nine genera and so far two hundred and thirty species on the basis of ribosomal gene and morphological characteristics.

1.6 MORPHOLOGY OF AM FUNGI

Apparently the AMF infected and non-infected roots show no morphological differences, unless studied microscopically. The root infection by AM fungi is a complex process. Prior to root colonization, the development of AM fungi is known as presymbiosis which take place in three stages; germination of spore, hyphal growth, and appresorium formation followed by penetration and colonization of root (Harrier, 2001). According to the form and structure, arbuscular mycorrhizal mycelium comprises the following parts (Brundrett, 1991);

i Extra radical hyphae

ii Intraradical hyphae

iii Arbuscules

iv Vesicles

v Auxillary cells

Extraradical hyphae

On the basis of functional behavior extraradical hyphae may be called infective, absorptive and fertile hyphae (Friese & Allen, 1991). Those hyphae which grow parallel along the root/radical surface are called „runner‟ or infective hyphae (Trividi, 2007); these develop new entry points but do not grow out of the roots. The absorptive hyphae form a network of absorbing surface in which the main hypha is rough and dichotomously branched, 8-2 µm in diameter. Tuft of fine thin walled hyphae arise from its surface (Mosse, 1959). The extraradical hyphae are therefore responsible for bringing increase in absorptive surface (Plenchette et al., 2005). Some sort of hyphal bridges may also develop for transferring nutrients between plants growing in close proximity (Newman, 1998). Some of the hyphae which produce spores are called fertile hyphae. 5

These spores are produced on extrametrical hyphae, either single or aggregated (Khachatourians & Arora, 2002). Intraradical hyphae

The intraradical hyphae are present in middle cortical layers of the radical/root. The infection is caused by germination of spores or from external hyphae emerging from the roots of other plants which harbor such fungus. They pass into the epidermis and then into the outer cortical layers intercellularly by degrading the middle lamella and proliferate over there (Trividi, 2007). The internal hyphae are mostly aseptate but in certain cases septate hyphae were also observed which seemed to be the hyphae of other parasitic fungi already present in the root (Ali, 1969). Intraradical hyphae which are straight may form H- or Y- shaped branches. Coil forms of hyphae have also been observed but their frequency varies at different locations of the root (Morton, 2002).

Arbuscules

The word arbuscule is derived from the Latin word „arbusculum‟ which means small tree. These are formed by the terminal arborization of the infection hyphae which penetrate in the inner root cortex and these are the major sites where exchange of nutrients between the fungus and host take place (Paszkowski et al., 2006). These are considered as the key sites where symbiotic phosphate is delivered (Yang & Paszkowski, 2011). Arbuscules are short lived; they vanish or become low in intensity with senescence (Sylvia, 2003).

Vesicles

Vesicles are hyphal swellings in the root cortex that contain oil globules and serve as a storage organ (Sylvia, 2003). These may be inter- or intracellular (Biermann & Lindeman, 1983). Vesicles develop at the latter stage when the fungus become older and is not very active (Trividi, 2007). It has been observed that number of vesicles increases when the plant is in the flowering stage (Iqbal & Bareen, 1986). Vesicles are not formed by all Glomus species (Morton & Redecker, 2001).

Auxillary Cells

The auxiliary cells are spore like structures which are formed from hyphae after spore germination formed in the family Gigasporaceae (order Diversisporales). These are produced before the formation of mycorrhizae and their outer wall have different ornamentations, like papillae, knobs, spines or sometimes smooth surfaces (Mueller et al., 2004). These spores are extra radical spores, containing lipids. Before sporulation they are found in abundance but disappear with the formation of larger spores. It is suggested that auxillary cells are also the source of carbon, supplied to the mycorrhiza independent of the host (Trividi, 2007).

1.7 SIGNIFICANCE OF AM FUNGI

1.7. i VAM as Bio-Fertilizer

AM fungi are known to be of great importance due to their high capability to increase growth, yield and quality of crops through efficient nutrient acquisition in infertile soils and consequently lessen the prerequisite for Phosphate-based fertilizers (Chen et al., 2005; Akhtar & Siddiqui, 2008; Giasson et al., 2008; Khalafallah & Abo- Ghalia, 2008; Sawers et al., 2008; Roy-Bolduc & Hijri, 2011). In turn, the fungi get carbon from the host plant. AM fungi have the capability of absorbing all essential macro and micro nutrients which are required for plant growth (Lester, 2009).

1.7. ii Phosphorus (P) uptake

Phosphorus (P) is one of the most important macronutrient of plants and makes 0.2% of a plant's dry weight (Daniel et al., 1998). It is essential component of cell membrane, nucleic acids (DNA, RNA), ATP, co-enzymes, phospholipids and phosphoproteins and plays vital role in many metabolic activities like energy transfer, photosynthesis and respiration etc (Ozanne, 1980). Being essential part of nucleic acid it has important role in cell division and development of new tissues which initiate development of lateral roots and fibrous roots (Brady & Weil, 2002). P is also helpful to bring increase in the leaf area and shoot dry weight of plants (Yahiya et al., 1995). Plants deficient in phosphorus shows stunted growth, dull green leaves with accumulation of 7 sugar which causes development of anthocyanin that impart purplish color to the leaves (Turk et al., 2006). Delayed flowering and maturity of plant is also one of the symptoms of P deficiency (Chauhan et al., 1992). Phosphorus is available in soil as inorganic P and organic P. The inorganic P may either be in soluble or insoluble form. It is poorly available and hard to absorb by plants, because phosphates of many elements like Iron, Aluminium and Calcium have poor solubility (Schachtman et al., 1998). Usually phosphorus concentration inside the plant cell is 1,000 times greater than the soil, so active transport is needed for their absorption and transportation and it is taken in the form of monovalent orthophosphate (H2PO4) (Vance, 2003; Bucher, 2007). To overcome the difficulty of low availability of phosphorus, plants have developed different strategies to increase the uptake of phosphorus (Marschner, 1995). Arbuscular mycorrhizal symbiosis is one of the strategies of the plants that convert unavailable soluble inorganic phosphorus to the available forms of orthophosphate thus increasing the uptake of P. The AMF inoculated plants which have fine hyphal network has been proved to be more successful rather than thick roots and root hairs in getting access to phosphate in the soil (Redecker, 2005). Moreover, mycorrhizal fungi discharge powerful chemicals (lytic enzymes and organic acids) in the soil that bring about dissolution of tightly bound soil nutrients including P (Jansa et al., 2011). Many workers have reported improvement of phosphate uptake and growth of plants by AMF (Henrike et al., 2007; Smith & Read, 2008; Giasson et al., 2008; Brundrett, 2009; Hijikata et al., 2010). On the contrary, high concentration of phosphate seems to induce low fungal colonization level by the plants (Redecker, 2005).

1.7. iii Rock Phosphate (RP) uptake

Pakistan is rich in rock phosphate mineral. It is in the form of tri-calcium phosphate [Ca3 (PO4)2] which is insoluble form and is not directly available to the plants as such, unless it is converted into soluble form (Blatt & Robert, 1996; Das, 2005). These reserves are mostly found in Hazara division of KP, Pakistan within latitudes 34º5/to 34º30/ N and longitudes 73º15/ to 73º20/ E, in different localities with different metamorphic forms e.g Tranawai (schist, shales and limestone), Kakul, Lagerban,Guldaman (dolomite and phosphorites) and at Thandiani (limestone) (Hasan, 8

1989; Chernoff, et al. 2002). Rock phosphate is the only raw material in the world which is used for the production of phosphate fertilizers (Khasawneh & Doll, 1978). These fertilizers are available in different forms, and are prepared by different acidulation treatments of rock phosphates e.g Superphosphate (9% P) is prepared by treating RP with sulphuric acid; Double superphosphate (17.5% P) and Triple superphosphate (20% P) by treating RP with phosphoric acid (Richards, 1954). In Pakistan 25% of phosphate fertilizer are produced from local resources and the demand is growing annually by 10% (web, 1). This gap of demand and supply can be met through import or by enhancing production capability of fertilizer, which needs huge amount of funding to this sector; it is therefore needed that low cost indigenous methods should be applied. RP is the natural and cheapest i.e about three times lesser in price than other phosphate fertilizers (Nye and Kirk, 1986), but unfortunately it is poorly soluble and need to adopt other strategies to increase its solubility and to make it available to the plants. In Pakistan, many microbiological organisms are present in the soil which helps in solubilization of mineral phosphates (Gadd, 1999). Among these, Arbuscular mycorrhizal fungi (AMF) are useful component which give sustainability to soil-plant system (Smith & Read, 1997; Schreiner et al., 2003). So, mycorrhizal inoculation can be helpful and effective for changing low grade rock phosphate into available form, which can be taken by the plants for their growth and development (Sabanavar & Lakshman, 2009). It is believed that AM fungi get access to such insoluble forms of rock phosphate by bringing alteration in the pH or by the organic acids secretion whose anions may be acting like chealating agents (Javaid, 2009).

1.7. iv Nitrogen (N) uptake

Nitrogen (N) is the essential macro-element and the basic component of amino acids which are the building blocks of proteins, enzymes and other cell parts (Swan, 1971). Nitrogen is also significant component of ATP (adenosine triphosphate), nucleic acids such as DNA which has vital role in metabolism, cell division, growth and development. It is also part of chlorophyll which is a green pigment in the plant cells and is responsible for photosynthesis, a carbon assimilation process, while its deficiency results in 9 yellowing of plant leaves, such plants remain stunted and weak in stature and their seeds are protein deficient (Uchida, 2000). Free atmospheric N cannot be utilized by plants, until and unless it is converted into nitrates. The N is supplied to the plants either in the form of chemical or biological fertilization. Biological fertilization includes the use of rhizobacteria and arbuscular mycorrhizal fungi (AMF) in the soil (Miransari, 2011). Nitrogen is taken from the soil in the form of nitrates (NO3−) & nitrite (NH4+) (Hodge et al., 2001). In the AMF symbiosis arginine synthesis take place in the extra radical mycelium which is then transferred to intraradical mycelium. In the intraradical mycelium the arginine break up into simpler components and N is released which is transferred to the host plant (Tian et al., 2011). Arginine is considered to be the most efficient mean for the transportation of such molecules to long distances through the plant (Govindarajulu et al., 2005). This has been confirmed by labeling experiments (Jin et al., 2005).

Plants which are infected by AMF show greater ability of the N-fixation (Barea et al., 2002), e.g. leguminous plants which are colonized by AMF have shown increase in nodules number (Rabie & Almadini, 2005; Garg & Manchanda, 2008).

1.7. v Potassium (K) uptake Like other macronutrients Potassium (K) does not form any organic compound with a vital role (Swan, 1971) but it activate certain enzymes to promote metabolism in the plants like protein synthesis etc (Uchida, 2000). Opening and closing of stomata is regulated by K+ ion pump. Thus, it reduces water loss from the leaves and help to develop tolerance against stress conditions like drought (Talbott & Zeiger, 1998). Its deficiency results in necrosis or interveinal chlorosis of leaves, stunted growth and yield reduction (Mengel & Kirkby, 2001; Brady & Weil, 1999). The tissues inhabited by mycorrhizal fungi have increased K concentration which clearly indicates an increased uptake of K by mycorrhizal plants (George et al., 1992). Higher K concentration was reported in sorghum colonized by Glomus fasciculatum (Raju et al., 1987). In contrast some other results showed that in mycorrhizal plant tissues, K concentrations can be reduced (Pinochet et al., 1997).

1.7. vi Carbon (C)

When the AM association develops, the fungus receives carbon from the plant (Johnson et al., 2002), while plant receives nutrients (particularly P) and water from the fungus (Harrison, 2005; Gosling et al., 2006). The fungus utilizes the C provided by the plant for its growth, development and physiological functions (Davies, 2011). But in cases where carbon supply is restricted (achlorophyllous plants), carbon flows in opposite direction i.e from fungus to the plant (Bidartondo et al., 2002).

1.7. vii Other Macronutrients uptake

Macronutrients such as magnesium (Mg), calcium (Ca) and sulfur (S) (Liu et al., 2002; Nasim, 2005) which are necessary for normal plant growth, are also transported by arbuscular mycorrhizal fungi, primarily by mass flow in soil (Hammer et al., 2011). By mycorrhizal colonization, nutrient contents like that of Mg & Ca can sometimes be increased while at other times it remains ineffective (Kothari et al., 1990; Alloush & Clark, 2001). AMF brings improvement in Mg++ uptake which helps maintain high chlorophyll concentration (Giri et al., 2003).

1.7. viii Zinc (Zn) uptake Zinc which is a micronutrient is necessary for chlorophyll production. It also activates many growth hormones and enzymes. Its deficiency adversely affects photosynthesis due to change in chloroplast pigments (Kosesakal & Unal, 2009). Symptoms of Zn deficiency are; interveinal chlorosis, short internodes and reduced leaf size (Brown et al., 1993). AMF symbiosis increase Zn absorption and accumulation in the roots which help in reducing its toxicity at high level in the soil (Chen et al., 2003).

1.7. ix Other Micronutrients uptake

AMF also enhance the absorption of certain micronutrients, such as copper (Li et al., 1991; George, 2000), Cadmium, (Gonzalez et al., 2002), Iron (Caris et al., 1998), Uranium (Cavagnaro, 2008), Zinc (Jamal et al., 2002; Habte & Osorio, 2002, Chen et al., 2003) and nickel (Jamal et al., 2002) resulting in better growth of the plants (Hodge et al., 2001; Ghazi & Al-Karaki, 2006). 11

1.7. x Disease and pathogen

AMF are recognized as one of the agent giving protection to plants against pathogens and other pests (Sharma & Dohroo, 1996; Bi et al. 2007) by reducing severity of diseases, caused by pathogenic bacteria, fungi and nematodes (Bhat & Mahmood, 2000; Shafi et al., 2002). This protection is produced in various ways e.g. in some cases mantle like structure is formed in the roots which are invaded by mycorrhiza which form a sort of physical barrier against the invasion of roots by pathogens or by formation of enzymes like chitinase, which stops formation of cell wall in the pathogenic fungi (Dehne et al., 1978). It has also been reported that in some cases AMF secrete a substance that act like antibiotics and competes with the pathogens or by increase in the formation of lignin content in the cell wall of such roots which make them tough and hard to be attacked by such organisms (Linderman, 1992; Ziedan et al., 2010).

1.7. xi Soil Aggregation

AMF helps in increasing soil stability and quality by contributing to soil aggregation, which in turn helps in decreasing soil erosion (Jeffries et al., 2003). These mechanisms are biological, biochemical and physical processes; as for example the mycelium secretes glomalin which is a glycoprotein (Kristine, 2008) and is glue like substance which helps in binding soil particles with eachother as well as on the hyphae, resulting in soil stability and soil aggregation. (Jeffries et al., 2003; Rillig & Mummey, 2006).

1.7. xii Hormone production

The arbuscular mycorrhizal fungi have enhancing effect on the production of plant growth hormones like Cytokinin and Indole acetic acid results in better growth and development of the plant (Allen et al, 1981). Moreover, AM fungi bring alteration in amount of Jasmonic acid and abssiccic acid which are necessary for establishment of AM symbiosis.

1.7. xiii Salinity

Salinization is a stringent problem of the arid and semi arid regions of the world and increasing day by day (Abdel-Latef, 2010). Nearly, 5% of the cultivated land has been affected by this menace and may increase up to 50% by the middle of 21st century (Sheng et al., 2008; Wang et al., 2003). In Pakistan, the estimated cultivated land is 20.2 million hacters out of which 14% i.e 6.3 million ha has already been affected by salinity and has caused almost 64% yield loss (Afzal et al., 2005; Anonymous, 2002). Salinity is of great significance in agriculture because it affects all vital process (physiological and biochemical processes) at the cellular and molecular levels, thus causing great losses in their productivity (Nawaz et al., 2010; Munns & Tester, 2008; Mathur et al., 2007). Salinity inhibit the germination of seeds (Saboora & Kiarostami, 2006), photosynthesis (Ragab et al., 2008) and spore germination (Juniper & Abbott, 2006). This adverse effect of salinity on spore germination can be reduced by preinoculation by AMF (Al-Karaki, 2006). Many workers reported that AMF work as bio-ameliorators of saline soils which combat salinity tolerance (Alguacil et al., 2003; Tian et al., 2004; Cho et al., 2006; Zhong-Qun et al., 2007; Miransari et al., 2008). AMF colonization enhances absorption of nutrient like P, N, Ca and Mg and also maintains the K+: Na+ ratio (Kaya et al., 2009). Physiological changes i.e relative permeability, photosynthetic efficiency, water status, nodulation and nitrogen fixation, abscissic acid accumulation and other biochemical, molecular and ultra-structural changes are boosted by AMF in the plants which increase tolerance against stresses of saline soils (Evelin et al., 2009; Abdel-Latef & Chaoxing, 2011).

1.7. xiv Drought Resistance

AMF also has a significant role for higher plants for combating drought. It has been reported that AMF give protection to the plants from the detrimental effects of drought and also against other abiotic stresses (Auge, 2004; Ruiz- Lozano, 2006; Garg & Chandel, 2010). The possible explanations for increase of drought resistance in AMF inoculated plants may be, due to enhanced water uptake by providing large surface area for water absorption by AMF and by getting excess to the smallest soil pores (Smith & Read, 2008), by increase in hydraulic conductivity (Robert et al., 2008) and by osmotic 13 adjustment which helps the plant to maintain the turgor pressure even at low tissue water potential (Auge et al., 1986). Similarly, under drought stress, mycorrhizal fungi bring decrease in abscisic acid concentration and increase indole acetic acid and gibberellins concentrations (Liu et al., 2000), which are helpful in relieving plants from drought stress.

1.7. xv Succession

Mycorrhizal plants can easily establish on dry and arid areas and thus AMF play an important role in plant succession (Allen, 1987). It may be due to drought resistance of mycorrhizal plants and having the specialty of enhanced water uptake due to the presence of mycorrhiza which help in reaching to the smallest sized pores in the soil and also providing large surface area for water absorption (Smith & Read, 2008). It might also be by extracting nutrients from nutrient deficient soils (Cripps & Eddington, 2005). On contrary, plants lacking mycorrhiza are slow in growth in early succession (Jeffries et al., 2003). Thus successful colonization of land by plants is indebted to mycorrhiza (Read et al., 2000; Brundrett, 2002).

1.7. xvi Phytoremediation

AMF are ubiquitous in the natural ecosystem and even in agricultural areas (Brundrett, 2002). Thus AM fungi are the integral part of plant roots and enhance their functioning in such a way to enable them to grow on degraded soils or even contaminated with heavy metals (Karimi et al., 2011). AM provides natural protection against heavy metals toxicity by decreasing the uptake of the heavy metals in the plants (Zaidi & Musarrat, 2004) or by storing them in vesicles with help of binding substance i.e glomalin (Khan, 2005). For maintainance of biotopes AMF is considered as an important biological component of the self sustaining ecosystems (Jeffries et al., 2003).

1.7. xvii) Land rehabilitation

Vesicular arbuscular mycorrhiza (VAM) fungi play an important role in land rehabilitation (Allen & Allen, 1988; Sylvia & Will, 1988; White et al., 1989). VAM inoculated plants have the power of establishment even in saline soils (Sannazzaro et al., 14

2007). VAM also help plants to grow in nutrient deficient soil or those which have eroded, including those soils which are contaminated with heavy metals (Karimi et al., 2011; Rahmanian et al., 2011). Mycorrhizal fungi also bring about aggregation of soil particles which control soil erosion. Most of the plants growing in sandy soils & dunes are mycorrhizal inoculated which help them in binding soil grains.

1.7. xviii Weed control

AM fungi are also useful in the eradication of weeds by release of allelopathic compounds in the agriculture soils (Francis & Read, 1994).

1.8 MYCORRHIZAL DEPENDENCY

Mycorrhizal dependency is “the ratio of the dry mass of a plant exhibiting mycorrhizal association to that of non-mycorrhizal plants (Lambers et al., 2008).” Relative mycorrhizal dependency is “the difference between mass of dry shoot of plants exhibiting mycorrhizal association and those of non-mycorrhizal plants, given as percentage of mycorrhizal plant‟s dry mass (Plenchette et al., 1983).” The plant and mycorrhizal species involved in the association and nutrient levels of the soil directly affect the Mycorrhizal dependency and this mycorrhizal dependency in turn affects the response of the plant to mycorrhizas which may help in natural selection (Costa et al., 2005; Zangaro et al., 2007; Nogueira & Cardoso, 2007). This dependence helps in classifying the plant species as obligatory or facultative mycotrophics (Janos, 2007).

1.9 TEST PLANT

Sunflower (Helianthus annuus L.)

1.9. i Local names Soraj mukhee (urdu), Nowar parast (Pushto). 1.9. ii Systematic position

Systematic Position of Sunflower (Helianthus annuus L.) is as;

Kingdom: Plantae

Division: Angiospermae

Subdivision: Eudicots

Class: Asteroids

Order: Asterales

Family: Asteraceae

Subfamily: Helianthoideae

Scientific Name: Helianthus annuus L. (Dwivedi & Sharma, 2014)

Sunflower (Helianthus annuus L.) is one of the important oilseed crop in the world and ranks fourth in production of vegetable oil (Weiss, 2000). About 18% of edible vegetable oil of the world is obtained from sunflower (Husain et al., 2010). Pakistan is the third largest importer of edible oil in the world (Meric et al., 2003). Pakistan being deficit in edible oil production spends a huge amount of foreign exchange 241.936 billion rupees (Pakistan economic survey, 2013-14). Only 30% of our requirement is met with domestic production from cottonseed, canola, sunflower, mustard and rapeseeds, while the rest of 70% is met through import (Amanullah & Hatam, 2001). Sunflower shares 22% in the domestic production of edible oil (Pakistan economic survey, 2013-14). Pakistan has good potential in making country self sufficient in edible oil production, it is therefore needed not only to introduce high yielding hybrids of sunflower in the country but also use various manipulations, including use of AMF to increase edible oil production under climatic conditions prevailing in Pakistan (Haq et al., 2006).

Sunflower has shortest growing season among the oil seed crops (FAO, 2002). In Pakistan it grows successfully under different climates. It can tolerate temperature ranging from 8 to 34°C, but the optimum temperature is 20-25°C for better crop (Shah et al., 2005).

1.9. iii Cultivation

Sunflower (Helianthus annuus L.) is cultivated on more than 22 million hectare (ha) of land worldwide with production of 26 million tonnes of seeds (Skoric et al., 2007). In Pakistan sunflower is cultivated on 0.7 million hectares area with production of 144 thousand tons of edible oil (Pakistan economic survey, 2013-14).

In the Khyber Pakhtoonkhwa (KP), during 2009-10 sunﬂower was cultivated on of 559 ha with production of 1608 kg/ha (MINFAL, 2009, 2010). The major sunflower growing areas are D.I. Khan, Mardan and Swabi districts, where suitable environmental conditions favor the production of sunflower (Web, 2). The present research work was conducted on the following four hybrids of sunflower, NKS-278, Hysun-33, SMH-0917 and SMH-0907. NKS-278 and Hysun-33 are imported hybrids, cultivated on commercial scale while SMH-0917 and SMH-0907 have been developed at NARC. These above mentioned hybrids are useful for the farming on the basis of higher yield, resistance to diseases and drought resistance.

1.9. iv Botanical Decription:

Helianthus annuus L. is herbaceous stiff plant which is 1-3 meters in height. It has tap root system in the beginning but at maturity it develops large fibrous lateral roots. The stem is round, unbranched and hispid. The upper leaves are alternate while lower leaves are opposite. They are mostly ovate, broad, 4-20cm long and 3-15cm wide, margins are serrate and apex is acute or acuminate. The inflorescence is large flower head, called capitulum which may be terminal or axillary in position. The head comprises of ray florets and disk florets. The ray florets are sterile, ligulate and mostly yellow in colour, while disk florets are tubuler, and perfect. Ovary is inferior. Fruit is an achene. The sunflower head has 1000-2000 individual florets born on a common receptacle, surrounded by green involucre (Bashir et al., 2015).

1.9. v Chemical Constituents

Sunflower seed has high nutritional value, as it contains, fatty acids (linoleic acid, oliec acid, palmitic acid & stearic acid), carbohydrates, proteins, calcium, 17 vitamin, iron, carotenoids, waxes, tochophherol, chlorogenic acid, caffeic acid, quinic acid, total minerals K, N, S, P, Ca, Mg, Na, Zn and Cu (Boriollo et al., 2014)

Phytochemical studies revealed that it contains a number of bioactive compounds which are non-nutritional and have curative properties for many diseases, including fixed oil, active proteins, flavonoids, phenol, tannins, terpene compounds, alkaloids, carbohydrates (non-starch), saponins and steroids (Bashir et al., 2015; Ibrahim & Ajongbolo, 2014).

1.9. vi USES

Sunflower is mainly cultivated for production of seeds (Khan et al., 2007) which is a rich source of edible oil and is used for human consumption and industrial use. Sunflower meal can be used as poultry and cattle feed, as it is a rich source of protein and also producing raw materials for a number of industries (Web 3). Sunflower is also a medicinal plant. Various parts of the plant are traditionally use for cure of various ailments. The seeds of sunflower are used in the treatment of pulmonary infections, coughs and colds. Helianthus tincture is very useful for the treatment of fevers (Suo & Yang, 2014).

Sunflower seed oil is used as an anti- inflammatory, anti-oxidant, antipyretic, antitumor, antiasthmatic, astringent, stimulant, diuretic, cathartic, vermifuge and in antimicrobial agent (Bashir et al., 2015)

1.9. vii Nutritional Values of Sunflower Oil:

Sunflower oil is used for cooking, frying, medicinal and canning purposes (Husain et al., 2010). This oil is light in colour and taste and is very beneficial for human health due to high amount of vitamin E, monosaturated and poly-saturated fats content and little amounts of saturated fat (Arshad & Amjad, 2012). Sunflower oil contains two types of fatty acids; saturated fatty acid like stearic acid (18:0), palmitic acid (16:0) and unsaturared fatty acid like oleic acid (18:1) and linoleic acid (18:2). Unsaturated fatty acid is further divided into monounsaturated fatty acids (oleic acid (18:1) and polyunsaturated fatty acid (linoleic acid; 18:2) (Lee et al., 2010; Baydar & Erbas, 2005). 18

Oil containg unsaturated fattyacids are nutritionally much better and has positive effect on human health, as they are responsible for lowering “bad” LDL cholesterol (low density lipoproteins) and simultaneously increasing the "good", HDL cholesterol (high density lipoprotein). On contrary, the saturated fatty acids behave otherwise (Izquierdo & Aguirrezabal, 2008) Sunflower oil is also used as basis for lubricants, paints, varnishes, candles, soap and cosmetics industries (web. 5).

1.10 OBJECTIVES OF PRESENT STUDY

Soils of Pakistan like most of the arid and semiarid soils of world are mostly Phosphorus (P) deficient due to their alkaline and calcareous nature, affecting plants adversely (Memon et al., 1992; NFDC, 2001; Gill et al., 2004). Phosphorus contents of an average soil is about 0.05%, out of which only 0.1% of the total P is brought in use by the plants because of its low solubility. To overcome this problem plants have adopted different strategies to acquire sufficient phosphorus (Sharma, 2004). Arbuscular mycorrhizal fungal association is one among these adaptations (Khade & Rodrigues., 2009; Coline et al., 2011). Due to scarcity of phosphorus content in the soil and its rapid utilization, efforts are being made to supplement plants with low grade rock phosphate. Mycorrhizal inoculation can help plants by solubilizing rock phosphate into available form, which helps in plant growth (Sabanavar & Lakshman, 2009).

Throughout the world scientists are now focused on developing alternative technologies to minimize dependence on chemical fertilizers. Although remarkable research work has been done on various aspects of AM, but in Asian countries including Pakistan, such soil nutrient deficiency and host growth responses of oil seed crops are least addressed. Therefore, in the present research work it is intended to evaluate the role of AM fungal technology on host growth responses, mineral nutrient acquisition and productivity of selected sunflower (Helianthus annuus L.) hybrids at various levels of rock phosphate with the following objectives. 19

 To find out the effect of AM Fungi on host growth responses of four hybrids of sunflower (Helianthus annus L.) under various levels of rock phosphate (RP).

 To examine the effect of different rock phosphate levels on AMF spore population in rhizospheric soils.

 To assess the role of AM technology on mineral composition, nutritional aspects and fixed oil (fatty acid) profile of selected sunflower hybrids under different RP amendments.

 To screen out the possible effects of rock phosphate amendments on percent colonization of AM fungi.

 To evaluate the M.D (Mycorrhizal Dependency) values for test species at various levels of rock phosphate.

 To encourage the use of AM fungi for soil fertility and productivity, which will in turn reduce and discourage the use of synthetic fertilizers, which are not only very costly, but also cause soil and water pollution.

1.11 EXPECTED OUTCOMES

This research work has substantial environmental and economic significance. It encompasses soil fertility management by using biological alternatives instead of using heavy application of synthetic fertilizers. Some effective and efficient AM endophytes associated with Helianthus species would be identified and exploited for enhancing productivity of fixed oil plant, which will reduce dependency on import of edible oil and will save foreign exchange. This will also improve the economic status of growers of this important crop.

CHAPTER: 2 LITERATURE REVIEW

2.1 Effect of AM fungal inoculation on plant growth, yield and nutrient acquisition Tjondronegoro & Agustin (2000) studied the influence of Glomus fasciculatnm and soil water condition on growth of maize and soybean. Results revealed that G. fasciculatum increased dry weights of both plants and grains but the response was not constantly statistically significant. Cruz et al. (2000) reported that there was considerable increase in plant fresh weight, leaf water potential and ethylene concentration in the roots of AMF inoculated than non-inoculated papaya plants under drought conditions. Estrada- Luna et al. (2000) examined that AMF inoculated micro propagated guava (Psidium guajava L.) plantlets showed greater shoot length, leaf production, dry matter content (shoot, root and leaf) and leaf tissue mineral like phosphorus, magnesium and copper levels than non-AMF plantlets. Abdel-Fattah & Mohamedin (2000) studied the effect of the interaction between AMF and Streptomyces coelicolor on the growth and nutrition of sorghum (Sorghum bicolor) plants. The result indicated that AMF inoculation (either in the presence or absence of Streptomyces coelicolor) significantly enhanced the growth and nutrient contents of sorghum as compared to non-inoculated plants. Al-Karaki (2000) reported that AM inoculated garlic (Allium sativum L.) plants show enhanced acquisition of mineral nutrients with low mobility, such as P, Zn, Cu and Fe by extending their fungal hyphae far from the rooting zone. Cigar et al. (2000) observed that AMF inoculation can increase cucumber growth through uptake of P, Mn and Zn. Chinnamuthu & Venkatakrishnan (2001) reported that dual soil application of vermicompost and AMF significantly improves the yield of sunflower. Fidelibus et al. (2001) showed that four Glomus species isolated from different geographic areas enhances the rate of transpiration, phosphorus content in leaves and root length of citrus seedlings in AMF inoculated than non-inoculated plants when grown under well-watered conditions and high level of phosphorus. Kelly et al. (2001) investigated that maize and soya bean plants inoculated with AMF (Glomus clarum) fungi shows improved phosphorus acquisition and dry mass as compared to non-AMF inoculated plants. Inoue et al. (2001) investigated the influence of AMF (Glomus species) colonization on growth 21 and P uptake of Baby Corn, in sandy soil. Results revealed that there was no significant interaction between phosphorus fertilizer and AMF inoculation on N, P and K uptake. Alloush & Clark (2001) determined that P uptake and growth were positively correlated with AMF (Glomus clarum) colonization. Marschner et al. (2001) found that arbuscular mycorrhizal (Glomus mosseae or G. intraradices) infection had no significant effect on growth or P content of shoot. Rao & Tak (2001) found positive effect of AMF on different mineral nutrient uptake. Feng et al. (2002) examined in green house experiment the influence of AMF (Glomus mosseae) colonization on growth of maize plants. It was found that mycorrhizal plants had greater P, dry mass and soluble sugars in roots in contrast to control (non- mycorrhizal) plants. Karasawa et al. (2002) conducted a pot experiment and evaluated the influence of previous crops on maize (Zea mays L.) growth and found that the AMF inoculums improved the shoot weight (17 to 49%), plant growth and AMF colonization of maize after sunflower cropping, as compared to that after mustard cropping. Nagaraja et al. (2002) described the positive effects of AMF inoculation on height, yield, total chlorophyll, shoot and root dry weight of sunflower (Helianthus annuus.L.). Among AMF spores Glomus deserticola is more effective in percent mycorrhizal colonization than other fungi (G. mosseae, G. aggregatum and Gigaspora margarita). Liu et al. (2002) conducted a field experiment and suggests that AMF enhanced maize biomass and nutrients (K, Ca and Mg) uptake in P low soil. Habte & Osorio (2002) reported that AMF inoculation enhanced the absorption of macro (P, K, Ca and S) and micronutrients (Cu, Fe, Zn and Mn) from the soil. Fisher & Jayachandran (2002) investigated that AMF significantly improve total phosphorus content and dry weight of two endangered plant species. McGonigle et al. (2003) reported that AMF inoculated maize (Zea mays L.) plants show better shoot phosphorus uptake and growth in undisturbed soil than the disturbed soils. Bergero et al. (2003) studied that AMF colonization bring increase in the dry weight and P uptake and also stimulates root and shoot growth in peanuts. Kaya et al. (2003) observed maximum biomass and fruit yield in AMF inoculated watermelon (Citrullus lanatus) plants, as compare to non-inoculated plants, whether there were water stressed or not. Caravaca et al. (2003) studied seedlings of Olea europaea L. and 22

Rhamnus lycioides L. under drought stress and found that mycorrhizal association have profound effect on foliar P concentration and other physiological parameters than the non-mycorrhizal seedlings. Jansa et al. (2003) studied the influence of AMF (Glomus intraradices), on the P and Zn uptake by maize. Results indicated that plants inoculated with AMF enhance uptake of P and Zn as compared to control. Bi et al. (2003) studied the influence of two AMF (Glomus versiforme and G. mosseae) on the maize growth and uptake of nutrients. They found that mycorrhizal inoculation enhances growth and nutrient aquisition of mazie plants in contrast to control (non-mycorrhizal) plants. Phiri et al. (2003) found that AMF colonization improved growth and nutrient uptake (P, N, K, Mg and Ca) in Tithonia diversifolia plants. Zandavalli et al. (2004) showed that Araucaria angustifolia seedling associated with AMF produce good results in respect of high root: shoot ratio and high concentration of P, K, Na and Cu in their leaves which suggests that this specie have high dependence on mycorrhiza. Porcel and Ruiz-Lozano (2004) reported that mycorrrhizal plants shows maximum amount of carbohydrates content in soybean plants than non- mycorrhizal. Nowak (2004) investigated that AMF significantly improve P, N and K content as compared to non-AMF geranium plants. Giri et al. (2005) studied the effect of AMF (Glomus fasciculatum and G. macrocarpum) on Cassia siamea in semiarid soil and found that it bring significant increase in their nutrient content (P, K, Cu and Zn ), stem height, dry root and shoot weights than the non-mycorrhizal plants. Karthikeyan et al. (2005) examined the effect of AMF inoculation in tea (Camellia sinensis L.) seedling under nursery conditions and found that this increased their growth and nutrition status. Moreover, the response of the plant was also different to different AMF species. Jalaluddin (2005) found that the combined inoculation of AMF and Bradyrhizobium japonicum increased root nodulation, fresh weight, dry weight and seed weight to a considerable amount than their separate application. Rabie (2005) found that AMF inoculation in mung bean plants brought maximum increase in growth, chlorophyll concentration, sugar contents and mineral concentrations of P, N, K, Mg and Ca than non-AMF inoculated plants.. This symbiosis also resulted in increase in the dry weight of root: shoot, height, protein content and nitrogenase activity in roots. Redecker (2005) found positive effect 23 of AMF on different mineral nutrient uptake. Sailo & Bagyaraj (2005) reported that P contents was higher in AMF treated Coleus plants than non-AMF treated plants. Davies et al. (2005) examined the effect of AMF on growth, leaf elemental concentration and yield on Solanum tuberosum L. and found positive effect of AMF on mineral nutrient (P, Fe and Mg) uptake than non-inoculated plants. Kytöviita (2005) reported that AMF improves the uptake of nutrient and water from the soil to the host plant. Subramanian et al. (2006) suggested that tomato plant with mycorrhizal infection showed better resistance when grown in field under drought stress by enhancement of nutritional status specially P and N and resulted in better growth and fruit quality than non-mycorrhizal plants. Mena-Violante et al. (2006) demonstrated that AMF has positive effect on fruit growth and fresh weight of Capsicum annuum L. exposed to drought. Adriano-Anaya et al. (2006) examined that AMF (Glomus intraradices) inoculation enhanced root and shoot dry mass of sorghum (Sorghum bicolor). Bittman et al. (2006) reported that P uptake and maize growth were positively correlated with AM colonization Hameeda et al. (2007) investigated that dual inoculation of three growth- promoting bacterial strains and AMF (Glomus spp.) improves plant biomass upto (17 to 20%) and mycorrhizal colonization (without growth-promoting bacterial strain) improves (25 to 35%) in sorghum plants. Joshee et al. (2007) studied the effect of inoculation of five different strains of AM fungi and found that all of them have positive effect on plant growth specially root development compared with non-inoculated control plants. Among the five strains, S3004 was more effective in increasing in plant height and fresh weights (root, shoot and seed). Liu et al. (2007) reported that AMF inoculation in liquorice (Glycyrrhiza uralensis) resulted in better growth, nutrient acquisition and glycyrrhizin production in comparison to control. Alizadeh & Alizadeh (2007) studied that mycorrhizal inoculated corn (Zea mays L.) plants has positive effect on nutrient absorption under various soil moisture condition. Henrike et al. (2007) reported that positive effect on leaf size may be attributed to AMF hyphae which enhances uptake of phosphorus (P). Bennett & Bever (2007) reported that AMF inoculation improved plant growth which was related with higher uptake of essential nutrients. Tawaraya et al. 24

(2007) reported that mycorrhizal symbiosis generally improves growth and nutrient uptake of Aloe vera plant. Kungu et al. (2008) found that AMF inoculated Senna spectabilis show good response towards drought resistance due to better growth of shoot and root, leaf number, leaf water content, and nutrient uptake than non inoculated plants. Das et al. (2008) performed an experiment on Stevia (Stevia rebaudiana) by studying combined and sole effect of biofertilizers (AMF+ Bacillus megatheriam (PSB) + Azospirillum). Their sole application brought increase in N, P and K contents and biomass yield of plant over control. These results were further enhanced when all the biofertilizers were used together. Roldan et al. (2008) determined that inoculation of Juniperus oxycedrus seedlings with mixture of three AM fungi (Glomus intraradices, Glomus deserticola, Glomus mosseae) along with organic residue show significant increase in plant growth and foliar nutrients (N, P, K) regardless of water regime. Khan et al. (2008) studied the effect of AMF inoculation on Cenchrus ciliaris at two water regimes (100% and 50% field capacity) and found that there was considerable increase in yield, NPK content, dry weight of root and shoot and water use efficiency in 100% field capacity than 50% field capacity as compare to non mycorrhizal plants. Manoharan et al. (2008) observed that the crude protein and crude fiber content is greater in the plants with AMF than the control treatment. Khalafallah and Abo-Ghalia, (2008) reported that mycorrrhizal plants shows maximum amount of carbohydrates content of wheat plant than non-mycorrhizal under well watered conditions. Bai et al. (2008) studied the role of AMF on uptake of P by maize plants. Results showed that AMF inoculated plants had significant positive effects on shoot and root dry weight and P contents in contrast to non-AMF treatment. Dasgan et al. (2008) determined that tomato cultivar M19 when grown hydroponically under open and re-cycled perlite substrate showed better results in fruit growth than vegetative parameters in AMF (Glomus fasciculatum) inoculated plants. Boby et al. (2008) found synergistic effect on growth, yield, N, P and chlorophyll content of cowpea by dual inoculation of AMF (Glomus mosseae) and six soil yeasts. Franco et al. (2008) demonstrated that AMF inoculated sorghum (Sorghum bicolor L.) along with plant growth-promoting rhizobacteria (Azospirillum brasilense) increase plant productivity, even in low moisture condition in the soil. Channashettar et al. (2008) observed that plant 25 shoot and root biomass was significantly increased in Euphorbia prostrate with inoculation of AMF mycorrhiza and vermicomposting. Sheng et al. (2008) conducted a greenhouse experiment and examined the effect of Glomus mosseae (AM) fungus on maize photosynthesis and water status under salt stress condition. It was observed that the increased maize dry shoot and root mass improving plant water status, chlorophyll concentration, and photosynthetic capacity, than non-mycorrhizal plants and hence alleviates the harmful effect of salt stress on plant growth. Pagano et al. (2008) found the positive symbiotic response of AMF on plant height, dry matter content and nutrient acquisition of the host plant. Mehrvarz & Chaichi (2008) reported that inoculated plants of Barely (Hordeum vulgare L.) exhibited higher level of total ash (8.05%) than non- mycorrhizal (7.84%). Kacprzak and Fijalkowski (2009) studied the effect of sewage sludge from pulp industry and mycorrhizal fertilization on biomass of sunflower (Helianthus annuus L.) plant grown at degraded soil from the terrain of zinc mill. It was found that the simultaneous addition of 20% and 30% (w/w) of sewage sludge and root mycorrhization caused increase of sunflower biomass production by approx. 38 and 18%, respectively. Aher (2009) demonstrated that groundnut grown in nutrient deficient soils give better result in terms of yield, growth and nutrition (P, N, K, Mg, Fe, Ca and Na) when inoculated with AMF (Glomus fasciculatum) than non-inoculated control plants. Bisht et al. (2009) evaluated the effects of tetrapartite symbiotic interaction (AMF, Rhizobium leguminosarum, Pseudomonas fluorescens and Dalbergia sissoo) on the growth and nutrient uptake in two different soils ( Mollisol and Entisol) and found enhanced plant growth in Entisol soil compared to uninoculated plants. He further showed that AMF and Pseudomonas fluorescens combination gave decrease in plant growth, suggesting that appropriate bacteria - AMF combination is required to get the desired plant growth. Bolandnazar (2009) studied the effect of three different AMF species (Glomus etunicatum, Glomus intraradices and Glomus versiforme) on red onion cv. Azar-Shahr which resulted increase in the survival rate of seedlings and similarly seedling establishment rate also increased by 21%. The yield of onion bulb was also increased by 3 times than the control plants. Carretero et al. (2009) investigated that effect of AMF (Glomus intraradices) on three clones of Manihot esculenta and found that for 26 improvement of yield AMF inoculation is necessary irrespective of clones under experiment. Castillo et al. (2009) studied the effect of two different AMF (Glomus intraradices and Glomus claroideum) on Capsicum annuum L.) and found that inoculation with Glomus claroideum a native fungi accelerated the maturation stage and resulted in better plant yield. Guralchuk et al. (2009) studied the growth of medicago sativa L. in highly polluted soil with arsenic and heavy metal. Those which are inoculated with metal tolerant strain of AMF (Glomus mosseae) resulted in better growth as compared to less heavy metal tolerant AMF strains. Gutierrez-Oliva et al. (2009) evaluated that effect of AMF (Glomus claroideum) inoculated Ananas comosus L. plantlets at different phosphorus level and found that growth rate was maximum at 0.9 mM P level. Mehraban et al. (2009) analyze the effect of different AMF on various cultivar of sorghum for better results of growth and yield and found that they behave differently on differ cultivars of sorghum. It was therefore suggested that AMF species with better results should be sorted out before using it on the plant species. Shrestha et al. (2009) conducted a field experiment and studied the influence of arbuscular mycorrhiza (AM) fungi in the maize productivity. It was observed that AM inoculation exhibits significant effect on plant yield and height. Parras-Soriano et al. (2009) reported that AMF inoculated plants enhanced growth and uptake of nutrients like, potassium and nitrogen in Olive trees. Lester (2009) reported that mycorrhizal fungi discharge powerful chemicals (lytic enzymes and organic acids) in the soil that bring about dissolution of tightly bound soil nutrients like P and Fe. Omomowo et al. (2009) found that inoculation with Glomus mosseae has higher fat content of cowpea than un-inoculated control. Amaya-Carpio et al. (2009) showed that mycorrhizal infection had positive effect on absorption of P, N, Cu, Fe, and Zn in Ipomoea carnea. Khade & Rodrigues (2009) reported that AMF inoculation results in the enhancement of total P and K content in Carica papaya plant as compared to non-AMF controls. Similar results were also reported by Aliasgharzad et al. (2009) for onion plants. Kafkas & Ortas (2009) observed that AMF inoculation can increase Pistacia specie seedling growth through uptake of P and Zn. Jin et al. (2010) reported that dual inoculation of AMF and rhizobia showed better growth performance in Lathyrus sativus. Yua et al. (2010) observed that plants inoculated with G. mosseae or G. etunicatum had positive effect on P uptake and maize biomass. 27

Rydlova et al. (2010) reported that AMF inoculated plants show enhanced uptake of phosphorus and micronutrients. Celebi et al. (2010) observed that AMF application improved silage yield and green herbage and dry matter yield, leaf and stem ratios as compared to non-AMF maize plants. Ortas (2010) reported that AMF inoculation significantly enhances plant growth; fruit yield and shoot P and Zn nutrient uptake in cucumber seedling. Naderi et al. (2010) examined the influence of AMF on absorption and uptake of some macro nutrients in sorghum plant. Results showed that AMF inoculated plants enhanced the absorption of N, P and K than non-inoculated plants. Alizadeh (2010) conducted an experiment on corn and determined the effect of mycorrhizae and non-mycorrhizae treatments on absorption of some macro and micro elements in water stress and different nitrogen rates. The results showed that with increase of nitrogen in mycorrhizae and non-mycorrhizae treatments absorption of nitrogen, phosphorus, manganese and a little potassium increased but iron decreased. Alizadeh et al. (2010) investigated that AMF inoculation significantly enhanced N, P and K absorption in shoot and root. Ratti et al. (2010) reported that Catharanthus roseus inoculated with Glomus mosseae showed enhancement of protein level and nutrients (P, S, Cu and Mn) than non-mycorrhizal plants. Rasouli-Sadaghiani et al. (2010) observed increase in leaf area in mycorrhizal plants as compared to control, supporting the fact that AMF inoculation increase leaf size and overall plant growth as compared to non- inoculated ones. Wu et al. (2010) reported that increase in photosynthesis results in increase in the level of glucose and sucrose in leaves of inoculated plants. Alizadeh et al. (2011) investigated the effect of AMF inoculation on grain yield of sorghum (sorghum bicolor) under drought stress condition. The results indicated that there was increase of 17.8% grain yield in inoculated sorghum crops as compared to control. Fan & Liu (2011) made studies on the use of AMF inoculation on Poncirus trifoliata seedlings for growth and drought tolerance which showed positive correlation between the two, it means that AMF bring positive biochemical and physiological changes that is helpful to the plant. Babu & Reddy (2011) found that AMF inoculated Bermuda grass produce maximum biomass. He & Zhong (2011) experimented on Cinnamomum camphora seedlings in drought stress condition by inoculating them with AMF which showed better result in term of increase in biomass, soluble sugar, proline and protein. 28

Haghighatnia et al. (2011) reported that AMF inoculated citrus plants under drought stress gave better results than non-inoculated plants for increase in phosphorus nutrition and dry weights of root and shoot. Campanelli et al. (2011) used three artichokes hybrids seedlings inoculated with mycorrhiza and studied their effect on the growth and physiological parameters. This symbiotic relationship enhanced their growth and proved to be good for propagation. Aslani et al. (2011) showed that symbiotic relationship of Ocimum basilicum L. with AMF enhances their growth. Asrar & Elhindi (2011) studied the effect of AMF (Glomus constrictum) inoculation in marigold under water stress condition and found enhancement of growth and P content by this association. Farzaneh et al. (2011) reported that arbuscular mycorrhizal fungal (AMF) inoculation enhances mineral nutrient uptake (N, P, K, Mg, Ca, Fe, Cu, Zn and Mn) in chickpea. Sajedi & Rejali (2011) found that mycorrhizal inoculation increased concentration of all nutritional elements (micronutrients) in maize plant. Sharif et al. (2011) studied the response of AMF inoculation on growth of different crops (maize, sorghum, millet, mash bean, and mung bean) in (P)–deficient soil. Result shows that crops inoculated with AMF enhanced root and shoot dry matter and micronutrient uptake considerably as compared to non- inoculated crops. Yaseen et al. (2011) investigated that the effect of AMF inoculation in two different varieties of Vigna unguiculata and found that china variety out performed than the watni variety in terms of vegetative productivity and nutrient uptake than the non-inoculated plants. Asrar et al. (2012) found that AMF inoculation not only causes vegetative growth but also affect the flower yield positively in Antirhinum majus cv. Butterfly irrespective of water supply. Bagheri et al. (2012) studied the effect of two AMF species (Glomus intraradices and Glomus mosseae) on two pistachio cultivars and found that it bring increase in the growth by 75% and plant-water relations by 100% even in the drought stress condition. Han et al. (2012) inoculated cucumber seedlings with four different kinds of AMF and found that Glomus mosseae was comparatively more effective in their growth i.e height, root and shoot fresh weights etc as compared to the control. Habibzadeh et al. (2012) studied the role of mycorrhizal fungi in mungbean Vigna radiate L. for growth and yield and found that Glomus intraradices produced good results bringing increase in the grain yield and dry weight in the vegetative parts of 29 the plant irrespective of water supply. Yaseen et al. (2012) reported that inoculated chickpea (Cicer arietinum) plants with AMF produced better results in vegetative and reproductive growth parameters. Jezdinsky et al. (2012) found that AMF inoculation in leek (Allium porrum L.) plants exhibited a pronounced effect on leaf area and fresh weight of the plant under drought conditions. Karti et al. (2012) showed that AMF inoculation in Stylosanthes seabrana bring increase in proteins and dry weight of root and shoot and enhancement of leaf water potential under water stress condtions. Ortas (2012) found that AMF inoculated maize plants shows better plant yield and uptake of nutrient, as compared to non-inoculated plants. Ramakrishnan & Selvakumar (2012) reported that AMF (Glomus fasciculatum and Glomus intraradices) inoculation in Lycopersicum esculentum Mill. significantly enhanced growth and available nutrient content (N,P,Zn and Cu) as compared to control treatment. Soleimanzadeh (2012) stated that AMF inoculated plants had significant positive effects on oil yield of sunflower (Azargol cultivar) than non-inoculated plants. About 7 % increase in number of seeds was also observed. Evelin et al. (2012) found that mycorrhizal colonization increases the uptake and efficiency of macronutrients nutrient (P, N and Mg) from the soil. Karagiannidis et al. (2012) found the positive symbiotic response of oregano and mint to three Greek AM fungi in terms of growth and macro and micronutrients acquisition. Zeng et al. (2013) showed that symbiotic relationship of medicinal plant and AMF resulted good yield. Gong et al. (2013) conducted a pot experiment under water stress condition and found that AMF symbiosis in Sophora davidii plants increases its growth and other physiological activities thus making seedling more resistant to the drought conditions. Gholamhoseini et al. (2013) investigated the role of AM Fungal (Glomus mosseae and Glomus hoi) inoculation on growth, uptake of nutrients and yield of sunflowers grown under drought stress conditions. Result shows that inoculated plants shows better plants growth, dry matter, seeds number and weight and oil yields as compare to non-inoculated plants irrespective of the AMF species. Vinichuka et al. (2013) studies revealed that AMF inoculated sunflower plant significantly enhanced plant biomass and 137Cs uptake than non-inoculated plants. Abdallah et al. (2013) examined the effect of certain vitamins (α-tocopherol, 30 nicotinamide) and AM fungi on growth, some yield components and biochemical aspects of sunflower (Giza 102) at different Water Regimes. The results revealed that sunflower inoculated with AM fungi and vitamins particularly α-tocopherol addition increased plant growth parameters, yield components, photosynthetic pigments, total carbohydrates, total N and some minerals at 60% water holding capacity. Patra et al. (2013) reported that an interaction of AMF +Phosphate Solubilizing Bacteria+Azotobacter significantly affected growth, yield and oil content over Azotobacter + AMF, PSB + AMF inoculation in sunflower (Helianthus annuus L.). Audet et al. (2013) conducted greenhouse stratified compartmental pot experiment using „dwarf‟ sunflowers and micronutrient zinc as a metal contaminant and evaluated the role of arbuscular mycorrhizal symbiosis toward plant growth and Zinc uptake. He concluded that mycorrhizal inoculated plants showed better plant growth and contained 40% lesser concentration of zinc in their shoots as compared to non-inoculated plants although the soil Zn level was at the highest i.e 200 and 400 mg Zn kg−1 dry soil). Kavitha & Nelson (2013) reported high number of seeds per head in AMF inoculated plants than the non-inoculated plants. Bera et al. (2014) reported that AMF inoculation significantly increased plant height, head diameter, 1000 seed weight, seed yield and oil content of sunflower (Helianthus annuus L.). Alqarawi et al. (2014) found that mycorrhizal symbiosis significantly increased K+ and P and in Ephedra aphylla under salt stress conditions. Heidaria & Karamiba (2014) evaluated the effect of AMF colonization on grain yield, nutrient uptake and oil content in sunflower under water stress conditions. From their results it is concluded that mycorrhizal plants had significantly higher grain yield, nutrient uptake and oil content by increasing water stress. Abeer et al. (2015) investigated the effects of AMF on the growth, plant growth regulators and inorganic nutrients in tomato (solanum lycopersicum l.) grown under salt stress condition. It was observed that AMF enhanced accumulation of phosphorus, potassium, magnesium and calcium but biomass and yield were negatively affected. Aher (2015) studied the influence of AMF on vegetative parameters of Arachis Hypogea L. and observed that mycorrhizal inoculated seedlings grew faster and healthier over control seedling. AbduAllah et al. (2015) reported that mycorrhiza plants performed better as compared to non-mycorrhizal plants regarding linoleic acid content. Arias et al. 31

(2015) reported that sunflower inoculated with arbuscular mycorrhizal fungi (AMF) absorbed equal or lower amounts of Iron (Fe), sodium (Na) and calcium (Ca) than controls. Halder et al. (2015) reported that inoculation of biofertilizers (AMF) increased the growth, macronutrient (N, P, K and Mg) and micronutrient upake (Fe, Mn and Zn) in Ipomoea aquatic.

2.2 Effect of AMF on spores density and root colonization Lahlali (2001) conducted an experiment with amplification of three vesicular arbuscular mychorrhizal (Sclerocystis spp., Glomus mosseae and G. intraradices) species in aeroponic conditions and showed that by Glomus mosseae, root colonization rate was 18.15 % in corn and 53.71 % in sorghum and they produced an average number of spores of 3.10 and 20.83 per centimeter of roots of corn and sorghum respectively. While G. intraradices showed 50.74 % and 34.12 % root colonization rates of sorghum and corn respectively. Galvez et al. (2001) studied that soil under low-input management had higher VAM fungus spore population, than soil under conventional management, spore populations and colonization of maize roots by VAM fungi were higher in no-tilled than in chisel-disked or moldboard plowed soil.

Karasawa et al. (2002) studied the influence of previous crops on maize (Zea mays) growth are relatively due to multiplication of indigenous AMF density. The AMF inoculum improved the shoot weight (17 to 49%), plant growth, AM colonization of maize after sunflower cropping, as compared to that after mustard cropping. Niranjan et al. (2002) observed that maximum value of AMF root colonization was recorded in Prosopis cineraria seedlings inoculated with Glomus mosseae. Hart & Reader (2002) studied the strategy of AMF colonization in three different families (Acaulosporaceae, Glomaceae and Gigasporaceae) and found that in Glomaceae the colonization rate was high in the plant than the soil, while Gigasporaceae gave opposite results but in the Acaulosporaceae the extent of colonization was low both in soil and host. Thus taxonomic differences do play role in their extent of colonization and biomass both in the plant and soil. Klironomos & Hart (2002) studied the colonizing strategy of AMF by using different inoculums e.g spores, infected roots and extraradical hyphae and found 32 great difference among the members of Glomineae and Gigasporineae to the extent of suborder level. In this study it was revealed that Glomineae isolates can be infected by all three types of inoculums while Gigasporineae mostly rely on spores and to some extent by root fragments but extraradical hyphae don‟t play any role of infection in this case. Lukiwat & Simanungkalit (2002) reported that dual inoculation (Glomus and Bradyrhizobium japonicum) resulted in better AMF colonization and spores number than others treatment in soybean plants. Jansa et al. (2003) investigated that the presence of the genus Scutellospora in maize roots was strongly reduced in chiseled and plowed soils. Fungi from the suborder Glominae were more dominant colonizers of maize roots growing in plowed soils. Kumar et al. (2004) found better AMF root colonization in chickpea plants. Duponnois et al. (2005) studied the effect of AM fungi in relation to microbial communities present around and found that the structure and functionalities differ among the microbes present in the rhizosphere than those in association with extrametrical hyphal network. Plenchette & Duponnois (2005) conducted a pot experiment on Atriplex nummularia inoculated with AMF (Glomus intraradices) in low phosphorus soil. The roots were well colonized with mycorrhizal hyphae and vesicles but no arbuscules were found, which suggested that arbuscules are not necessary for growth stimulation. Jefwa et al. (2006) studied the diversity and frequency of occurrence of glomale mycorrhizal fungi on three farming (maize/sesbania intercrops and maize monocrop) systems in a drought prone and nitrogen deficient site in southern Malawi. Twelve glomale mycorrhizal species were recorded, four species in Glomus, four in the genus Acaulospora, two in Gigaspora and two in Scutellospora. It was found that occurrence and persistence of glomale species are influenced by agroforestry combinations, and that the spores of most species are tolerant to dry conditions. Reddy et al. (2006) isolated and multiplied spores of the mycorrhizal fungus (Glomus fasciculatum) in sterilized sandy soils using sorghum roots as collateral host. They suggested that the sorghum roots were greatly effective for culturing and multiplication of G. fasciculatum spores. Adriano- Anaya et al. (2006) investigated that dual inoculation of AM (Glomus intraradices) fungi and Gluconacetobacter diazotrophicus has synergistic effect on root colonization of maize (Zea mays). 33

Perner et al. (2007) studied the effect of AMF along with two levels of compost and found that root colonization was higher upto 36% in inoculated as compared to non- inoculated pelargonium plants. Valsalakumar et al. (2007) conducted a field experiment to determine the AMF spores associated with green gram and found that Glomus mosseae (81%) was in abundance followed by Glomus microcarpum, (24%) Gigaspora margarita, (24%) and Scutellospora specie (5%). Moreover, the percent colonization was negatively correlated with soil phosphorus and positive correlation existed between pH & nitrogen with potassium which shows that some other factors are responsible for distribution and sporulation of fungi rather than soil nutrients. Isobe et al. (2008) collected the roots along with rhizosphere soil from soybean and maize fields. The soil samples were examined for chemical properties and the density of AM fungal spores while roots for infection ratio. It was concluded that in soybean and maize fields there exists a significant difference in the spore density. The density of AM fungi spores is generally higher in soils with a higher phosphate absorption coefficient. Muok et al. (2009) determined the influence of AMF on intercropping Sclerocarya birrea (marula), millet (Pennisetum glaucum) and corn (Zea mays) in the presence of AMF. The result reveals that intercropping marula with millet or corn helps in AM fungal spores propagation in the soil which would enhance marula establishment especially in low nutrient soil. Wiseman et al. (2009) investigated that colonization rarely exceeded 5% when corn (Zea mays) and sorghum (Sorghum bicolor) were treated with commercial AMF inoculants while 38-61% colonization is reported with viable lab- cultured inoculant. Moreover, commercial inoculants usually increased soil nutrient and shoot growth. Shrestha et al. (2009) studied that AM spore density was improved in the mycorrhizal inoculated plants as compared to control in both maize and finger millet crops. Powell et al. (2009) gave explanation for the variation of root colonization by AMF as a result of great genetic divergence of species in two fungal clades i.e Diversisporales and Glomerales. The later showed extensive colonization than the former. Gutjahr et al. (2009) reported that roots colonized by AM fungi change its form and structure in such a way that it gets profusely branched. Nasrullah et al. (2010) suggested that under different agro-ecological conditions spores density in soil and roots infection intensity varied from one site to another and in 34 soil with low organic matter contents higher AM infections rates were observed. Hua et al. (2010) studied that AMF inoculation led to significantly higher root colonization of AMF in maize. Mustafa et al. (2010) investigated that in sweet corn there was a positive relationship between roots colonization and spores density. Alizadeh et al. (2011) investigated the effect of AMF inoculation on grain yield of sorghum (sorghum bicolor) under drought stress condition. The results indicated that there was increase of 17.8% grain yield in inoculated sorghum crops as compared to control. Borde et al. (2011) analyzed growth, photosynthetic activity, proline and antioxidant responses of AMF (Glomus fasciculatum) on inoculated and non-inoculated millet (Pennisetum glaucum) crops under salt stress condition. Results indicated that AMF inoculated plants perform better in response to growth and nutrient uptake under moderate salt levels by improving the accumulation of proline and antioxidant activity as compared to control (non- AMF inoculated) millet plants. Sharif et al. (2011) observed that AMF inoculation shows maximum root infection intensity and soil spore density in millet crop followed by mash beans i.e. 35% and 32% respectively in P-deficient soil. Poomipan et al. (2011) investigated that reintroduction of a native Glomus enhanced AMF spore density and root colonization in maize. Ortas & Akpinar (2011) conducted a greenhouse experiment on a growth medium to assess the impact of maize genotypes to several mycorrhizal inoculums on spore production. Pagasso and Darva genotypes produce high root colonization percentages than others. Tian et al. (2011) studied the spatio-temporal dynamics of native arbuscular mycorrhizal fungal community in an intensively managed maize agro ecosystem in North China and analyzed the percent root colonization and spore density. Twenty seven AMF species belonging to five genera such as Glomus aggregatum, Glomus claroideum, Glomus etunicatum, Glomus geosporum and Glomus mosseae were isolated. It was concluded that the effects of growth stage, soil depth and management system on spore relative abundance differed among AMF species. The highest species richness and diversity occurred at the R1 stage. Murat et al. (2011) found that AMF inoculated plants produce good yield, root colonization and increase in phosphorus in seed and shoot. Kavitha & Nelson (2013) investigated the root colonization and species diversity of arbuscular mycorrhizal fungi in the rhizospheric soil of sunflower, collected from 35 different areas. It was concluded that the higher colonization was observed at Arasadipatti, Thirukkanurpatti (Thanjavur), Thalaimalaipatti (Trichy) and the lower colonization was observed at Thanjavur. The colonization, ranged from 20%-70%, this may due to some stresses like abiotic and biotic. Overall 14 species of arbuscular mycorrhizal fungi were isolated, of which 7 species were contributed by Glomus spp., 3 by Gigaspora spp. and 2 by Scutilospora spp. and Acalospora spp. The spore densities of soils varied from 210 to 740/100g soils. Gholamhoseini et al. (2013) examined the effect of two species of mycorrhizal fungi (Glomus mosseae and Glomus hoi) on root colonization in sunflower under different drought stress conditions and showed that G. mosseae is more effective in root colonization under water stress. Kumar et al. (2013) investigated that diversity of AMF, spore density and root colonization varied in different seasons irrespective of the host species. Maximum root colonization and AMF spore density was recorded in rainy season followed by winter and minimum in summer season. Moreover, eleven AMF species belonging to four genera viz, Glomus, Acaulospora, Sclerocystis and Gigaspora were isolated in which Acaulospora and Glomus were more predominant all species of plants studied. Gunwal et al. (2014) studied that AMF spore density and root colonization decreases in heavy metals contaminated soils. Moreover, Glomus species were more predominant than other AM fungal spores. Urcoviche et al. (2014) assessed the spore density and root colonization by AMF in medicinal and seasoning plants (Rosmarinus officinalis L., Tropaeolum majus, Mentha crispa L., Peumus boldus, Origanum vulgare and Matricaria chamomilla) and found that AMF root colonization ranges from 17 to 48% and among AMF spores, Glomus spp were more predominant followed by Acaulospora sp. in all species of plants studied. Sarkar et al. (2014) studied the species composition and diversity of AM across the soil of degraded forest and rubber plantation in India and found 1880 spores‟ i.e 1380 spores from degraded forest and 500 from rubber plantation, representing 27 species. Acaulospora and Glomus were present in 1/3rd and 1/4rth of the total spore density respectively. Moreover the rubber forest was showing decrease of Gigaspora by 14.30 %, Acaulospora by 21.33 %, Glomus by 33.47 % and Scutellospora by 77.42 % as compared to degraded forest. 36

Abeer et al. (2015) investigated that in tomato the spore population and AMF colonization were negatively affected grown under salt stress condition. Aher (2015) studied the influence of AMF (Glomus fasciculatum, Glomus geosporum, Scutellispora nigra and Scutellispora Sp.) on root percent root infection in Arachis Hypogea L. and found that among all Glomus fasciculatum was most effective over control.

2.3 Effect of AM fungal inoculation on plant growth, yield and nutrient acquisition in relation to fertilizer application

Bagayoko et al. (2000) studied the influence of mycorrhiza on growth and nutrient uptake of pearl millet (Pennisetum glaucum L.), cowpea (Vigna unguiculata) and sorghum (Sorghum bicolor L.) on a West African soil. Result shows that dual inoculation of AM fungi and P led to increase root and shoot dry matter and acquisition of P, Ca, K, Zn and Mg in sorghum and cowpea. Meshram et al. (2000) reported that chickpea seeds inoculated with AM fungi along with rock phosphate exhibit maximum values for shoot and root dry matter, grain yield as compare to control plants. Davies et al. (2000) demonstrated that AMF (Glomus intraradices) enhanced Capsicum annuum L. vegetative and reproductive growth under low phosphorus (P) level. Inoue et al. (2001) investigated that there was no significant interaction between phosphorus fertilizer and AMF inoculation on N, P and K uptake in Baby corn plant. Fusconi et al. (2001) concluded that AM fungi significantly improve Allium porrum growth in soils with low phosphorus (P) availability. Prasad & Bilgrami (2002) reported that AMF (Glomus fasciculatum) inoculated Saccharum officinarum show effective results in chlorophyll content increment at low dose of phosphate fertilizer as compare to control. Liu et al. (2002) conducted a field experiment and suggests that arbuscular mycorrhizal (AM) fungi enhanced maize biomass in P low soil and micronutrients uptake (Zn, Cu, Mn, and Fe) varied with P levels added to soil. Singh et al. (2002) investigated that influence of soil P on arbuscular mycorrhizal symbiosis is governed by the host genotype. AM colonization, at various P levels improved vegetative maize plant growth, root and shoot phosphorus. However, the pattern of phosphorus and zinc uptake in mycorrhizal inoculated and non-AMF inoculated shoots was genotype specific. Bressan & Vasconcellos (2002) worked under 37 greenhouse conditions, and evaluated the effect of AMF (Glomus etunicatum and Glomus clarum) inoculation and P levels on root system morphology, of maize (Zea mays L.). Results show that plants inoculated with mycorrhizal fungi enhanced P concentration, root dry weight and no. of first and second order lateral roots in the plant. Kapoor et al. (2003) reported that AM inoculation of Foeniculum vulgare mill. along with phosphorus fertilization significantly enhanced growth, essential oil yield and P-uptake of plants. Liu et al. (2003) evaluated the influence of AMF under various levels of P by maize (leafy normal stature, leafy reduced stature and Pioneer 3979) hybrids on P uptake. After 9 weeks of growth, plant biomass and total P content was determined. Results revealed that mycorrhizal inoculated plants improved plant biomass and total phosphorus content in leafy normal stature and leafy reduced stature hybrids than pioneer 3979. Kalipada & Singh (2003) investigated that bio-fertilizer inoculation enhanced the yield and nutrient (P, N and K) uptake with increasing phosphorus level in Chickpea plant. Mahboob et al. (2003) found that seed inoculation with biofertilizers and different fertilizer levels significantly affected yield and yield attributes of rice and mungbean crops. Kerur & Lakshman (2004) observed enhanced level of nutrients in two different VAM inoculated floricultured plants along with phosphate fertilizer, in comparison to non-inoculated plants. Tufenkci et al. (2005) concluded that AMF (Glomus intraradices) inoculated Chickpea (cv. Aziziye-94.) plants with P (50 mg/kg) and N (100 mg/kg) fertilizer significantly enhances its growth, nutrient contents (P, K, Ca and Zn) and yield parameters. Bhat et al. (2005) reported that VAM inoculation along with different phosphorus rates recorded the highest seed yield over the control in Vigna radiata L. Rahman et al. (2006) conducted a pot experiment separately in sterile and non- sterile soil and examined the effects of AM (Glomus mosseae) fungus inoculation and phosphorus (P) on growth and nutrient uptake (N & P) of maize both under drought stressed and unstressed conditions. The results showed that the inoculation of maize plants with Glomus mosseae alone or with Phosphorus enhanced plant height, dry shoot and root weight, and nitrogen and phosphorus content of the plant. Sharathbabu & Manoharachary (2006) reported that dual inoculation of AMF (Glomus fasciculatum) and 38 rock phosphate significantly enhanced Tylophora indica (Burm.f.) plant growth, dry weights and nutrient uptake than in single inoculation or in control plants. Xia et al. (2007) examined that maize inoculated with AM fungi and phosphorus addition increased shoot and root P, Mn, Cu, and Zn uptake than control plants. Rashid et al. (2008) suggested that inoculated brinjal (Solanum melongenum L) seedling with AM fungi and phosphorus fertilization enhanced plant dry biomass and essential nutrient uptake under low level of P (15 and 30 kg ha-1) and further higher levels of P (above 30 kg P ha-1), showed decreasing trend. Subramanian et al. (2008) conducted a greenhouse experiment in a red sandy loam soil to study the responses of maize to AMF (Glomus intraradices) colonization at varying levels of P. The results revealed that mycorrhizal symbiosis improves root morphology, nutritional status and CEC of maize plants than non-mycorrhizal plants. Mycorrhizal inoculated plants had higher P concentrations in roots, shoots, and grains, regardless of P levels. Sabanavar & Lakshman (2009) reported the enhanced growth and nutrient uptake as compared to control plants at high rock phosphate levels (100%) in varieties of Sesamum.. Jambotkar & Lakshman (2009) assessed the influence of AMF along with phosphorus amendment on the growth parameters of Brassica juncea. Result has shown that growth parameters, major macronutrients (P, N and K) and moisture content are highly promising in AMF inoculated plants as compare to P inoculated or control ones. Suparno (2009) studied the effectiveness of AMF inoculation in enhancing the potency of Papuan Crandallite phosphate rock to the growth of Cocoa seedling and found that AMF inoculated seedling results in increase of shoot dry weight (50.14%) and P uptake (64.88%) at high dosages of phosphate rock, as compared to the control. Rakshit & Bhadoria (2010) analyzed the effect of arbuscular mycorrhizal (Glomus mosseae) fungi on maize growth and P nutrition with low soluble phosphate fertilization in Oxisol pot experiment. Results indicated that the dry weight and percentage of P of mycorrhized plants with added phosphate (P) were higher than non-mycorrhized plants. Thenua et al. (2010) results revealed that chickpea plants inoculated with rock phosphate and biofertilizers (phosphate solubilizing bacteria and vesicular arbuscular mycorrhizae) has significantly positive effect on seed yield. Sani & Farahani (2010) examined that AMF inoculated coriander plants with different levels of superphosphate show better oil 39 content, nutrient in shoot and root yield as compare to control plants. Ibiremo (2010) investigated that organic fertilizer (ground cocoa pod husk) amended with phosphate fertilizers (single super phosphate and sokoto rock phosphate) and arbuscular mycorrhizal fungal inoculation has significantly increased growth of cashew (Anacardium occidentale, L.) in Nigeria. Jalaluddin & Hamid (2011) studied the effect of microbial (VAM- fungal spores), organic and inorganic fertilizers on germination of seed and seedling growth of 4 sunflower varieties (SA-278, Helico-250, Hysun-33 and Hysun-38) and it was observed that VAM inoculated plants show better seedling growth as compared to organic and inorganic fertilizers. Ardakani & Mafakheri (2011) demonstrated that low application of P fertilizer i.e 30kg / ha with AMF inoculation produced the same grain yield as obtained by use of 90 kg /ha in non-mycorrhizal wheat plants. Ghorbanian et al. (2011) examined that AMF symbiosis and different levels of phosphorus had significant positive effects on yield and nutrient uptake of maize (Zea mays L.) under water stress condition than non AMF conditions. Fernández et al. (2011) evaluated the effect of indigenous AMF inoculation and P levels (0, 12, and 52 mg P kg–1) on P-uptake efficiency in soybean and sunflower. Result shows that mycorrhizal inoculation enhanced phosphorus-acquisition efficiency in soybean, in contrast to sunflower which didn‟t show apparent effect to AMF symbiosis. Soleimanzadeh (2012) conducted an experiment in 2010 in farm of Islamic Azad University, Parsabad Moghan and determined the effect of two levels of AM Fungal inoculation (with and without Mycorrhiza ) and different levels of phosphorus fertilizer (25%, 50% 75% and 100% P recommended ) on growth and yield Azargol cultivar of sunflower. The results showed that AMF inoculated plants had significant positive effects on head diameter, seed yield and oil yield of sunflower (Azargol cultivar) than non- inoculated plants. However, this positive effect of AMF inoculated plants decreased with increasing P levels. It is suggested that 50% P recommended could enhance seed yield and oil production to an adequate level. Prasad et al. (2012) investigated the impact of AMF and Pseudomonas fluorescens with different levels of superphosphate on plant growth parameters of Chrysanthemum indicum L. and found that growth parameters were significantly improved in plants inoculated with biofertilizers (AMF + P. fluorescens) 40 and recommended dose of superphosphate. Babaei et al. (2012) reported that dual inocualtion of AM fungi along with microbial inoculant (Pseudomonas fluorescens) significantly increased the growth and oil content of Sunflower under low phosphorus level. Vaseghmanesh et al. (2013) conducted a field experiment in Boyerahmad area,

Iran and studied the effects of two levels of mycorrhiza M1 and M0 (with and without mycorrhiza) and four levels of triple phosphorus (0, 50, 100 and 200 kg/ha) on yield of sunflower (Progress cultivar). The results showed that, application of mycorrhiza and phosphorus fertilizer had significant effect on the seed yield, biological yield and seed weight as compared to control. However, the maximum Biological yield and seed yield

(35806 kg/ha and 2372.2 kg/ha respectively) was recorded with M1P0 treatment. Hassan et al. (2013) studied the impact of manure and mineral fertilization on the mycorrhizal community structure and productivity of sunflower plants and found that sunflower inoculated with AM fungi and inorganic fertilizer increased Phosphorus and nitrogen contents than manure-fertilized plants which had got distinct AMF community structure. Yaseen et al. (2013) reported that arbuscular mycorrhizal (AM) fungal inoculation along with rock phosphate and rhizobium significantly increased the growth parameters over non inoculated burgundy plant. Tanwar et al. (2013) assess the efficiency of AM fungi along with microbial inoculant (Pseudomonas fluorescens) on growth and yield of Capsicum annuum at different levels of superphosphate (P fertilizer). The outcomes showed that growth parameters and yield is highly promising in plants inoculated with biofertilizers (an interaction of AMF + P. fluorescens) at low concentration of superphosphate. While, at high P level all the growth parameters noticeably decrease. Patil et al. (2013) showed AMF inoculated maize plants has positive influence on all growth parameters, macro and micronutrients uptake at low concentration of superphosphate. Adavi & Tadayoun (2014) conducted an experiment in 2013 in Fereidoonshahr, Esfahan, Iran and determined the effect of two levels of AM Fungal inoculation (with and without Mycorrhiza ) and four levels of phosphorus fertilizer (25%, 50% 75% and 100% P recommended ) on growth and yield of potato (Solanum tuberosum L.). The results showed that AMF inoculated plants had significant positive effects on tuber size, number 41 of tuber per plant and yield (tuber and starch) of potato than non-inoculated plants. However, this positive effect of AMF inoculated plants decreased with increasing P levels. It is suggested that 50% P recommended could enhance tuber yield and starch production to an adequate level. Yadav & Aggarwal (2014) assessed the impact of AMF (Glomus mosseae and Acaulospora laevis) and phosphate solubilizing bacteria (Pseudomonas fluorescens) with different levels of superphosphate (P fertilizer) on plant growth, yield and nutrient uptake of Soybean. The results revealed that growth parameters and yield were the highest in plants inoculated with biofertilizers (AMF + P. fluorescens) at the low concentration of superphosphate. While, at oversupply of superphosphate all the growth parameters markedly decrease. Jan et al. (2014) studied the influence of AMF inoculation with compost (fresh animal dung and rock phosphate) on yield and P uptake of wheat (Triticum aestivum L. c.v. Atta Habib). Results revealed that grain, root and shoot yield were significantly affected by inoculation of AMF with compost

2.4 Effect of AM Fungal inoculation on percent root colonization and spores density in relation to fertilizer application

Joner (2000) found that additions of NPK fertilizers led to reduced AMF colonization in subterranean clover. Satpal & Kapoor (2000) studied the root colonization in Vigna radiata in phosphorus deficient soils, and found that the AMF inoculated plants with rock phosphate stimulated root colonization and increase in biomass and nutrient uptake of plant as compared to those without rock phosphate. Gryndler et al. (2001) studied the effect of organic and mineral fertilization on the mycelium growth and sporulation of AMF and found that the manure treatment showed positive signs of growth in mycelium and sporulation with the increasing quantity of mineral fertilization while in unmanured soil showed negative results with such increase of minerals. The Correlation analysis showed that positive results were due to available soil phosphorus along with luxurious supplies of mineral fertilizer. Aryal et al. (2003) studied the effect of dual inoculation of bean plant with rhizobium and AMF (Glomus spp) under organic fertilization which increased its root colonization, spore population nodulation and shoot N& P. 42

Pragatheswari et al. (2004) investigated that addition of Glomus mosseae spores to the soil bring increase in root colonization in all plant species. But this increase is negatively affected by incraseing level of phosphorus in the soil. Caravaca et al. (2004) studied the effectiveness of mycorrhiza (Glomus intraradices and Glomus deserticola) with certain amendments in the soil by the addition of rock phosphate, sugarbeet and Aspergillus niger for the provision of better conditions and found that Glomus deserticola inoculated Dorycnium pentaphyllum L. seedlings showed highest mycorrhizal colonization than Glomus intraradices in a semiarid soils. Linderman & Davis (2004) evaluated the effect of organic and inorganic fertilizer on AMF establishment and functions formed by Glomus intraradices and found that organic fertilizers favors more than inorganic fertilizers. Scagel (2005) studied the effect of different ericoids mycorrhizal fungi (EMF) on seven bush blueberry cultivars grown in containers with the addition of organic or inorganic fertilizer. The root colonization was better when organic fertilizer was used which shows that the nutrients have a role in colonization of plant by EMF. However it should be noted that host specificity to the inoculums and its availability in the container has also a vital role in root colonization. Manna et al. (2006) examined the role of P fertilizer and farmyard manure (FYM) application on AMF inoculated soybean and wheat plants, in rhizosphere with P solubilizing/mineralizing enzyme producing microbes and showed that microbes with their enzyme activity enhances organic and inorganic P in the system, thus increase in root colonization by AMF was recorded and was almost twice in soybean than the wheat plants. Aryal et al. (2006) reported that in Phaseolus vulgaris L. plants AMF infection was higher in organic fertilized (OF) plants than chemically fertilized (CF) plants. Panwar & Tarafdar (2006) evaluated the distribution of three endangered medicinal plant species (Withania coagulans, Mitragyna parvifolia and Leptadenia reticulate) and their colonization with AMF. They observed high AMF diversity towards plant species and negative correlation of soil phosphorus with AMF spores, which suggests that abiotic/edaphic factors were more important than the biotic factors. Gryndler et al. (2006) studied the effect of organic and inorganic fertilizers on development of external mycelium in term of hyphal length. The use of only organic manure resulted increase in 43 the hyphal length while the mineral fertilizers reduced their growth. Sharathbabu & Manoharachary (2006) reported that dual inoculation of AMF (Glomus fasciculatum) and rock-phosphate significantly enhanced percentage of mycorrhizal colonization than in single inoculation or in control Tylophora indica plants. Takács et al (2006) examined that in red clover roots arbuscule contents was more sensitive to rock phosphates with different solubility than the infection. Moreover, mycorrhizal dependency of the host eliminated at high soil P concentration. Sabanavar & Lakshman (2009) performed an experiment on two varieties of Sesamum indicum L. by suppling different doses of rock phosphate along with phosphate solubilizing bacteria and AMF and found that these bacteria help a lot in solublization of rock phosphate and thus promoting root colonization and spore density. Fernández et al. (2011) evaluated the effect of indigenous AMF inoculation and P supply (0, 12, and 52 mg P kg–1) on AMF root colonization in soybean and sunflower. Both crops produced positive results of root colonization in the field. But it was also observed that mycorrhizal colonization was significant at medium and high level of soil phosphorus in soybean while no such trend was found in sunflower. Soleimanzadeh (2012) studied the response of sunflower (Azargol cultivar) to mycorrhizal inoculation under different levels of phosphorus fertilizer (25%, 50% 75% and 100%) and results showed that positive effect of percent root colonization decreases with increasing P levels i.e 50% P recommended. Prasad et al. (2012) reported that AM root colonization and AM spore density were significantly improved in Chrysanthemum indicum L. inoculated with biofertilizers (AMF + Pseudomonas fluorescens) and recommended dose of superphosphate. Hassan et al. (2013) studied the effect of organic and inorganic fertilization on the productivity and AM Fungal community structure of sunflower (Helianthus annuus L.). By using PCR-DGGE and sequencing of an 18S rRNA gene fragment, 12 AM fungal spores as different taxa of uncultured Glomus, Acaulospora, ClaroideoGlomus, Funneliformis and Rhizophagus were identified in soil samples. Results showed that sunflower plants grown in organic fertilizers had a distinct AMF community structure than inorganic fertilizers. Tanwar et al. (2013) reported that AM root colonization was 44

significantly improved in Capsicum annuum inoculated with biofertilizers (AMF + Pseudomonas fluorescens) with half of the recommended dose of superphosphate. Jan et al. (2014) studied the influence of AMF inoculation with compost (fresh animal dung and rock phosphate) on mycorrhizal colonization of wheat (Triticum aestivum L. c.v. Atta Habib). Results showed that maximum roots infection and spores density (26 spores / 20 gm soil) were observed by the inoculation of AMF-I than AMF-II with full dose of compost. Yadav & Aggarwal (2014) reported that AMF spore number and percent root colonization were found highest in combination of Glomus mosseae+Acaulospora laevis + Pseudomonas fluorescens at half of the recommended dose of superphosphate as compared with non-mycorrhizal Soybean plants.

2.5 Mycorrhizal dependency (MD)

Zangaro et al. (2000) examined the mycorrhizal dependency (MD) of some native woody species of South Brazil and found that it was 90% for pioneer, 48% for early secondary, 12% for late secondary and 14% for climax species. Yao et al. (2001) found that AMF significantly influence mycorrhizal dependency at moderate phosphorus level. Ortas et al. (2002) investigated that sour orange has significant mycorrhizal dependency (MD) in relation to phosphorus and zinc nutrition. Vander-Heijden (2002) investigated that there is positive relationship between the mycorrhizal dependency (MD) of a plant species and vary greatly in responses to different arbuscular mycorrhizal fungal species.

Tawaraya et al. (2003) studied mycorrhizal dependency of various plant species and cultivars and observed that cultivated plant species exhibited a lower mycorrhizal dependency value than the wild plant species. Giri & Mukerji (2004) studied that, with plant‟s age the mycorrhizal dependency (MD) of Sesbania aegyptiaca and Sesbania grandiflora improves. At day 61 infection activities peaked under salinity conditions. Ryan et al. (2004) studied the host mycorrhizal dependency in relation to phosphorus concentration in the soil and reported that there exists negative correlation to high P levels. 45

Oliveira et al. (2006) determined the influence of various AMF species on mycorrhizal dependency (MD) of Conyza bilbaoana (56%) and Salix atrocinerea (44%) in alkaline anthropogenic sediment. They found that Conyza bilbaoana exhibit great mycorrhizal dependency (MD) than Salix atrocinerea. Rabie & Almadini (2005) invesitigated the role of dual inoculation of AMF (Glomus clarum) and Azospirillum brasilense on the host plants (Vicia faba) under salinity stress and found increase in salt tolerance, mycorrhizal dependency (MD) and phosphorus level as compared to non- inoculated faba plants. Zhang et al. (2006) reported that AMF inoculated Oryza sativa L. reduces the negative effect of chlorothalonil fungicide used in different concentrations (0, 50 and 100 mg kg-1soil). Mycorrhizal dependency was greater at 50 mg kg-1 chlorothalonil level. Takács et al (2006) studied the host mycorrhizal dependency in relation to phosphorus concentration in the soil and reported that mycorrhizal dependency eliminated at high soil P concentration. Sensoy et al. (2007) studied relative mycorrhizal dependency (RMD) in various Capsicum annuum L. genotypes with different AMF species. They found that N52 genotype had maximum and the Karaisali genotype had minimum relative mycorrhizal dependency (RMD) value. Zangaro et al. (2007) found that all plant species and even cultivers differ in their mycorrhizal dependency. Cardoso et al. (2008) determined the mycorrhizal dependency (MD) of mangaba tree (Hancornia speciosa) tree under increasing phosphorus fertilizer levels. They found that Mangaba tree showed significant dependence on AM associations, but it varies according to fungal inocula and phosphorus levels. Yucel et al. (2009) studied that wild emmer wheat (Triticum turgidum) showed a wide range of mycorrhizal dependency i.e. 56.8%-90.5%. Mariana et al. (2009) revealed that soybean had greater AM fungal colonization and mycorrhizal dependency than maize and sunflower under various growth conditions. Radovich & Habte (2009) evaluated the effect of arbuscular mycorrhizal (Glomus aggregatum) dependency on three Moringa genotypes under different soil solution P levels. The effect of three genotypes differs greatly and was dependent on soil solution P. They found that Moringa oleifera considered as moderately and Moringa stenopetala as marginally dependent on AM 46 associations Garg & Manchanda (2009) investigated that AMF inoculated pigeonpea plant showed high mycorrhizal dependency (MD) in saline soil. Kumar et al. (2010) evaluated the effect of AMF against salinity stress on seedling growth, mycorrhizal colonization and mycorrhizal dependency (MD) of Jatropha curcas L. and reported that with increasing salt stress growth and root colonization decreases while mycorrhizal dependency increases from 12.13 to 20.84% at 0–0.4% of NaCl than the non-inoculated plants. Yaseen et al. (2011) investigated that the effect of AMF inoculation in two different varieties of Vigna unguiculata and found that china variety out performed than the watni variety in terms of vegetative productivity and mycorrhizal dependency than the non-inoculated plants. Miranda et al. (2011) determined the mycorrhizal dependency and relative field mycorrhizal dependency of Physalis peruviana L. They found that the relative field mycorrhizal dependency index improved with the experiment's duration and peaked (42.5%) at day 61under salinity conditions. Ramakrishnan & Selvakumar (2012) studied the single and combined effect of Glomus fasciculatum and Glomus intraradices inoculation in Lycopersicum esculentum Mill. on growth and mycorrhizal dependency (MD) which was found highest (106.35%) in combined treatment as compared to single or control treatment. Mekahlia et al. (2013) investigated that Olea europaea grown under different climatic gradients (arid, semi-arid and sub-humid) and variety of seasons show greater mycorrhizal dependency. It varies with the bioclimatic zones and is more pronounced in spring rather than winter season.

CHAPTER: 3 MATERIAL AND METHODS

The pot experiment was carried out to find out the effects of arbuscular mycorrhizal fungi (AMF) along with application of various levels of rock phosphate fertilizer in order to know its effect on growth performance, mycorrhizal dependency values, fatty acid profile, proximate and elemental composition and also to know AMF infection in roots and spore density in the rhizospheric soils of selected sunflower hybrids. The present research work consist of two parts 1. Field work 2. Laboratory work

3.1 FIELD WORK

3.1. i Equipment and Reagents Seeds of selected sunflower hybrids, Earthen pots (89 cm diameter and 48 cm depth), rock phosphate fertilizer, clay soil, sand, mixed constorium of different AMF species (Glomus fasciculatum, G.mosseae, G. aggregatum, Sclerocystis pakistanica, Gigaspora gigantea along), Rhizobase inoculums (roots of wheat and maize), Urea (100 mg/ pot) Hoagland nutrient solution without phosphorus (-P), measuring tape, filter paper, electric grinder, Flasks, conical flasks, petri-dishes with lid,crucible, electric balance, Kjeldahl flask, muffle furnace, fume hood, atomic absorption spectrometer, heating digester, flame photometer, thermospectronic apparatus, cathode lamp, air acetylene flame, Soxhlet‟s apparatus, extraction thimble, Near Infrared Reflectance Spectroscopy model 6500 visible (Foss NIRSystems Inc. Silver Spring MD), cuvettes sealed with plastic and aluminum foils

K2SO4, CuSO4, Selenium, conc. H2SO4 distilled water, concentrated HNO3, perchloric acid, NaOH (40% W/V), boric acid solution, methyl indicator, 0.005 N. HCl , HCL (2%) and solvent (Petroleum Ether) .

3.1.ii Plant Material Authentic and highly disease resistant seeds of four hybrids of sunflower i.e NKS- 278, Hysun-33, SMH-0917 and SMH-0907 were obtained from Oilseeds Research Program, NARC Islamabad, Pakistan.

3.1.iii Experimental site The experimental and field work was conducted at the Department of Botany, University of Peshawar. The site lies from 33°-27˝ to 34° -27˝ northern latitude. The climate of Peshawar is extreme with very hot summers and mild winters. Winter in Peshawar, starts in mid-November and ends in late March. Summer months are May to September. The mean maximum temperature in summer surpasses 40° C (104° F) during the hottest month with mean summer minimum temperature of 25° C (77° F). The mean minimum temperature during winter is 4° C (39° F) and maximum is 18.35° C (65.03° F) (web, 6).

3.1.iv Soil Clay soil and sand used during the experiment were obtained from the ground (100cm depth) of Botany Department, University of Peshawar. Chemical analysis of the soil and sand sample was carried out at N.I.F.A (Nuclear Institute for Food and Agriculture). Nitrogen concentration of soil sample as determined by Kjeldhal method of Bremmer & Mulvaney (1996). ABDTPA extractable P, Cu, Fe, Zn and Mn were analyzed by the method as described by Soltanpour & Schawab (1977), soil pH by Richards (1954), electrical conductivity by Chapman (1965), soil organic matter by Nelson & Sommer (1982). The clay soil having PH 7.8, electric conductivity 0.675 ds/m2, Nitrogen 0.032% and Phosphorus 0.001 mgkg-1. All 96 pots having 89 cm diameter and 48 cm depth were filled with 6 Kg of this nutrient deficient sandy loam textured soil (2:1 soil/sand).

3.1.v Application of AMF inoculum

In the experimental work, mixed constorium of different AMF species i.e. Glomus fasciculatum, G.mosseae, G. aggregatum, Sclerocystis pakistanica, Gigaspora gigantea along with roots of wheat and maize infected with arbuscular mycorrhiza were used as 49 rhizobase inoculum. The roots were cut into 1cm pieces, which along with soil base inoculum (rhizospheric soil) were spread uniformly in pots, at a depth of 3cm and 6cm in layers before sowing. Inoculum for each pot consisted of 160 gm. of mycorrhizal infected roots and adhering soil. Mycorrhizal inoculum preparation, placement and application were done by the method given by Brundrett et al. (1996).

3.1.vi Fertilizer application

Fertilizers were applied by following Krishna & Bagyaraj (1982) method. Urea (100 mg/ pot) as nitrogen source was added to all the pots irrespective of treatments. Moreover 20ml of Hoagland nutrient solution (-P) was provided to all pots 15 days after seed sowing (Hoagland solution composition is given in Appendix I).

Rock phosphate fertilizer (P2O5) are obtained from Hazara deposits (Recommended dose=80kg P2O5/ha. Agricultural University Peshawar, KPK). Following four levels of Rock phosphate were applied in combination with without AMF, each as given below.

i. RP0 No Phosphate added (Control)

ii. RP1 25% of the recommended dose (100mg P2O5/ kg soil) iii. RP2 50% of the recommended dose (200 mg P2O5/kg soil) iv. RP3 100% of the recommended dose (500mg P2O5/kg soil)

3.1.vii Experimental design, treatments and replications The field experimental work was carried out in a randomized complete block design (RCBD) along with eight treatments; each treatment was replicated three times with five plants in each pot (8 treatments x 4-hybrids of sunflower x 3 replicates), so each treatment includes 3 pots with 5 seeds cultivated in each pot. Seeds were placed at 5 cm depth from the soil surface. For the AMF treatments, the appropriate AMF inoculum was placed in soil over which seeds were planted. Rock phosphate fertilizer was applied in each pot according to treatment as basal dose. Pots were randomly placed and maintained under natural condition in the net house (Plate 1, Table, 1). There were following treatments (each with three replicates) in a Randomized Complete Block Design (RCBD).

i. MRP0 Mycorrhiza without Rock phosphate (Control)

ii. NMRP0 Non-mycorrhizal without Rock phosphate (Control) 50

iii. MRP25 Mycorrhizal + Rock phosphate level 1

iv. NMRP25 Non-mycorrhizal + Rock phosphate level 1

v. MRP50 Mycorrhizal + Rock phosphate level 2

vi. NMRP50 Non-mycorrhizal + Rock phosphate level 2

vii. MRP100 Mycorrhizal + Rock phosphate level 3 viii. NMRP100 Non-mycorrhizal + Rock phosphate level 3 In order to test the significance of the sunflower hybrids, treatments and interactions probability test (p < 0.05) was carried out.

3.1.viii Evaluation The following parameters were recorded after plant harvesting. 1. Plant height 2. Length of roots 3. Number of fresh leaves/ plant 4. Number of wilted leaves/ plant 5. Length and width of leaves 6. Dry wt. of plant 7. Head diameter 8. No. of seeds /Head 9. Weight of seeds /Head 10. Mycorrhizal dependency (MD) 11. AMF spores density 12. REC index in the roots 13. Proximate analysis 14. Oil yield 15. Fixed oil (Fatty acid) profile a) Unsaturated fatty acid (Linoleic acid and oleic acid) b) Saturated fattyacid (Palmitic acid and stearic acid) 16. Nutrient uptake a. Macro-nutrients (P, N, K) b. Micro-nutrient (Zn) 51

3.1.ix Mycorrhizal dependency (MD) Mycorrhizal dependency (MD) value of selected sunflower hybrid was calculated by formula given by Plenchette et al. (1983). MD = Dry wt. of mycorrhizal plants - Dry wt. of non-mycorrhizal plants x 100 Dry wt. of mycorrhizal plants 3.1.x Statistical analysis Experimental data was statistically analyzed by applying ANOVA test, procedures outlined by Steel and Torrie (1980) and Least Significant Difference (LSD) was used for any significant difference among the treatments. Standard error (±) was also measured for different parameters.

3.2. ANALYSIS OF PLANTS 3.2.i Mineral analysis After harvest, dry weight of plant was determined by drying these samples at 70°C for 48h. Plant parts such as shoots and roots were grinded homogenously to powder with the help of electric grinder and the powders were digested. Ashing was done by wet digestion method and the resultant residues were subjected for mineral analysis through atomic absorption spectrometer by Perkin Elmer A Analysts 700 device for quantitative detection of P, N, K & Zn elements. The elemental analysis for micronutrient (Zn) was conducted at CRL Department of Physics, University of Peshawar while macro nutrients analysis was carried out at the Department of Soil Sciences and water management, University of Agriculture, Peshawar, KPK. Wet digestion method for Nitrogen Take 0.2 gm of each dried powdered seed sample in a flask and 1.1 g of digestion mixture [K2SO4 (100gm) + CuSO4 (10g) + Selenium (2g)] and 5ml of conc. H2SO4 was added. The flask was then placed on heating digester for 30 minutes till bluish green color was obtained. Then the samples, after cooling were diluted up to 100ml by distilled water and then filtered. These filtrates were used for detection of nitrogen (N) through KJeldhal method of Bremmer & Mulvaney (1996).

Wet Digestion for Other Elements One gram each dried crude powdered seed sample was taken in a beaker and 10 ml concentrated HNO3 (Nitric Acid) was added. It was allowed to stand overnight. After 24 hours 4ml perchloric acid was added to each sample and kept for 25 minutes. The sample was heated and evaporated till small volume was left behind. After cooling samples were diluted upto 100ml solution and filtered. The stock solutions were marked with names and coded numbers and were analyzed through atomic absorption spectrometer for quantitative detection of the Zn element while K and P were detected through flame photometer and thermospectronic apparatus respectively (AOAC, 2000; Benton et al., 1991). Preparation of stock/ standard solutions (100ppm) Ten ml of 1000ppm standard stock solutions of each sample was taken in the 100 ml volumetric flask and was diluted by doubling its volume with distilled water to prepare 100ppm solution. Standard solutions (2.5, 5, 10 ppm) Working standard solutions (2.5, 5, 10 ppm) were prepared by taking 2.5, 5, and 10 ppm of 100 ppm standard stock solutions in flasks of 100ml and each was diluted by doubling its volume with distilled water. Instrumental conditions for different elements in the samples Procedure The instrument was set according to the instrumental conditions required for respective elements. The respective cathode lamp was turned on and allowed to warm up for 10 minutes and the air acetylene flame was ignited. The instrument was calibrated and standardized with working standard of 2.5, 5, and 10ppm of the respective minerals (Parkin Elmer). Working standards were run as unknown to verify the standardization. The sample stock solution was aspired into the flame and concentration of respective element was calculated in ppm.

3.2.ii Proximate analysis Moisture content, ash, crude protein, crude fibers and fats contents of dried powdered seed samples was analyzed by using standard method of Association of Official Analytical Chemists (AOAC, 2000) at National Institute Of Food And Agriculture, Tarnab, Peshawar. Determination of Moisture

Take 2.0g of powdered seed sample in a clean, weighed petri-dish (W1), and covered it with lid. Then it was placed in oven for 4-6 hours at 105oC. The petri-dishes were then shifted to desiccators for 30 minutes for cooling. After cooling, these were again weighed (W2). Moisture content was determined by using method of (AOAC, 2000). Percent moisture was calculated as follow;

% Moisture= W1 - W2 ×100 Sample weight

Determination of Ash To measure ash content 2.0 gram of powdered seed sample was taken in a preweighed dried and clean crucible (W1). First the sample was burnt on fire and then placed in a muffle furnace at 5500C for 6 hours for complete incineration of sample. The o crucible was then cooled to 200-300 C and weighed again (W2). Percent ash was calculated as follow; % Ash = W1-W2 × 100 (AOAC, 2000) Weight of samples W1= Weight of crucible+sample W2 = Weight of crucible + Ash

Determination of Crude Protein For the determination of crude protein the samples were digested by taking 0.5 gm of powdered seed sample in a Kjeldahl flask and by adding 4-5 g of digestion mixture

[K2SO4 : CuSO4 (8:1)] and 10ml of conc. H2SO4. The flask were then placed on heating digester for 30 minutes under a fume hood to start digestion and continued till the mixture becomes clear. After digestion, the heater was turned off and the flasks cooled. 54

The digests were then transferred to 100ml volumetric flask from which 10 ml was introduced in to distillation flask. Then 10ml of NaOH (40% W/V) was added gradually to the funnel and the funnel was plugged firmly. The distillation continued for at-least 5-

10 minutes and NH3 produced was collected as NH4OH in a conical flask containing 5ml of boric acid (2%) solution and few drops of methyl indicator. During distillation pink color was changed to yellowish. The tip of condenser was washed in the conical flask. The distillate was then titrated against standard 0.005 N. HCl till the restoration of pink color (a reagent was run as blank, through all the steps as mentioned above). Percent crude protein was determined as follow; i. % Crude Protein = 6.25 × %N ii. % N = (S-B) ×N×0.014×D×100 Wt. of sample × V (AOAC, 2000) S= Sample titration reading B= Blank titration reading N = Normality of HCl D = Dilution of the sample after digestion V = Volume taken for distillation 0.014 = Miliequivalent Wt of Nitrogen

Determination of Crude Fiber Take 2.0 g powdered seed sample in a clear beaker. 200 ml of HCL (2%) was added to it and the sample was boiled for about half an hour. It was next drained into a beaker. The fiber residue was again digested in a similar manner like that of acid digestion in 200ml of NaOH and filtered. The residue was washed with hot water to make it acid free and then transferred to a dried preweighed (W1) crucible to remove moisture content. The crucible was ignited in a muffle furnace at 550oC for 30 minutes and then cooled in desiccators for an hour and weighed (W2). Percent crude fiber was determined as follow; % Crude fiber = W1-W2 × 100 Sample weight (AOAC, 2000)

W1 = Weight of dry crucible with sample W2 = Weight of crucible with sample after heating

Determination of crude fats Soxhlet‟s apparatus was used for determination of crude fats. First one gram of moisture free powdered seed sample was wrapped in filter paper and placed in clean and dried extraction thimble. Then the thimble was placed in an extraction tube. Preweighed, round bottom flask was filled with 200 ml of solvent (Petroleum Ether) and connected to the extraction apparatus. The apparatus was turned on for extraction for 5-6 hrs, siphoning occurred after 5-6 min at the condensation rate of 3-4 drops per second. The thimble was removed after complete drying of solvent. Finally the flask is cooled and weighed again (AOAC, 2000). Percent crude fat was calculated as follow;

% Crude Fat= (Weight of flask + Ether extract) - (Empty weight of flask) × 100 Weight of Sample

Determination of Carbohydrate: Carbohydrates were determined by subtracting the weights of protein, fats, crude fibers, ash and moisture content from 100 (AOAC, 2000). Total Carbohydrates= (100 - % moisture - (% Ash+ % fibers+ % fats+ % protein).

3.2. iii Oil Fatty Acid Profile: Fatty acid composition and oil content analysis of whole seed samples were analyzed by Near Infrared Reflectance Spectroscopy model 6500 visible (Foss NIRSystems Inc (Silver Spring MD) at National Institute Of Food And Agriculture, Tarnab, Peshawar by using standard method of Association of Official Analytical Chemists (AOAC, 1990). In this method seed samples were placed in cuvettes with diameter of about 4.7 cm sealed with plastic and aluminum foils and scanned on the NIRS equipped with computer. Light from the light source, reflected from the sample as diffuse reflectance, was recorded by the detector. In the reflectance mode, the light source and the detector were on the same side of the sample cell. The instrument was operated using Vision® software. In fatty acid profile, the percentage of protein content, oil 56 content, saturated and unsaturated fattyacids, like linoleic acid, oleic acid, steric acid and palmitic acid were calculated directly from the system.

3.3. LABORATORY WORK Equipment and Reagents Rhizospheric soil samples, sunflower roots, alcohol, distilled water, 10 % KOH; lactic acid, glycerin, Canada balsam, filter paper, spirit lamp, test tubes, test tube holders, forecep, dissecting needles, glass bottles with lids, slides, cover slips, , binocular microscope, compound microscope, petridishes, beaker, different size sieves (50, 80 and 200 um) The laboratory work was carried out in two steps. A: Assessment of roots B: Extraction of spores.

3.3. i ASSESSMENT OF ROOTS Collection and preservation of roots Roots of selected sunflower hybrids were cut, collected and preserved in sthe fixative solution. Preparation of preservative For preservation 70% alcohol was used. 70% alcohol was prepared by taking 70 ml of alcohol and 30ml of distilled water. Preservation Plant roots were thoroughly rinsed with water to remove soil particles and cut into small segments and preserved in glass bottles containing 70% alcohol. The bottles were labeled and kept at room temperature in the laboratory. Preparation of KOH solution (10%) KOH solution was prepared by mixing 10gm. of KOH in 90ml of distilled water. The solution was stirred thoroughly and kept at ordinary room temperature. Preparation of acid fuchsin stain To prepare acid fuchsin stain, 0.025 gm. of acid fuchsin was dissolved in 220ml of lactic acid and then mixed with 10ml of distilled water and 16ml of glycerin. The solution was stirred well and used for staining (Brundrett et al. 1984). 57

Staining procedure The staining procedure given by Phillips & Hayman (1970) was used for staining fungal structures for non-pigmented roots. The roots preserved in 70% alcohol were thoroughly washed with tap water and then heated for 5-7 minutes in 10% KOH to remove the host cytoplasm and most of the nuclei. It was again washed with water and dried on filter paper. These were then stained by heating in 0.025% acid fuchsin for 3 – 5 minutes. Assessment of root colonization For this purpose, a procedure referred by Giovannetti & Mosse (1980) as + slide method was followed. Twenty five segments of roots of individual plant each approximately 1 cm long were randomly selected for microscopic study. Morphology of AMF entophyte was studied and expressed in percentage (%). The infection percentage was calculated by using the following formula:

% age mycorrhizal infection = No. of infected segments × 100 Total No. of segments studied Microphotographs of the best selected slides were taken.

3.3.ii EXTRACTION OF SPORES Collection of soil samples “Wet sieving and decanting technique” of Gerdemann & Nicolson (1963) was used to extract the spores from each collected soil sample as following; Rhizospheric soil samples were collected from the studied sunflower plants at flowering stage. 100gm of soil sample was weighted and suspended in water and vigorously agitated to detach soil debris and aggregates in order to get uniform mixture, which was passed through 2mm sieve to screen out large debris. The residue was washed several times. Small amount of the residue was put into petridishes for studying the spores. All liquid and soil suspension was recovered in a beaker that passed through the first sieving. The soil suspension was agitated and passed successively through sieves of 50, 80 and 200 um size (Dalpe, 1993). The residue from each sieve was collected on the 58 filter papers in Petri dishes and examined under microscope. Material collected on each sieve was examined separately. Mounting of spores With the help of needle the spores were picked up and mounted on a slide in a drop of Canada balsam for microscopic studies. Calculation of density of spores Density is defined as “the mean number of spores per 100 gm. of soil”. Density of spores in each soil sample was calculated by following Stahl & Christensen (1982) standard method. Identification of spores The observed spores were micro photographed at two magnifications (4x, 10x). These were identified with the help of keys following Hall & fish (1978), Trappe (1982) and Schenck & Perez (1990).

Table 1: Biological materials, time schedule and cultural practice

Biological Time schedule and cultural practice material Seeds Hybrids Seeds Source Rock AMF Time of phosphate Inoculation seeds Fertilizer sowing application 1.NKS-278 Oilseeds Helianthus Research 1.4.2013 11.4.2013 12.4.2013 annuus L. 2.HYSUN- Program, (Sunflower) 33 National Agricultural 3.SMH-0917 Research Centre 4.SMH-0907 (NARC), Islamabad Watering Thinning Spray Harvesting

The plants were The weak watered plants were Cypermethrine 07.07.2013 regularly. removed and 10% only two 5ml/lit. healthy plants were left in each pot allowed to grow for agronomic studies

CHAPTER: 4 RESULTS AND DISCUSSION

In the present research work, four hybrids of sunflower (Helianthus annuus L.), were grown in the phosphorus deficient soil with and without mycorrhiza at various levels of rock phosphate (P fertilizer) to study their response to AMF inoculation and to find out the mycorrhizal dependency (MD) value. The response varied in different hybrids which are in accordance with the work of Declerck et al. (1995) and Linderman & Davis (2004). In response to AMF inoculation the growth performance was measured in terms of agronomic parameters (Plant height, root length, number of fresh leaves/plant, number of wilted leaves/plant, leaf length and width, number of seeds/head, seeds weight/ head, head diameter, dry weight, MD, proximate and nutrient composition and fixed oil profile) of the hybrids. Moreover, spore density in the rhizospheric soils and AM colonization in the roots of the studied plants were also measured. All plants need phosphorus during growth period, since it is essential component of cell membrane, nucleic acids (DNA, RNA), ATP, co-enzymes, phospholipids and phosphoproteins and plays vital role in many metabolic activities like energy transfer, photosynthesis and respiration etc (Ozanne, 1980). Pakistan soil, being alkaline and calcareous nature is mostly phosphorus (P) deficient, affecting plants adversely (Memon et al., 1992; NFDC, 2001; Gill et al., 2004). Phosphorus contents of an average soil is about 0.05%, out of which only 0.1% of the total P is brought in use by the plants because of its low solubility. Phosphorus is available in soil as inorganic P and organic P. The inorganic P may either be in soluble or insoluble form. It is poorly available and hard to absorb by plants, because phosphates of many elements like Iron, Aluminium and Calcium have poor solubility (Schachtman et al., 1998). Due to scarcity of phosphorus reserves in the soil and their rapid utilization, efforts are being made to supplement plants with certain chemical fertilizers. But the use of heavy amount of chemical fertilizer is not only very costly, but also causes soil and water pollution. It is therefore needed that low cost indigenous methods should be applied. Rock phosphate is the natural, cheapest i.e about three times lesser in price and eco-friendly than other phosphate fertilizers (Nye & Kirk, 1986), but unfortunately it is 61 poorly soluble and need to adopt other strategies to increase its solubility and to make it available to the plants. So, to overcome this problem plants have adopted different strategies to acquire sufficient phosphorus (Sharma, 2004). Arbuscular mycorrhizal fungal (AMF) association is one among these adaptations (Coline et al., 2011). Mycorrhizal inoculation can be helpful and effective for changing low grade rock phosphate into available form, which can be taken by the plants for their growth and development (Sabanavar & Lakshman, 2009). Arbuscular mycorrhizal association plays an integral role in growth and development of most of the agricultural plants (Safir, 1980; Mosse, 1981; Harley & Smith, 1983; Cooper, 1984; Khan, 1994; Siddiqui & Mahmood, 2001; Gosling et al., 2006) especially in phosphorus deficient soils. AMF is the common biotic factor of ecosystem which establishes symbiotic relationship with terrestrial plants, and increase plant growth, plant protection, quality of the soil and mineral and water uptake (Mahmood & Rizvi, 2010; Maillet et al., 2011).

4.1 Growth Parameters In the present study among all measured parameters, the mycorrhizal plants showed significant differences at P<0.05 as compared to non-mycorrhizal plants at various rock phosphate (RP) levels. 4.1.i Plant height The growth of a plant is reflected by its height which can be taken as one of the morphological attribute indicating crops growth behavior. Beside genes many other factors are also involved in controlling this character e.g. environment, soil nutrients and seed vigor play vital role in this regard. The results of Means, ANOVA and LSD test of plant height of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in table 2 and Figs.1a,b,c. The ANOVA results showed that hybrids and treatments were highly significant (P < 0.0000) regarding plant height at various levels of rock phosphate in mycorrhizal (M) and non-mycorrhizal (NM) plants, while replications and interactions between hybrids × treatments were non-significant (Appendix 2, Fig. c), as recorded values of all interaction data are statistically at par with each other in terms of plant height. 62

It is evident from the results that in terms of plant height Hysun-33 (112.16cm) responded better, followed by NKS-278 (94.60cm), SMH-0917 (89.96cm) and SMH- 0907 (84.13cm), in mycorrhizal plants as compared to non-mycorrhizal plants (Fig. 1a). These results are in agreement with Boyetchko & Tewari (1995) who reported that the degree of host specificity exists in AM fungi. Treatment means data (Table 2, Fig.1b, Plate 2-4) also indicated that mycorrhizal treated plants along with rock phosphate showed better performance as compare to non-mycorrhizal treated plants at control (RP0) and low levels of rock phosphate (RP1 and RP2). In the current research study variation in plant height of various sunflower hybrids may probably be due to their different genetic makeup. In our results maximum shoot length in mycorrhizal plants (111.63cm) was recorded at RP0 (0%) and low (25%) rock phosphate level (RP1) followed by RP2 (50%) as compared to non-mycorrhizal plants. These results are in conformity with the findings of Fusconi et al. (2001), who observed that AM fungi significantly improved Allium porrum growth in soils with low phosphorus (P) availability. Increased plant height in mycorrhizal plants have also been reported by Estrada- Luna et al. (2000) in Psidium guajava, Nagaraja et al. (2002) in sunflower, Giri et al. (2005) in Cassia siamea, Rahman et al. (2006) in maize, Jambotkar & Lakshman (2009) in Brassica juncea, Shrestha et al. (2009) in maize, Hani et al. (2009) in Prosopis chilensis, Ramakrishnan & Selvakumar (2012) in tomato, Bera et al. (2014) in sunflower and Aher, (2015) in Arachis Hypogea L. At high P level (RP3), the non-mycorrhizal plants outperformed (103.33cm) than mycorrhizal plants (92.05cm) (Table 2, Fig. 1b, Plate, 5). This result supports the previous findings of Abdel-Fattah (1997) and Menge et al. (1978) who reported that high level of phosphatic fertilizers inhibited AM colonization and consequently the mycorrhizal benefits. The AMF inoculated plants show increase in cytokinin contents in the shoot that results in mitotic activity and cell expansion responsible for their growth (Bass & Kuiper, 1989; Tarafdar & Marschner, 1995). Moreover, the acquisition of low mobility nutrients, predominantly phosphorus also results in better plant growth (Rejali et al., 2008; Colard et al., 2011). The mycorrhizal mycelia have the capability of converting phosphorus from non- available to available form to the root (Vance, 2003). The extra-radical mycelia play 63 an important role in absorption of low mobility nutrients (P, Cu, Zn and Fe) (Al-Karaki, 2000; Chandanie et al., 2009) by providing large surface area (Sylvia et al., 1993). As these are thinner in diameter than root hairs therefore, can get easy access to thin pores of soil, from where they recover important nutrients, which are beyond the reach of root hairs (Mengel & Kirkby, 2001). Table-2: Plant Height (cm) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS-278 Hysun- SMH- SMH- Rock phosphate 33 0917 0907 T levels HxT RP0 108.45 133.30 104.44 100.33 111.63a M RP1 103.29 129.54 93.37 91.18 104.34a RP2 87.21 112.86 87.20 76.79 91.01b RP3 99.73 99.65 81.45 87.37 92.05b

RP0 88.05 106.76 94.29 86.36 93.61b NM RP1 93.98 103.54 84.41 72.55 88.62b RP2 69.85 95.67 76.07 66.88 77.12c RP3 106.25 115.99 99.48 91.60 103.33a Mean (H) 94.60b 112.16a 89.96bc 84.13c

LSD value at 5% level of significance for factor H= 6.377, factor T = 9.018 Key: H=Hybrids T = Treatments M=Mycorrhizal NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 64

Fig. 1a: Mean Plant height (cm) in different hybrids of Helianthus annuas L.

Fig. 1b: Effect of RP treatment on plant height (cm) in four hybrids of Helianthus annuas L.

Fig. 1c: Effect of mycorrhiza on plant height (cm) under different RP levels in four hybrids of Helianthus annuas L. 65

4.1.ii Root Length The results of Means, ANOVA and LSD test of root length of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 3, table 3 and Figs. 2a,b,c. The ANOVA (Appendix 3) showed that the effect of replications was found to be significant (P < 0.0145). The effect among hybrids, treatments and hybrid x treatments interaction were highly significant (P < 0.0000) regarding root length at various levels of rock phosphate in mycorrhizal and non- mycorrhizal plants (Table 3). Hybrid mean data (Table 3, Fig. 2a) showed significant differences; Hysun-33 had maximum (13.09cm) root length followed by SMH-0917 (12 cm), SMH-0907 (11.07cm) and NKS-278 (10.39 cm) at various rock phosphate levels. The effect of treatments also gave significant results. Mycorrhizal plants showed better performance at RP1 (14.84 cm) and RP2 (13.05 cm) levels as compared to non- mycorrhizal plants (Table, 3, Fig. 2b). Similar results were also observed for Withania somnifera and Spilanthes somnifera (Rai et al., 2001). Under control condition better performance was exhibited by mycorrhizal plants (11.40 cm) as compared to non- mycorrhizal control plants (8.39cm). These results are in line with work of Moradi et al. (2013), who reported that AMF inoculated chickpea plant showed greater root length as compared to non-inoculated plants. Similar results were also reported by Hajbagheri & Enteshari (2011) in basil, Taylor et al. (2008) in maize and Dash & Panda (2001) in cotton and bean. Mean values for Hybrids × Treatment interaction data (Table. 3, Fig.2c) revealed that mycorrhizal response varied in different hybrids at various rock phosphate levels. Statistical analysis of interaction data showed that maximum root length was 19.03cm in Hysun-33 and 18.13cm in SMH-0917 at RP1, 12.20cm in NKS-278 & SMH-0907 at RP0 as compared to non-mycorrhizal plants. Similarly, it is also clear from mean data that the non-mycorrhizal treatments with increasing RP level showed significant increase in root length over the control in all hybrids (Table. 3, Fig.2c). Mycorrhizal infected roots are many folds better in their length and show good physiological activities than ordinary roots. They stimulate root development (Kumar et al., 2007) and increase the formation of lateral roots (Berta et al., 2002). The extensive network of extra-radical hyphae present on roots provides large surface area (Smith & 66

Read, 1997) so as to enhance increased water and nutrients uptake (Copetta et al., 2006; Chandanie et al., 2009; Hashem et al., 2015). This mycorrhiza also produces certain enzymes which are helpful in converting insoluble nutrients into soluble forms (Abdel-hafez & Abdel-Monsief, 2006). At high level of rock phosphate (RP3) reverse result was noticed (Table 3, Fig. 2b). The non-mycorrhizal plants performed better (12.61cm) than mycorrhizal plants (10.10cm). Soleimanzadeh (2012) reported that there is declining trend in AMF colonization at high phosphorus levels. Because of the fact that high level of phosphorus brings anatomical changes in roots which causes resistance for the penetration of fungal hyphae and its colonization (Bressan & Vasconcellos, 2002, Subramanian et al. 2008). Similar trend was also observed in the present study.

Table-3: Root Length (cm) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means

Percent (%) NKS- Hysun-33 SMH- SMH- Rock phosphate 278 0917 0907 levels HxT T M RP0 12.20ef 11.10fg 10.10gh 12.20ef 11.40c RP1 10.06gh 19.03de 18.13a 12.16ef 14.84a RP2 11.03fg 16.13b 15.03bc 10.03gh 13.05a RP3 9.16hi 13.03de 8.20i 10.00gh 10.10d

NM RP0 9.13hi 9.20hi 6.16j 9.10hi 8.39 i RP1 9.13hi 11.20fg 10.06gh 11.03fg 10.35d RP2 9.23hi 12.00ef 14.16cd 12.00ef 11.85bc RP3 11.16fg 13.10de 14.16cd 12.03ef 12.61b Mean (H) 10.39c 13.09a 12.00 a 11.07b

LSD value at 5% level of significance for factor H= 0.5616, factor T = 0.7942 HxT interaction= 1.588 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 2a: Mean Root length (cm) in different hybrids of Helianthus annuas L.

Fig. 2b: Effect of RP treatment on root length (cm) in four hybrids of Helianthus annuas L.

Fig. 2c: Effect of mycorrhiza on root length (cm) under different RP levels in four hybrids of Helianthus annuas L

4.1.iii Number of Fresh Leaves AMF inoculation resulted in increase in number of fresh leaves. The results of Means, ANOVA and LSD test of number of fresh leaves/plant of the four hybrids following different treatments are given in the Appendix 4, table 4 and Figs.3a, b,c. The ANOVA (Appendix 4) showed that no significant difference exists among the replications but the difference is highly significant (P < 0.0000) among hybrids and treatments regarding number of fresh leaves at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants. The effect among hybrids x treatments interactions was also found to be significant (P < 0.0388) (Table, 4, Fig, 3c). It is evident from mean data of hybrids that Hysun-33and NKS-278 showed significantly better response than SMH- 0917 and SMH-0907 in terms of number of fresh leaves (Fig, 3a). However, mycorrhizal response varied in different hybrids at various rock phosphate levels. The statistical results revealed that mycorrhizal (M) plants responded better over non-mycorrhizal (NM) plants at low rock phosphate levels (RP1 and RP2) regarding number of fresh leaves/plant (Fig, 3b,c). Among the control plants, mycorrhizal plants showed better performance in comparison to non-mycorrhizal plants in all hybrids (Plate 2-4). Findings of present study are in conformity with that of Kavitha & Nelson (2013) who reported that AMF inoculated plants significantly enhanced number of leaves than non-inoculated plants. Aher (2015) studied the influence of AMF on vegetative parameters of Arachis Hypogea 69

L. and observed that mycorrhizal inoculated seedlings grew faster and healthier over control seedling. The present results are also in line with (Estrada-Luna et al., 2000; Silveira et al., 2006; Devachandra et al., 2008; Boureima et al., 2008; Gutierrez-Oliva et al., 2009; Jambotkar and Lakshman, 2009). More efficient absorption of nutrients and water in inoculated plants might have resulted in larger number of leaves, which facilitate better development of the aerial part of the plant in relation to the uninoculated plants. Similar observations have observed by Silveira et al., 2006. Chlorophyll a, chlorophyll b, caroteniod, nitrate, nitrogen, phosphorus, potassium contents and cytokinin increase in AMF inoculated plants (Manoharan et al. 2008). Cytokinin stimulates protein synthesis, cell division and cell expansion which bring increase in the number of leaves (Van-Staden & Davey, 1979). However at high levels of rock phosphate (RP3) reverse results were noticed i.e the non-mycorrhizal plants performed better than mycorrhizal plants (Table, 4, Fig, 3b). This might be due to the fact that addition of P beyond certain limit adversely affects plant colonization with AMF (Ammijee et al., 1990).

Table-4: Number of Fresh Leaves/plant in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatments Hybrids Means Percent (%) Rock NKS-278 Hysun-33 SMH- SMH- T phosphate 0917 0907 levels HxT M RP0 14.00abcde 16.00ab 13.00bcdefg 15.00abcd 14.00a RP1 15.00abcd 13.00bcdef 9.00ijk 9.00ijk 11.00cd RP2 15.00abc 17.00a 11.00defghij 10.00ghijk 13.00ab RP3 15.00abcd 11.00efghij 10.00fghijk 12.00cdefghi 12.00bc

NM RP0 13.00bcdef 11.00efghij 7.00kl 8.00jkl 10.00d RP1 14.00abcde 12.00bcdefgh 9.00ijk 5.00l 10.00d RP2 14.00bcdef 16.00ab 12.00cdefghi 9.00hijk 13.00 abc RP3 15.00abcd 16.00ab 12.00cdefghi 12.00cdefghi 14.00a Means (H) 14.00a 14.00a 10.00b 10.00b

LSD value at 5% level of significance for factor H= 1.264, factor T = 1.788 HxT interaction= 3.576 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 3a: Number of fresh leaves/plant in different hybrids of Helianthus annuas L.

Fig. 3b: Effect of RP treatment on number of fresh leaves in hybrids of Helianthus annuas L.

Fig. 3c: Effect of mycorrhiza on number of fresh leaves/plant under different RP levels in Helianthus annuas L

4.1. iv Number of Wilted Leaves The results showed that AMF inoculation resulted in decrease in the number of wilted leaves. The results of Means, ANOVA and LSD test of number of wilted leaves/plant of the four hybrids following different treatments are given in the Appendix 5, table 5 and Figs.4a,b,c. The results showed that hybrids and treatments were highly significant P < 0.0000, considering number of wilted leaves at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants. The hybrids x treatments interaction also showed significant results (P < 0.0243) (Fig. 4c), while replications were non-significant (Appendix 5). It is evident from the mean data that the non-mycorrhizal plants wilted rapidly as compared to mycorrhizal plants. Among hybrids SMH-0907 and SMH-0917 showed significant number of wilted leaves than Hysun-33 and NKS-278 (Fig.4a). Similarly, table,5 also show that the number of wilted leaves at RP3 level is greater in mycorrhizal plants as compared to non-mycorrhizal plants, However, non-mycorrhizal plants showed more number of wilted leaves as compared to mycorrhizal plants (Fig 4b). As mycorrhiza increase root proliferation and efficiency, so there is more water uptake by mycorrhizal plants as compared to non-mycorrhizal ones. The present results are in line with the similar findings of Arora et al. (1991); Marulanda et al. (2003) who reported that AMF inoculation increased the capability of water uptake in plants.

Table-5: Number of Wilted Leaves/plant in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phosphate 0917 0907 levels HxT M RP0 5.00fghijk 6.00defghi 6.00efghij 4.00ijk 5.00bc RP1 3.00jk 7.00bcdefg 6.00efghij 9.00abc 6.00abc RP2 3.00k 4.00hijk 6.00 efghij 8.00bcde 5.00c RP3 5.00ghijk 8.00bcdef 6.00 efghij 8.00 bcde 7.00ab

NM RP0 6.00defghi 7.00cdefgh 10.00ab 8 bcdef 8.00a RP1 5.00ghijk 7.00bcdefg 7.00cdefg 11.00 a 8.00a RP2 6.00efghij 6.00efghij 8.00bcdef 9.00 abcd 7.00a RP3 4.00hijk 6.00efghij 6.00efghij 6.00 defghi 6.00bc Mean (H) 5.00c 6.00b 7.00b 8.00a

LSD value at 5% level of significance for factor H= 1.000, factor T = 1.415 HxT interaction= 2.830 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 4a: Number of wilted leaves/plant in different hybrids of Helianthus annuas L.

Fig. 4b: Effect of RP treatment on number of wilted leaves/ plant in hybrids of Helianthus annuas L.

Fig. 4c: Effect of mycorrhiza on number of wilted leaves under different RP levels in Helianthus annuas L.

4.1 v Leaf Length Overall results show that AMF inoculated plants have greater leaf length as compared to non-mycorrhizal plants. The results of Means, ANOVA and LSD test of leaf length of four hybrids following different treatments are given in the Appendix 6, table 6 and Figs.5a,b,c. The ANOVA (Appendix 6) showed that effect of treatments was highly significant P < 0.0011 regarding leaf length at various levels of RP fertilizer in mycorrhizal and non-mycorrhizal plants, while the effect among hybrids, replication and interaction between hybrids and treatments were non-significant (Tabe 6), as recorded values are statistically at par with each other in terms of leaf length (Fig, 5a,c). The data revealed that the mycorrhizal plants showed better performance as compared to non-mycorrhizal plants at control. It is clear from mean data (Table 6, Fig. 5 b) that at RP1 the mycorrhizal plant showed maximum (10.10 cm) leaf length followed by RP2 (9.75 cm) as compared to non-mycorrhizal plants, while at high levels of rock phosphate (RP3) reverse result was noticed i.e the non-mycorrhizal plants performed better (9.37cm) than mycorrhizal plants (8.22cm) (Table 6, Fig. 5b). Our results are in line with the work of Prasad & Bilgrami (2002) who reported that AMF (Glomus fasciculatum) inoculated Saccharum officinarum show effective results in chlorophyll content increment at low dose of phosphate fertilizer as compare to control. The positive effect on leaf size may be attributed to AMF hyphae which enhances uptake of water (Faber et al., 1991) and phosphorus (P) (Henrike et al., 2007). Ghazi & Zak, (2003) also reported that increase in leaf length and leaf turgidity is due to better absorption of water and nutrients especially zinc and copper. AMF inoculation also results in increase of cytokinin, which is helpful in the enhancement of other physiological activities, like cell division, cell expansion, chlorophyll and protein formation which in turn results in improvement of photosynthetic activities (Van-Staden & Davey, 1979). Increase in photosynthesis results in increase in the level of glucose and sucrose in leaves of inoculated plants. Similar results are also reported by Wu et al. (2010).

Table-6: Leaf Length (cm) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- T Rock phosphate 278 0917 0907 levels HxT M RP0 8.7 9.14 8.89 8.71 8.86bcde RP1 11.1 8.89 10.41 9.98 10.10a RP2 10.49 10.16 9.72 8.71 9.75ab RP3 9.29 8.12 6.60 8.89 8.22e

NM RP0 8.12 8.53 8.96 8.02 8.40de RP1 8.45 8.89 8.89 8.63 8.71cde RP2 9.47 9.04 8.89 9.55 9.24abcd RP3 8.96 10.31 9.04 9.14 9.37abc Means(H) 9.32 9.14 8.91 8.94

LSD value at 5% level of significance for factor T= 0.3613 Key: H=Hybrids T = Treatments M=Mycorrhizal NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 77

Fig. 5a: Leaf length in different hybrids of Helianthus annuas L.

Fig. 5b: Effect of RP treatment on leaf length (cm) in four hybrids of Helianthus annuas L.

Fig. 5c: Effect of mycorrhiza on leaf length (cm) under different RP levels in four hybrids of Helianthus annuas L. 78

4.1. vi Leaf width Like leaf length there was significant increase in leaf width in response to RP in both inoculated and non-inoculated plants.The results of Means, ANOVA and LSD test of leaf width of four hybrids following different treatments are given in the Appendix 7, table 7 and Figs. 6a,b,c. The ANOVA (Appendix 7) showed that effect of treatments was highly significant P < 0.0011 regarding leaf width at various levels of RP fertilizer in mycorrhizal and non-mycorrhizal plants, while the effect among hybrids, replication and interaction between hybrids and treatments were non-significant (Table, 7, Fig. 6a,c). Recorded values of all hybrids are statistically at par with each other in terms of leaf width i.e maximum being in SMH-0917 (2.26cm) and NKS-278 (2.25cm) hybrids followed by SMH-0907 (2.22cm) and Hysun-33 (2.08cm) (Fig, 6a). Treatments data gave significant results. Mycorrhizal plants at RP2 level showed maximum leaf width (2.58cm), followed by RP1 (2.26cm) as compare to non-mycorrhizal ones (Table 7, Fig. 6b). This may be due to combined effects of fertilizer and AMF inoculation. However at high levels of rock phosphate (RP3), reverse results were noticed i.e the non-mycorrhizal plants performed better (2.20cm) than mycorrhizal plants (2.11cm) in terms of leaf width (Table 7, Fig 6b). Among the control plants mycorrhizal plants showed better performance (2.16cm) in comparison to non-mycorrhizal plants (2.01cm). Habibzadeh et al. (2012) also reported similar results in Vigna radiata (L.) plants which were inoculated with AMF (Glomus intraradices) inoculated plants. Similarly, Chaitra (2006) and Rasouli-Sadaghiani et al. (2010) also observed increase in leaf area in mycorrhizal plants as compared to control, supporting the fact that AMF inoculation increase leaf size and overall plant growth as compared to non-inoculated ones.

Table-7: Leaf Width (cm) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- T Rock phosphate 278 0917 0907 levels HxT M RP0 5.84 5.08 5.91 5.15 5.48 b RP1 6.35 5.15 5.74 5.74 5.74 b RP2 7.01 5.91 6.60 6.68 6.55 a RP3 5.84 4.82 4.64 6.17 5.35 b

NM RP0 4.90 5.08 5.41 5.08 5.10 b RP1 4.82 5.91 5.66 5.15 5.38 b RP2 5.48 5.33 6.17 5.33 5.58 b RP3 5.58 5.28 5.74 5.91 5.58b Means(H) 5.71 5.28 5.74 5.63

LSD value at 5% level of significance for factor factor T = 0.2827 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 6a: Leaf width in different hybrids of Helianthus annuas L.

Fig. 6b: Effect of RP treatment on leaves width (cm) in four hybrids of Helianthus annuas L.

Fig. 6c: Effect of mycorrhiza on leaves width (cm) under different RP levels in four hybrids of Helianthus annuas L.

4.1.vii Dry weight of plant Overall results showed that mycorrhizal inoculation increased dry weight of plants at moderate levels of RP. The results of Means, ANOVA and LSD test of dry weight of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 8, table 8 and Figs.7 a,b,c. The ANOVA results showed that effect among hybrids, treatments and interaction between hybrids x treatments were highly significant (P < 0.0000) regarding dry weight of plant at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Table 8, Figs. 7a,b,c). The effect of replications was also found to be significant (P < 0.0145) (Appendix 8). In hybrid response, Hysun-33 gave maximum dry weight of plant (112.67 g) followed by SMH-0917 (97.28 g), SMH-0907(92.78g) and NKS-278 (92.72g) (Table, 8, Fig. 7a). Treatments also gave significant results. With increasing rock phosphate levels upto 50% dry weight of mycorrhizal plants also increased. So, as compare to non- mycorrhizal plants mycorrhizal plants at RP2 level showed maximum dry weight (121.46gm), followed by RP1 (104.65gm) (Table 8, Fig. 7b). This may be due to combined effects of fertilizer and VAM. Sharathbabu & Manoharachary (2006) reported that dual inoculation of AMF (Glomus fasciculatum) and Rock-phosphate significantly enhanced plant growth, dry weights and nutrient uptake as compare to single inoculation or in control plants. The present results are also in agreement with findings of Verma & Arya (1998); Chaurusia et al. (2005); Toussaint et al. (2006) and Shadi et al. (2007). For high mycorrhization of plants certain standard amount of P fertilizer is required. If this level increases beyond that optimum limit, the AMF growth is inhibited. Thus some amount of P fertilizer is neseccarily required for the establishment and growth of AM fungal strain (Abbott et al. 1984). At higher P level (RP3), the non-mycorrhizal plants outperformed (Table 8, Fig.7b). Our results favor findings of Stephen (1980) and Menge et al. (1978), who showed that mycorrhizal plants have advantage over non-mycorrhizal plants in term of growth and dry weight but at high level of phosphorus incompatibility developed between plant and mycorrhizal colonization. 82

Mean values for hybrids × treatments interaction data (Table. 8, Fig.7c) revealed that among all hybrids maximum dry weight was found at moderate level of rock phosphate (RP2) i.e 113.06gm in NKS 278, 158.26gm. in Hysun-33, 107.30gm. in SMH-0917 and 107.23gm. in SMH-0907 and minimum in all hybrids at RP3 under mycorrhizal condition. Among control plants better performance was exhibited by mycorrhizal plants as compare to non-mycorhhizal plants (table, 8). Dry weight of mycorrhizal plants at RP0 was 101.89gm as compared to non-mycorrhizal plants (88.90gm). Our results agree with those of Tjondronegoro & Agustin (2000); Costa et al. (2001); Fisher & Jayachandran (2002); Feng et al. (2002); Paradi et al. (2003); Mohammad et al. (2004); Giri et al. (2005); Rahman et al. (2006); Schreiner, (2007); Turkmen et al. (2008); Sheng et al. (2008); Jambotkar and Lackshman (2009); Rakshit and Bhadoria (2010); Zhu et al. (2010); Sharif et al. (2011) and Laei et al. (2011). In AMF inoculated plants there is not only enhanced uptake of phosphorus and micronutrients (Bresinsky et al., 2008; Rapparini et al., 2008; Rydlova et al., 2010) but there is also enhanced uptake of other nutrients like, potassium and nitrogen (Siqueira et al. 2002; Freitas et al. 2004; Parras-Soriano et al., 2009), resulting in overall increase in plant metabolism. It has also been observed that mycorrhizal inoculation helps to increase transpiration, photosynthesis and chlorophyll concentration (Manoharan et al., 2008). It also enhances hormone production like Cytokinin and Indole acetic acid as compared to control ones (Allen & Allen, 1980), which give positive results in the growth and development of plant.

Table-8: Dry Weight (gm) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phosphate 0917 0907 levels HxT

M RP0 99.13g 119.06b 96.10h 107.23d 105.38c RP1 107.06d 120.20b 95.26hi 96.10h 104.65b RP2 113.06c 158.26a 107.30d 93.10j 117.93a RP3 87.03n 74.20p 96.10h 91.10k 87.10h

NM RP0 73.06p 107.06d 87.26n 89.06lm 89.11g RP1 90.03kl 106.03d 90.10kl 83.26o 92.35f RP2 74.23p 112.26c 104.10e 88.20mn 94.69e RP3 98.13g 104.30e 102.06f 94.20ij 99.67d Mean (H) 92.72c 112.67a 97.28b 92.78c

LSD value at 5% level of significance for factor H= 0.5613, factor T = 0.7937 HxT interaction= 1.587 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 84

Fig. 7a: Dry weight of plant (gm) in different hybrids of Helianthus annuas L.

Fig. 7b: Effect of RP treatment on dry weight (gm) of plant in Helianthus annuas L. hybrids

Fig. 7c: Effect of mycorrhiza on dry weight (gm) under different RP levels in four hybrids of Helianthus annuas L.

4.1.viii Head (Capitulum) Diameter Head diameter of AMF inoculated plants showed increase in diameter at RP1 and RP2 levels, and at RP0 level diameter of mycorrhizal plants was greater than non- mycorrhizal plants (Plate 6).The results of Means, ANOVA and LSD test of head diameter of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 9, table 9, and Figs.8a,b,c. The ANOVA results showed that effect among hybrids, treatments and interaction between hybrids and treatments were highly significant (P < 0.0000), regarding head diameter at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Fig. 8a,b,c), while replications were non-significant (Appendix 9). Hybrids mean data showed significantly different responses among of themselves i.e. Hysun-33 gave maximum head diamter (5.97cm), followed by SMH- 0917 (5.80cm), SMH-0907 (4.83cm) and NKS-278 (4.74cm) (Table, 9, Fig, 8a). Mean values for Hybrids × Treatment interaction data (Table. 9, Fig.8c) revealed that mycorrhizal response varied in different hybrids at various rock phosphate levels. Statistical analysis of interaction data showed that maximum head diameter was recorded for Hysun-33 (9cm) and SMH-0917 (7.13cm) at RP1, followed by SMH-0907 (5.30cm) and NKS-278 (5CM) at RP0 as compared to non-mycorrhizal plants. Treatment mean data (Table 9, Fig, 8b) revealed that at low and moderate levels of rock phosphate (RP1 and RP2), the mycorrhizal plants showed better performance as compared to non-mycorrhizal plants. Among the control plants, mycorrhizal plants (M) exhibited better performance (5.11cm) as compared to non-mycorrhizal (NM) plant (4.08cm) (Table 9, Plate 6). The present results are strengthened by the findings of of Kavitha & Nelson (2013) and Bera et al. (2014), who reported that in sunflower plants, head diameter in mycorrhizal plants was significantly increased as compared to uninoculated control. However reverse result were noticed when fertility level was increased (RP3), where non-mycorrhizal plants out performed (Table 9, Fig, 8b). Our results are in line with Curtis (2004). It might be due to detrimental effect of high level of P on the AMF activity and hence on plant growth as a whole (Fries et al., 1998). Our results is also supported by the work of Soleimanzadeh (2012) who observed that AMF inoculated plants had significant positive effects on head diameter, seed yield and oil 86 yield of sunflower (Azargol cultivar), than non-inoculated plants. However, this positive effect of AMF inoculated plants decreased with increasing P levels. It is, therefore presented that low and moderate P doses could enhance seed yield and oil production.

Table-9: Head (capitulum) diameter (cm) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH-0917 SMH- Rock phosphate 0907 levels HxT T M RP0 5.00de 4.16ef 6.00cd 5.30de 5.11bc RP1 4.16ef 9.00a 7.13bc 5.00de 6.32a RP2 4.20ef 8.10ab 6.00cd 4.00ef 5.57a RP3 4.30ef 4.16ef 6.16cd 4.00ef 4.65cd

NM RP0 5.00de 4.16ef 3.00f 4.16ef 4.08d RP1 3.00f 5.06de 6.00cd 6.23cd 5.07bc RP2 7.26bc 6.16cd 5.00de 5.00de 5.85ab RP3 5.00de 7.00bc 7.16bc 5.00de 6.04a Mean (H) 4.74b 5.97a 5.80a 4.83b

LSD value at 5% level of significance for factor H= 0.5718; T= 0.8087 HxT interaction= 1.617 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate 87

RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 8a: Head diameter in different hybrids of Helianthus annuas L.

Fig. 8b: Effect of RP treatments on head diameter (cm ) in four hybrids of Helianthus annuas L. 88

Fig. 8c: Effect of mycorrhiza on head diameter (cm ) under different RP levels in two four hybrids of Helianthus annuas L.

4.1.ix Number Of Seeds Per Head The results of Means, ANOVA and LSD test of number of seeds/head of four hybrids of sunflower (Helianthus annuus L.), following different treatments are given in the Appendix10, table 10 and Figs. 9a,b,c. The ANOVA results showed that hybrids, treatments and interaction between hybrids x treatments were highly significant (P < 0.0000) regarding number and weight of seeds per head at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while the effect of replications was found to be non-significant (Appendix10,). The hybrid response regarding number of seeds showed that Hysun-33 gave maximum number of (205) seeds followed by SMH-0917 (157), SMH-0907(149) and NKS-278 (125) (Table 10, Fig. 9a). However, interaction data (Table. 10, Fig.9c) revealed that mycorrhizal response varied in different hybrids at various rock phosphate levels. Statistical analysis of interaction data showed that maximum number of seeds was recorded for Hysun-33 (289) at RP1, followed by SMH-0917 (216) at RP2 level, NKS- 278 (206) and SMH-0907 (194) at RP0 as compared to non-mycorrhizal plants. Similar results were also observed by Mahboob et al. (2003) in rice and mungbean crops. It suggests that the effect of different levels of phosphorus on the seed yield is genotype dependent (Lickfett et al., 1999). The effect of treatments also gave significant results (Table 10 and Figs. 9b). Treatment mean data show that at RP0, RP1 and RP2 levels, the mycorrhizal plants showed best performance as compared to non-mycorrhizal plants, 89 while the non-mycorrhizal (NM) plants at RP3 level showed slightly better performance in terms of number of seeds per head as compared to mycorrhizal (M) plants (Table 10 and Fig.9b,c). In nutrient deficient soils, the amount of phosphorus which has to be applied to non-mycorrhizal plants can be very large. Ardakani & Mafakheri (2011) demonstrated that low application of P fertilizer i.e 30kg / ha with AMF inoculation produced the same grain yield as obtained by use of 90 kg /ha in non-mycorrhizal wheat plants. Chandrashekara et al. (1995) also reported that high level of phosphorus is needed to increase the seed yield in un-inoculated AMF plants. AMF inoculation compensate for high dose of phosphorus fertilizer. The present results also show that among the control plants (RP0), mycorrhizal plants performed better (170) as compared to non-mycorrhizal plant (139) (Table 10and Fig 9b). Similar results were obtained by Kavitha & Nelson (2013) regarding the high number of seeds per head in AMF inoculated plants than the non-inoculated plants. About 7 % increase in number of seeds was observed in such cases by Soleimanzadeh (2012).

Table-: 10 Number of seeds/ Head in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- Rock phosphate 0917 0907 level HxT T M RP0 206.00 ef 160.0 kl 120.0 pq 194.0 gh 170.00 b RP1 147.00 mn 289.0 a 201.0 fg 190.0 hi 207.00 a RP2 115.0 qr 270 b 216.0 d 65.0 u 166.00 bc RP3 106.0 s 162.0 k 125.0 p 65.0 u 129.5 f

NM RP0 100.0 st 160.0 kl 108.0 rs 187.0 hij 139.00 e RP1 92.0 t 152.0 lm 142.0 no 180.0 j 141.00 e RP2 116.0 qr 230.0 c 133.0 o 126.0 p 152.00 d RP3 120.0 pq 214.0 de 195.0 gh 183.0 ij 181.00 c Mean (H) 125.00d 205.00 a 157.00 b 149.00 c

LSD value at 5% level of significance for factor H= 3.001, factor T = 4.243 HxT interaction= 8.487 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 91

9a: Number of seeds/ head in different hybrids of Helianthus annuas L.

Fig. 9b: Effect of RP treatments on number of seeds/ head in four hybrids of Helianthus annuas L.

Fig. 9c: Effect of mycorrhiza on number of seeds/head under different RP levels in four hybrids of Helianthus annuas L. 92

4.1. x Weight Of Seeds Per Head The results of Means, ANOVA and LSD test of weight of seeds/head of four hybrids of sunflower (Helianthus annuus L.), following different treatments are given in the Appendix11, table 11 and Figs. 10a,b,c. The ANOVA results showed that hybrids, treatments and interaction between hybrids x treatments were highly significant (P < 0.0000) regarding weight of seeds per head at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Fig. 10 a-c), while the effect of replications was found to be non-significant (Appendix11). As evident from the result (Table 11, Fig. 10b) that mycorrhizal plants showed better response (4.54gm) as compared to control non-mycorrhizal plants (2.87gm) regarding seeds weight. The hybrid response regarding seeds weight showed that Hysun-33 gave maximum seeds weight (6.58gm.) followed by SMH-0917 (4.62gm.), NKS-278 (3.3.0gm) and SMH-0907 (2.96gm.) (Table 11, Fig. 10a). However, interaction data (Table. 11, Fig.10c) revealed that mycorrhizal response varied in different hybrids at various rock phosphate levels. Statistical analysis of interaction data showed that maximum seeds weight was recorded for Hysun-33 (11.50gm.) and SMH-0917 (9gm.) at RP1, followed by NKS-278 (5.46gm.) and SMH-0907 (5.20gm.) at RP0 as compared to non- mycorrhizal plants. The effect of treatments also gave significant results (Table 11 and Figs.10b). Treatment mean data show that at RP0, RP1 and RP2 levels, the mycorrhizal plants showed best performance as compared to non-mycorrhizal plants, while the non- mycorrhizal (NM) plants at RP3 level showed slightly better performance in terms of weight of seeds per head as compared to mycorrhizal (M) plants (Table 11and Fig., 10b,c). Vaseghmanesh et al. (2013) reported that application of mycorrhiza and phosphorus fertilizer had significant effect on the seed yield, biological yield and seed weight of Progress cultivar of sunflower as compared to control. Similar results were also observed by Meshram et al. (2000) in chickpea and Ghorbanian et al. (2011) in Maize. In nutrient deficient soils, the amount of phosphorus which has to be applied to non-mycorrhizal plants can be very large. According to Howeler et al. (1987), large quantities of phosphorus is required to be added to plants without mycorrhiza ranging 93 upto 1,600kg/ha to achieve the level of growth rate of mycorrhizal plants. AMF inoculation compensate for high dose of phosphorus fertilizer.

Table11 Seeds weight/ Head in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- Rock phosphate 278 0917 0907 levels HxT T M RP0 5.46 cde 4.50 ef 3.00 fgh 5.20 de 4.54 b RP1 4.50 ef 11.50 a 9.00 b 3.50 fg 7.12 a RP2 3.50 fg 9.00 b 6.00 cde 2.50 gh 5.25 a RP3 2.50 gh 4.50 ef 3.50 fg 1.50 h 3.00 c

NM RP0 2.50 gh 4.50 ef 3.00 fgh 1.50 h 2.87 cd RP1 2.50 gh 5.20 de 4.50 ef 1.50 h 3.42 c RP2 2.50 gh 4.50 ef 3.00 fgh 4.50 ef 3.62 c RP3 3.00 fgh 7.00 c 6.00 cde 3.50 fg 4.87 b Mean (H) 3.30c 6.58 a 4.62 b 2.96 c

LSD value at 5% level of significance for factor H= 0.5547, factor T = 0.7845 HxT interaction= 1.569 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 94

10a Seeds weight/ head (gm.) in different hybrids of Helianthus annuas L.

Fig. 10b: Effect of RP treatments on seed weight/ head (gm) in four hybrids of Helianthus annuas L.

Fig. 10c: Effect of mycorrhiza on seeds weight/head (gm.) under different RP levels in four hybrids of Helianthus annuas L. 95

4.2 Oil Fatty Acid Profile The results showed (Tables 12-16) that mycorrhizal plants rose without rock phosphate fertilizer (RP0) and at low RP levels had significantly better performance as compared to non-inoculated plants in terms of oil content and unsaturated fatty acid like linoleic acid and oleic acid, while at high RP level (RP3) the non mycorrhizal plants outperformed. On contrary, the saturated fatty acids behave otherwise. Mycorrhizal response varied with increasing rock phosphate in different hybrids. Soxhlet apparatus is used for chemical determination of oil contents in oil seed crops by solvent extraction technique using ether or n-hexane (IAL, 2008). Although the technique is very efficient but the solvents used are dangerous to human health and environment. Moreover, large number of seeds are required which are destroyed in this process therefore it become costly and time consuming method. This methodology is also inapplicable where the seeds available are in very small quantity e.g seed obtained by breeding tests for specific experiments etc (Robertson & Barton, 1984). An alternative method Near-infrared reflectance spectroscopy (NIRS) has been useful to the traditional chemical extraction for several plant species because it is fast, effective, non-destructive, and allows the simultaneous analyses of many components like oil, proteins, fibers etc (Batten, 1998; Matthäus & Bruhl, 2001; Sato, 2002 and Biskupek-Korell & Moschner, 2006) also reported the efficiency of this technique especially in sunflower seeds. Baydar and Erbas (2005) reported that sunflower oil is very beneficial for human health due to high amount of monosaturated and poly-saturated fats content and little amounts of saturated fat (Arshad & Amjad, 2012). Oil containg unsaturated fattyacids are nutritionally much better and has positive effect on human health, as they are responsible for lowering “bad” LDL cholesterol (low density lipoproteins) and simultaneously increasing the "good", HDL cholesterol (high density lipoprotein). On contrary, the saturated fatty acids behave otherwise (Izquierdo & Aguirrezabal, 2008 4.2. i Oil Content (%) Fatty acid profile of sunflower oil is one of the essential parameter through which its nutritional value and cooking quality can be determined. Overall results showed that mycorrhizal inoculation increased oil content of sunflower seeds at low level (25%) of rock phosphate (RP1). The results of Means, ANOVA and LSD test of oil content of four 96 hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 12, table 12 and Figs.11a,b.c. The ANOVA showed that effect among hybrids, treatments and hybrids x treatments interactions were highly significant (P < 0.0000), regarding oil content at various levels of rock phosphate in mycorrhizal and non- mycorrhizal plants (Table12, Fig. 11c) while the effect of replications was found to be non-significant (Appendix 12). These results are supported by the findings of Tiwari & Banafar (1995) and Figueira (1996), who reported that oil yield and fatty acid profile is greatly influenced by application of fertilizers. It is clear from treatment mean (Tale 12, Fig. 11b) data that at low level of rock phosphate (RP1) the mycorrhizal plant showed better oil content (34.32%) as compared to non-mycorrhizal plants (32.55%), while at high levels (RP2 and RP3) the non- mycorrhizal plants out performed. Our results are in aggrrement with the work of Chandrashekara et al. (1995), who found that AMF inoculated plants save 25-50 percent of phosphorus fertilizer as compared to non-inoculated plants for production of same amount of total biomass and oil contents. Comparison between four sunflower hybrids viz. NKS-278, Hysun-33, SMH- 0917 and SMH-0907 for their oil contents at various rock phosphate levels revealed that maximum oil contents was recorded in Hysun-33 (34.10%) followed by SMH- 0917 (33.68%), SMH-0907 (32.66%) and least in NKS-278 i.e (30.77%) (Table 12, Fig.11a). Hybrid differed significantly for seed oil content as reported by Cheema et al. (2001). The present results also showed that among control plants (RP0), mycorrhizal plants performed better (32.55%) than non-mycorrhizal plants (29.22%). Our results agree with Soleimanzadeh (2012), who stated that AMF inoculated plants had significant positive effects on oil yield of sunflower (Azargol cultivar) than non- inoculated plants. Increased oil content in mycorrhizal sunflower plants have also been reported by Heidari & Karamiba, (2014); Gholamhoseini et al. (2013) and Bera et al. 2014. Mean values for Hybrids × Treatment interaction data (Table. 12, Fig.11c) reveales that as compared to non-mycorrhizal plants, mycorrhizal response varied with increasing rock phosphate in different hybrids. Results clearly showed that Maximum oil 97 content was recorded for Hysun-33 (36.80%) and SMH-0917 (35.80%) at RP1, followed by SMH-0907 (34.50%) at RP2 level and NKS-278 (32.90) at RP0. The use of low doses of rock phosphate with AMF gave better results; while high levels of rock phosphate is antagonistic to AMF. Similarly, it is also clear from the table 12 that among the non-mycorrhizal treatments at various rock phosphate levels showed significant increase in oil content compared to control (RP0) in all hybrids (Table. 12, Fig.11c). The oil percentage increases at high P level is also reported by Bailey & Grant (1990) and Tamak et al. (1997) but these results negate the findings of Holmes & Ainsley (1977) and Cheema et al. (2001).

Table-12: Oil content (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock 0917 0907 phosphate HxT levels M RP0 32.90ijkl 31.90kl 35.50bcdef 29.90m 32.55b RP1 32.20kl 36.80ab 35.80cdefg 32.50jkl 34.32a RP2 31.60l 36.43abc 33.30ghijk 34.50efghi 33.95a RP3 27.50no 35.20abcd 31.20kl 31.60defgh 31.37b

NM RP0 28.80mn 26.90o 31.60l 29.60m 29.22c RP1 32.20kl 33.00hijkl 32.60jkl 32.40jkl 32.55b RP2 32.10kl 36.00bcde 35.50bcdef 33.10hijkl 34.17a RP3 28.90mn 36.60ab 34.00fghij 36.70a 34. 05a Means(H) 30.77c 34.10a 33.68a 32.66b

LSD value at 5% level of significance for factor H= 0.5848, factor T = 0.8270 98

HxT interaction= 1.654 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 11a: Oil content (%) in different hybrids of Helianthus annuas L.

Fig. 11b: Effect of RP treatments on oil content (%) in four hybrids of Helianthus annuas L. 99

Fig. 11c: Effect of mycorrhiza on oil content (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

4.2. ii Fatty Acid Profile Unsaturated Fatty Acids Over all results showed that the use of AMF along with low doses of rock phosphate promote monounsaturated (oleic acid) and polyunsaturated fatty acids (linoleic acid) which are more beneficial for health as they are responsible for lowering “bad” LDL cholesterol (low density lipoproteins) and simultaneously increasing the "good", HDL cholesterol (high density lipoprotein), while high levels of rock phosphate is antagonistic to AMF. Linoleic Acid (C18:2) High level of linoleic acid is considered good for oil quality. Sunflower seed has high concentration of linoleic acid a poly unsaturated fatty acid is essential for human beings and cannot be synthesized by animal body (Seiler, 2007). Over all results showed that the use of AMF along with low doses of rock phosphate promote mono unsaturated (oleic acid) and polyunsaturated fatty acids (linoleic acid), The results of Means, ANOVA and LSD test of linoleic acid of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 13, table 13 and Figs.12a,b,c. The ANOVA showed that effect among hybrids, treatments and interaction between hybrids and treatments were highly significant P < 0.0000 regarding linoleic acid at various levels of rock phosphate in mycorrhizal and 100 non-mycorrhizal plants, while the effect of replications was found to be non-significant (Appendix 13). The present results show that among control plants (Table, 13, Fig.12b) mycorrhiza plants performed better (71.15%) as compared to non- mycorrhizal plants (70.22%) regarding linoleic acid content. Increased linoleic acid content in mycorrhizal plants has also been reported by AbduAllah et al. (2015). Statistical analysis of data also showed that rock phosphate levels had significant effect on linoleic acid content in sunflower. It is clear from mean data that among the mycorrhizal and non-mycorrhizal treatments at rock phosphate levels showed significant increase in linoleic acid content over the control. However, at low level of rock phosphate (RP1) the mycorrhizal plants showed maximum (74.12 %) linoleic acid as compared to non- mycorrhizal plants (70.47%), while at RP2 and RP3 levels the non-mycorrhizal plants out performed (Table 13, Fig 12b). Hybrids also exhibited significant response in terms of linoleic acid i.,e Hysun-33 has higher (73.32%) linoleic acid content followed by SMH-0917 (73.02%), SMH-0907 (71.70 %) and NKS-278 (71.6%) and (Table 13, Fig.12a) as reported by Ahmad and Hassan (2000). However, interaction data revealed that mycorrhizal response varied with increasing rock phosphate in different hybrids. Results (Table. 13, Fig.12c) clearly showed that maximum linoleic acid (%) was recorded for Hysun-33 (75.7%) followed by SMH-0907 (75.30%) and SMH-0917 (74.30%) at RP1 level, while least was recorded for NKS-278 (72.10%) at RP0 as compared to non- mycorrhizal plants. Rajendran and Veeraputhiran (2001) observed that sunflower being an oilseed crop, showed better performance with P fertilization.

101

Table-13: Linoleic acid (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock 0917 0907 phosphate HxT levels M RP0 72.10fghi 72.10fghi 71.70hij 68.70lm 71.15cd RP1 71.50hijk 75.7abc 74.30bcde 75.30abcd 74.2b RP2 72.00ghi 74.10bcde 73.90cde 74.30bcde 73.57b RP3 68.00mn 72.90efgh 73.80cde 71.50hijk 71.55c

NM RP0 72.00ghi 70.30jkl 71.60hijk 67.00n 70.22e RP1 70.00kl 73.40efg 70.70ijk 67.80mn 70.47de RP2 74.3a 74.30bcde 74.20bcde 75.30abcd 74.2a RP3 74.20bcde 73.80cde 74.00ab 73.70def 73.92ab Means(H) 71.6b 73.32a 73.02a 71.70b

LSD value at 5% level of significance for factor H= 0.5851, factor T = 0.8274 HxT interaction= 1.655 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

102

Fig. 12a: Linoleic acid content (%) in different hybrids of Helianthus annuas L.

Fig. 12b: Effect of RP treatments on linoleic acid (%) in four hybrids of Helianthus annuas L.

Fig. 12c: Effect of mycorrhiza on linoleic acid (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 103

Oleic acid (C18:1 cis-9) The results of Means, ANOVA and LSD test of oleic acid of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 14, table 14 and Figs.13a,b,c. The ANOVA showed that effect among treatments and interaction between hybrids and treatments were highly significant P < 0.0008 and P < 0.0005 respectively (Tale 14, Fig. 13c), regarding oleic acid at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while the effect among replications and hybrids were found to be non-significant (Appendix 14). The present results show that among control plants (Table, 14, Fig.13b) mycorrhiza plants performed better (17.02%) as compared to non-mycorrhizal plants (14.47%) regarding oleic acid content. The perusal of data regarding oleic acid revealed that in sunflower maximum amount was recorded in RP1 (17.05%) which is statistically at par with RP0 (17.02%). However at high levels of rock phosphate (RP2 & RP3) reverse result noticed i.e the non- mycorrhizal plants performed better than mycorrhizal plants (Table, 14, Fig 13b), as high levels of rock phosphate is antagonistic to AMF. Similarly, among the non-mycorrhizal treatments at various rock phosphate levels results showed significant increase in oleic acid content over the control. However the response varied in different hybrids (Table, 14, Fig 13b,c). Findings of Khan et al. (1997) and Zakaria et al. (2006) also support the present study. Khan et al. (2013) reported that temperature may have positive or negative affects on the production of oleic acid in different sunflower hybrids. Recorded values of all hybrids are statistically at par with each other in terms of oleic acid (%) i.e maximum being in Hysun-33 (16.33%) followed by SMH-0917 (15.8%), SMH-0907(15.47%) and NKS-278 (15.32%) (Table14, Fig. 13a).

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Table-14: Oleic acid (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phophate 0917 0907 levels HxT M RP0 17.7 a 17.5 ab 17.4 abc 16 bcd 17.02 ab RP1 16 bcdefgh 18.8 a 17 bcd 16.4 bcdef 17.05 a RP2 15.2 efghi 14.7 hij 14.9 fghij 16 bcdefgh 15.2 cd RP3 13.4 j 15.4 defghi 14.5 hij 14.8 ghij 14.52 d

NM RP0 15.7 cdefghi 15.8 bcdefgh 15.3 defghi 15.1 efghij 14.47 de RP1 14.5 hij 16.1 bcdefgh 15.2 efghi 16 bcdefgh 15.45 bcde RP2 15.3 defghi 16 bcdefgh 16.6 bcde 14 ij 15.47 bcd RP3 14.8 ghij 16.4 bcdefg 15.5 defghi 15.5 defghi 15.55 bc Means(H) 15.32 16.33 15.8 15.47

LSD value at 5% level of significance for factor T = 0.8644 ; HxT interaction= 1.729 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 105

Fig. 13a: Oleic acid (%) in different hybrids of Helianthus annuas L.

Fig. 13b: Effect of RP treatments on Oleic acid (%) in four hybrids of Helianthus annuas L.

Fig. 13c: Effect of mycorrhiza on Oleic acid (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 106

Saturated Fatty Acids Overall results showed that the use of AMF along with high doses of rock phosphate bring increase in the production of saturated fatty acids like Palmitic acid and stearic acid in sunflower seeds. This research work has substantial health significance as saturated fatty acids are less beneficial for health as they are responsible for increasing “bad” LDL cholesterol (low density lipoproteins). Palmitic Acid (C16:0) The results of Means, ANOVA and LSD test of palmitic acid of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 15, table 15 and Figs.14a,b,c. The ANOVA showed that effect among replications, hybrids, treatments and interaction between hybrids and treatments were non-significant regarding palmitic acid at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Appendix 15), as recorded values are statistically at par with each other in terms of Palmitic acid (Fig, 14a,c). Khan et al. (2013) reported that temperature may have positive or negative effects on the production of palmitic acid in different sunflower hybrids. As evident from the result (Table 15) that non-mycorrhizal plants (4.23%) showed better response as compared to mycorrhizal control plants (3.75%) regarding the palmitic acid content. The perusal of treatment means data (Table 15 Fig. 14b) regarding palmitic acid revealed that the mycorrhizal responses were most pronounced at high level of applied fertilizer (RP2 & RP3). However at RP0 & RP1 levels of rock phosphate the non- mycorrhizal plants outperformed. (Table 15, Fig 14b). Recorded values of all hybrids are statistically at par with each other in terms of palmitic acid (%) i.e maximum being in NKS-278 (3.89%) followed by SMH-0907 (3.87%), SMH-0907 (3.82%) and Hysun-33 (3.74%) (Table 15, Fig. 14a).

107

Table-15: Palmitic acid (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates.

Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- T Rock phosphate 278 0917 0907 levels HxT M RP0 2.40 3.50 4.50 4.60 3.75 RP1 4.00 2.70 3.60 3.60 3.47 RP2 4.80 3.80 3.40 2.80 3.70 RP3 4.53 4.50 3.90 4.30 4.30

NM RP0 3.70 4.43 4.80 4.00 4.23 RP1 4.60 3.90 3.30 4.80 4.15 RP2 3.70 3.70 3.60 3.20 3.30 RP3 3.40 3.40 3.50 3.70 3.50 Means(H) 3.89 3.74 3.82 3.87

Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 108

Fig. 14a: Palmitic acid (%) in different hybrids of Helianthus annuas L.

Fig. 14b: Effect of RP treatments on Palmitic acid (%) in four hybrids of Helianthus annuas L.

Fig. 14c: Effect of mycorrhiza on Palmitic acid (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

109

Stearic Acid (C18:0) The results of Means, ANOVA and LSD test of stearic acid of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 16, table 16 and Figs.15a,b,c. The ANOVA (Appendix 16) showed that effect among treatments and interaction between hybrids and treatments were highly significant P < 0.0008 and P < 0.0037 respectively, regarding stearic acid at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while the effect among replications and hybrids were found to be non-significant. The perusal of treatment mean data (Table 16, Fig. 15b) regarding stearic acid revealed that the mycorrhizal responses were most pronounced at high level of applied fertilizer (RP3). However at RP0, RP1& RP2 levels of rock phosphate the non- mycorrhizal plants outperformed. (Table 16, Fig 15b). Hybrid means elucidated that stearic acid contents were maximum in SMH-0907 (5.84%) followed by NKS-278 (5.56%), SMH-0917 (5.40%) and Hysun-33 (4.9%) at various levels of rock phosphate (Table 16, Fig.15a). Mean values for Hybrids × Treatment interaction data (Table. 16, Fig.15c) revealed that mycorrhizal response varied with increasing rock phosphate in different hybrids. Results clearly showed that minimum stearic acid content was recorded for Hysun-33 (3.70%) and SMH-0917 (3.90%) followed by SMH-0907 (4.86%) at RP1 level and NKS-278 (4.305) at RP0 as compared to non-mycorrhizal plants.

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Table-16: Stearic acid (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock 0917 0907 phosphate HxT levels M RP0 4.30ghi 4.80efghi 4.90efghi 5.30cdefghi 4.82d RP1 5.20cdefghi 3.70bcdefg 3.90i 4.86efghi 4.41d RP2 5.60bcdefgh 4.80efghi 4.00hi 7.00ab 5.35bcd RP3 7.80a 4.50fghi 5.60bcdefgh 5.40bcdefghi 5.82abc

NM RP0 6.30abcde 7.00ab 6.70abcd 5.80bcdefg 6.45a RP1 5.10adefghi 4.30ghi 6.63abcd 6.10bcdef 5.53bcd RP2 5.40bcdefghi 5.90bcdefg 6.30abcde 6.80abc 6.10ab RP3 4.80efghi 4.60fghi 5.20cdefghi 5.50bcdefghi 5.02cd Means(H) 5.56 4.9 5.40 5.84

LSD value at 5% level of significance for factor T = 0.8136 ; HxT interaction= 1.627 Key: H=Hybrids T = Treatments M=Mycorrhizal; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate 111

Fig. 15a: Stearic acid (%) in different hybrids of Helianthus annuas L.

Fig. 15b: Effect of RP treatments on Stearic acid (%) in four hybrids of Helianthus annuas L.

Fig. 15c: Effect of mycorrhiza on Stearic acid (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 112

Plate 1

Experimental site of sunflower 113

PLATE 2

Control NKS-278(RP0) Control Hysun-33(RP0)

Control SMH-0917 (RP0) Control SMH-0907(RP0) a)Effect of mycorrhiza on plant height (cm ) in four hybrids of Helianthus annuas L. at RP0 (Control) 114

PLATE 3

NKS-278(RP1) Hysun-33(RP1)

SMH-0917 (RP1) SMH-0907(RP1)

B.Effect of mycorrhiza on plant height (cm ) in four hybrids of Helianthus annuas L. at 25% rock phosphate

115

PLATE 4

NKS-278(RP2) Hysun-33(RP2)

SMH-0917 (RP2) SMH-0907(RP2) c.Effect of mycorrhiza on plant height (cm ) in four hybrids of Helianthus annuas L. at 50% rock phosphate 116

PLATE5

NKS-278(RP3) Hysun-33(RP3)

SMH-0917 (RP3) SMH-0907(RP3) d.Effect of mycorrhiza on plant height (cm ) in four hybrids of Helianthus annuas L. at 50% rock phosphate

117

PLATE 6

A. Effect of mycorrhiza on seeds formation of sunflower

B. Effect of mycorrhiza on sunflower head 118

4.3 Proximate Analysis One of the basic requirements and necessity for the fulfillment of food regulators is the calculation of carbohydrate, crude fats, crude protein and crude fibers along with moisture and ash contents (Omomowo et al., 2009). Therefore, in the present research work proximate analysis (nutritional composition) of AMF inoculated selected sunflower hybrids have been made. 4.3.i Crude Protein (%) Proteins are macromolecules, consisting of long chains of amino acid. Besides building block of the body, they also perform many other vital functions in the living organisms. Many of the physiological functions depend on the enzymes and catalyst which are protein in nature. Protein is therefore needed for growth and maintenance in plants. Overall results showed that mycorrhizal inoculation increased crude protein content of seeds at moderate levels of RP. The results of Means, ANOVA and LSD test of crude protein content of four hybrids following different treatments are given in the Appendix 17, table 17 and Figs.16a,b,c. The ANOVA showed that effect among hybrids, treatments and interaction between hybrids and treatments were highly significant (P < 0.0000) regarding crude protein content at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while the effect of replications was found to be non-significant (Appendix 17). It is evident from the mean data (Table 17, Fig.16b) that the effect of treatments gave significant results. With increasing rock phosphate levels crude protein content of mycorrhizal plants also increases upto recommended 50% rock phosphate applied. So, mycorrhizal plants at RP2 level showed maximum (20.47%) crude protein followed by RP1 (20.20%) as compare to non-mycorrhizal ones. This might be due to combined effects of fertilizer and AMF. Jalali & Thareja (1985) also noticed that the mycorrhizal responses were most pronounced at low and intermediate levels of applied fertilizer levels. However at high levels of rock phosphate (RP3) the non-mycorrhizal plants outperformed. There was great variation among all hybrids. Hybrid means elucidated that crude protein contents were maximum in SMH-0917 (20.50%) followed by SMH- 0907 (20.00%), Hysun-33 (19.52%) and NKS-278 (18.28%) at various levels of rock 119 phosphate (Table 17, Fig.16a). Findings of Zaidi & Saghir (2006) also support the present study. The variation among hybrids in the present study could be attributed to genetic differences (Farshadfar & Farshadfar, 2008; Ereifej et al., 2001). Some authors reported negative correlation between oil and protein content of plants. (Poustavoyt & Dyakov, 1972; Pritchard et al., 2000; Radić, et al., 2009; Drumeva et al. 2011). Mean values for Hybrids × Treatment interaction data (Table 17, Fig. 16c) revealed that mycorrhizal response varied with increasing rock phosphate in different hybrids. Statistical analysis of interaction data showed that maximum crude protein content was recorded for SMH-0917 (22.50%) and Hysun-33 (20.50%) at RP2, followed by SMH-0907 (20.90%) and NKS-278 (19.20%) at RP0 as compared to non-mycorrhizal plants. The present results also show that among control plants better performance was exhibited by mycorrhizal plants (19.20%) than non-mycorrhizal control plants (17.60%) (Table 17, Fig.16b). Our results are supported with the findings of Khalafallah and Abo- Ghalia (2008) who reported that AM inoculation increased crude proteins contents of wheat plant by 6% than non-mycorrhizal. Similarly, Ratti et al. (2010) reported that Catharanthus roseus inoculated with Glomus mosseae showed higher protein level than non-mycorrhizal plants. He & Zhong (2011) also reported the same results in Cinnamomum camphora. The present results are also in agreement with the finding of other workers Ruiz-lozano et al. (1996); Subramanian and Charest (1998); Wu et al. (2006) and Manoharan et al. (2008) who observed that the crude protein content is greater in the plants with AMF than the control treatment. However our results are not in agreement with those of Nemec & Meredith (1981). Increase in protein contents of AMF inoculated plants might be attributed to accumulation of phosphorus and some other nutrients which are constituents of the metabolically active compound (Shadi et al. 2007; Marschner, 2002). The normal synthesis and degradation of protein help maintain the constant and steady level of soluble protein in plant cytoplasm (Guo et al., 1999). Thus quantitative increase in soluble protein contents is due to mediation of certain genes in AMF mediated plants (Jahromi et al., 2008; Ouziad et al., 2006).

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Table. 17 Crude Protein (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH-0917 SMH-0907 Rock phosphate HxT T levels M RP0 19.20efgh 17.60hijk 19.10efghi 20.90abcd 19.20c RP1 19.10efghi 19.30defg 22.30a 20.10bcdefg 20.20ab RP2 18.80fghi 20.50bcde 22.50a 20.10bcdefg 20.47a RP3 16.70k 20.40bcdef 21.30abc 19.30defg 19.42bc

NM RP0 17.50ijk 17.60hijk 16.10k 19.20efgh 17.60d RP1 18.60ghij 18.90efghi 19.50defg 19.20efgh 19.05c RP2 19.40defg 19.50defg 21.50abc 21.20abc 20.40a RP3 17.00jk 22.40a 21.70ab 20.00cdefg 20.27a Means (H) 18.28c 19.52b 20.50a 20.00ab

LSD value at 5% level of significance for factor H= 0.5825, factor T = 0.8238 HxT interaction= 1.648 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

121

Fig. 16a: Crude Proteins (%) in different hybrids of Helianthus annuas L.

Fig. 16b: Effect of RP treatments on Crude Protein (%) in four hybrids of Helianthus annuas L.

Fig. 16c: Effect of mycorrhiza on Crude Protein (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 122

4.3.ii Ash Content (%) The results of Means, ANOVA and LSD test of ash content of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 18, table 18 and Figs.17a,b,c). The ANOVA (Appendix 18) showed that effect among replications, treatments and interaction between hybrids and treatments were highly significant P < 0.0071, P < 0.0000 and P < 0.0007 respectively, regarding ash content at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants while the effect among hybrids were found to be non-significant (Fig.17b), as recorded values of all hybrids are statistically at par with each other in terms of ash content i.e maximum being in NKS-278 (3.31%) hybrid followed by Hysun-33 (3.20%), SMH-0917 (3.03%) and SMH-0907 (2.86%) (Fig, 17a). As evident from the result that mycorrhizal plants (3.98%) showed better response as compared to non-mycorrhizal control plants (1.97%) regarding the ash content (Table 18, Fig17b). According to Mehrvarz & Chaichi (2008) inoculated plants of Barely (Hordeum vulgare L.) exhibited higher level of total ash (8.05%) than non-mycorrhizal (7.84%). Similarly, Elsheikh & Mohamedzein (1998) reported that AM inoculation increased ash content of groundnut seeds. It is clear from mean data (Table 18, Fig. 17 b) that at low level of rock phosphate (RP1) the mycorrhizal plant showed better (4.63%) ash content as compared to non-mycorrhizal plants (2.69%). While the non-mycorrhizal plants at high level (RP2 and RP3) showed slightly better performance as compared to mycorrhizal plants, regarding the % ash content (Table 18, Fig 17b). Stacey et al. (2006) stated that chemical composition may vary with time. Mean values for Hybrids × Treatment interaction data (Table 18, Fig. 17c) revealed that mycorrhizal response varied with increasing rock phosphate in different hybrids. Results showed that maximum ash content (%) was recorded for SMH-0917 (5.76%) at RP1, followed by NKS-278 (5.56%) at RP0, Hysun-33 (5.41%) at RP1 level & SMH-0907 (3.84%) at RP0 as compared to non-mycorrhizal plants.

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Table-18: Ash content (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- T Rock phosphate 278 0917 0907 levels HxT M RP0 5.56ab 3.19bcde 3.35abcd 3.84abcd 3.98ab RP1 3.56abcd 5.41ab 5.76a 3.80abcd 4.63a RP2 2.35de 3.35abcd 3.49abcd 3.08bcde 3.06bcd RP3 2.15de 2.28de 0.79e 1.59de 1.70e

NM RP0 1.74de 1.96de 2.71cde 1.50de 1.97de RP1 2.79cde 2.85cde 3.35abcd 1.78de 2.69cde RP2 3.40abcd 3.24abcde 3.39abcd 3.58abcd 3.40abc RP3 4.94abc 3.39abcd 1.44de 3.74abcd 3.37abc Means (H) 3.31 3.20 3.03 2.86

LSD value at 5% level of significance for factor factor T = 1.270; HxT interaction= 2.540 Key: H=Hybrids T = Treatments M=Mycorrhizal; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

124

Fig. 17a: Ash content (%) in different hybrids of Helianthus annuas L.

Fig. 17b: Effect of RP treatments on Ash Content (%) in four hybrids of Helianthus annuas L.

Fig. 17c: Effect of mycorrhiza on Ash content (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 125

4.3.iii Moisture Content (%) The results of Means, ANOVA and LSD test of moisture content of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 19, table 19 and Figs.18a,b,c. The ANOVA showed that effect among treatments and interaction between hybrids and treatments were highly significant P < 0.0000 and P < 0.0016 respectively, regarding moisture content at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while the effect among replications and hybrids were found to be non-significant (Appendix 19), as recorded values of all hybrids are statistically at par with each other in terms of moisture content i.e maximum being in SMH-0917 (6.05%) followed by Hysun-33 (5.85%), NKS-278 (5.78%) and SMH-0907 (5.64%) (Table 19, Fig.18a). It is clear from mean data that at low rock phosphate (RP1) level the mycorrhizal plants showed maximum (6.76%) moisture content as compared to non- mycorrhizal plants (5.50%), while at high level (RP2 and RP3) the non-mycorrhizal plants out performed (Table 19, Fig. 18b) Palta et al. (2010), explained that accumulation of moisture, proteins, oil, ash and fibers varied with time. Mean values for Hybrids × Treatment interaction data (Table 19, Fig. 18c) revealed that mycorrhizal response varied with increasing rock phosphate in different hybrids. Results showed that maximum moisture content (%) was recorded for SMH-0917 (8.22%) and Hysun-33 (7.57%) at RP1, followed by NKS-278 (6.80%) and SMH-0907 (6.62%) at RP0 as compared to non- mycorrhizal plants.

126

Table-19: Moisture content (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH-0917 SMH- T Rock 0907 phosphate HxT levels M RP0 6.80abcd 4.92ghijklm 5.11efghijkl 6.62abcdef 5.86bc RP1 6.23bcdefghij 7.57ab 8.22a 5.04fghijkl 6.76a RP2 4.76ijklm 6.53bcdefg 6.87abc 5.19defghijkl 5.83bc RP3 4.83hijklm 6.52bcdefg 5.91cdefghij 4.16klm 5.35c

NM RP0 4.11lm 3.33m 4.88ghijklm 4.72jklm 4.26d RP1 5.79cdefghijk 4.65jklm 5.17defghijkl 6.40bcdefghi 5.50bc RP2 6.77abcd 6.18bcdefghij 5.53cdefghijkl 6.42bcdefgh 6.22ab RP3 6.97abc 7.17abc 6.72abcde 6.60abcdef 6.86a Means(H) 5.78 5.85 6.05 5.64

LSD value at 5% level of significance for factor factor T = 0.8262 ; HxT interaction= 1.652 Key: H=Hybrids T = Treatments M=Mycorrhizal; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

127

Fig. 18a: Moisture content (%) in different hybrids of Helianthus annuas L.

Fig. 18b: Effect of RP treatments on Moisture content (%) in four hybrids of Helianthus annuas L.

Fig. 18c: Effect of mycorrhiza on Moisture content (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 128

4.3.iv Crude Fats (%) The results of Means, ANOVA and LSD test of fat contents of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 20, table 20 and Figs.19a,b,c. The ANOVA (Appendix 20) showed that effect among replication was found to be highly significant P < 0.0000, while hybrids, treatments and interaction between hybrids and treatments were found to be non- significant regarding fat contents at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, as recorded values are statistically at par with each other in terms of crude fats (Figs.19a,c). In hybrid response, SMH-0907 gave maximum crude fats (35.90%) followed by SMH-0917 (34.96%), Hysun-33(34.80%) and NKS-278 (34.52%) (Table20, Fig. 19a). Our research studies revealed that high amount of fat contents was observed in mycorrhizal plants (35.69%) than the non-mycorrhizal control plants (31.77%). Our results agreed with the work of Cooper & Losel (1978), according to them infected roots contained more total lipid than uninfected roots. Moreover, Omomowo et al. (2009) found that inoculation with Glomus mosseae has higher fat content of cowpea than un-inoculated control. It is clear from treatment mean data (Table 20, Fig 19b) that at low rock phosphate (RP1) level the mycorrhizal plants showed maximum (35.36%) crude fats content as compared to non- mycorrhizal plants (32.55%), while at RP2 and RP3 levels the non-mycorrhizal plants out performed. Among hybrids fat content varied greatly. These results are supported by the work of Elsheikh & Mohamedzein (1998) and Bhaty & Christison (1984)

129

Table-20: Crude Fats (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates.

Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phosphate 0917 0907 levels HxT M RP0 38.63 32.76 35.66 35.71 35.69 RP1 34.10 34.10 36.28 36.97 35.36 RP2 32.18 37.77 37.52 36.02 35.87 RP3 35.61 35.83 36.43 35.13 35.75

NM RP0 31.00 32.00 31.10 33.00 31.77 RP1 32.00 33.00 31.20 34.00 32.55 RP2 35.10 36.00 35.00 38.02 36.03 RP3 37.60 37.00 36.53 38.40 37.38 Means (H) 34.52 34.80 34.96 35.90

Key: H=Hybrids T = Treatments M=Mycorrhizal; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

130

Fig. 19a: Crude Fat content (%) in different hybrids of Helianthus annuas L.

Fig. 19b: Effect of RP treatments on Crude Fat content (%) in four hybrids of Helianthus annuas L.

Fig. 19c: Effect of mycorrhiza on Crude Fat content (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 131

4.3.v Crude Fiber (%): Crude fibers are non starch polysaccharides. Chemically it contains all organic compounds present in the cell membrane and other cell structures except crude protein, fats and nitrogen extract. The results of Means, ANOVA and LSD test of crude fibers of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 21, table 21 and Figs.20a, b,c. The ANOVA (Appendix 21) showed that effect among hybrids and interaction between hybrids and treatments were found to be highly significant P < 0.0019 and P < 0.0048 respectively, regarding crude fibers at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while effect among replications and interaction between hybrids and treatments were found to be non- significant. As the data (Table 21) shows that mycorrhizal plants (9.22%) have high crude fibers as compared to non-mycorrhizal control plants (7.60%). Similar results were also found by Manoharan et al. (2008). On the contrary, Adewole & Ilesanmi (2011) according to them the control treatment (non-mycorrhizal) gave maximum values of crude fiber (27.33%) as compared to the mycorrhizal treatments. It is evident from the mean data that the effect of treatments gave significant results. With increasing rock phosphate levels crude fiber content of mycorrhizal plants also increases upto recommended 50% RP applied. So, mycorrhizal plants at RP2 level showed maximum crude fiber i.e (10.47%) followed by RP1 (10.20%) as compare to non-mycorrhizal ones (table 21, Fig 20b). This might be due to combined effects of fertilizer and VAM. Jalali & Thareja (1985) also noticed that the mycorrhizal responses were most pronounced at low and intermediate levels of applied fertilizer levels. While, at RP3 level the non-mycorrhizal plants outperformed. Hybrid means also showed significant differences i.e SMH-0917 had maximum (10.76%) crude fiber contents followed by SMH-0907 ( 9.87%) and Hysun-33 (9.51%), while minimum (8.41 %) in NKS- 278 (Table 21, Fig. 20a). The variation in the crude fiber of AMF inoculated plants seems to be varietal and climatic response (Kaya & Yalçın, 1999). Mean values for Hybrids × Treatment interaction data (Table 21, Fig. 20c) revealed that mycorrhizal response varied with increasing rock phosphate in different hybrids. Statistical analysis of interaction data 132 showed that maximum crude fiber content was recorded for SMH-0917 (12.50%) and Hysun-33 (10.50%) at RP2, followed by SMH-0907 (10.90%) and NKS-278 (9.20%) at RP0 as compared to non-mycorrhizal plants.

Table-21: Crude fibers content (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- T Rock phosphate 278 0917 0907 levels HxT M RP0 9.20 7.60 9.20 10.90 9.22abc RP1 9.10 9.30 12.30 10.10 10.20ab RP2 8.80 10.50 12.50 10.10 10.47a RP3 7.70 10.40 11.30 9.30 9.67ab

NM RP0 7.60 7.50 7.10 8.20 7.60c RP1 7.60 8.90 9.50 9.20 8.80bc RP2 9.40 9.50 11.50 11.20 10.40ab RP3 7.90 12.40 12.70 10.00 10.75a Means(H) 8.41c 9.51bc 10.76a 9.87ab

LSD value at 5% level of significance for factor H= 1.166 ; factor T = 1.649 Key: H=Hybrids T = Treatments M=Mycorrhizal ; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

133

Fig. 20a: Crude Fiber content (%) in different hybrids of Helianthus annuas L.

Fig. 20b: Effect of RP treatments on Crude Fiber content (%) in four hybrids of Helianthus annuas L.

Fig. 20c: Effect of mycorrhiza on Crude Fiber content (%) under different rock phosphate levels in four hybrids of Helianthus annuas L. 134

4.3.vi Carbohydrates contents (%)

The results of Means, ANOVA and LSD test of carbohydrate contents of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 22, table 22 (Fig.21a,b,c). The ANOVA (Appendix 22) showed that effect among replications and treatments were highly significant P < 0.0001 and P < 0.0000 respectively, regarding carbohydrate content at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while hybrid response and interaction between hybrids and treatments were found to be non-significant (Fig. 21a,c). As the treatment data (Table 22, Fig.21b) revealed that as compared to non- mycorrhizal plants there was decrease of carbohydrates content in mycorrhizal plants by - 25.63% and -24.08% at RP0 and RP1 level respectively, while at high RP levels mycorrhizal plants outperformed.. Similar results were also found by Manoharan et al. (2008) who reported that the total soluble sugar contents decrease in mycorrhizal seedlings in the leaves of all the selected tree species viz., Cassia siamea, Samanea saman, Erythrina variegata, Delonix regia, and Sterculia foetida than non-mycorrhizal seedlings. It is commonly accepted that the decrease in soluble sugars might be due to the fact that carbohydrates are transferred from host to the fungal partner (Lewis, 1975; Fitter, 1991). These results also match with the work of Harrison (2005) who reported that in such type of symbiotic association the fungus has a constant access to the sources of glucose and sucrose for procurement from the host plant. Moreover, Allen (1991) reported that for symbiotic life VAM fungi consumed 10 -20% of net photosynthate. Johnson et al. (1997) investigated that as AM fungi are obligate symbionts, the host should have a significant effect on the VAM fungi through the regulation of C supply (Kozlowski, 1992; Hampp & Schaeffer, 1995; Schaeffer et al., 1997). The osmotic balance in the root tissue of the host plant is affected by the presence of mycorrhizal fungi and hence the composition of carbohydrates and amino acids are influenced (Rosendahl & Rosendahl, 1991). Pearson & Schweiger (1993) stated that with the increase in AMF root colonization reduction in the percentage of carbohydrate concentration is reported. However, the present results differ from the findings of Wu et al. (2010) who showed that the sole AMF inoculation markedly increased leaf sucrose content and leaf 135 and root glucose content, compared to the non-AMF + PA treatments. Similarly, our results are contradictory to Khalafallah and Abo-Ghalia (2008) who reported that mycorrrhizal plants shows maximum amount of carbohydrates content of wheat plant than non-mycorrhizal under well watered conditions. Al-Garni (2006) and Porcel & Ruiz-Lozano (2004) also reported the same results in Phragmites australis and soybean plants inoculated with Glomus species.

Table-22: Carbohydrates content (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05)

Treatment Hybrids Means Percent (%) NKS- Hysun-33 SMH- SMH- T Rock phosphate 278 0917 0907 levels HxT M RP0 19.00 33.93 27.58 22.03 25.63bcd RP1 27.91 27.31 16.14 24.99 24.08cd RP2 34.11 21.35 18.12 25.51 24.77bcd RP3 34.01 25.57 24.93 31.52 29.00bc

NM RP0 38.05 37.61 38.11 52.58 41.58a RP1 32.22 31.70 31.28 29.42 31.15b RP2 25.93 25.58 32.08 19.58 23.54cd RP3 25.59 17.64 19.57 21.36 21.04d Means(H) 29.60 27.58 24.85 28.37

LSD value at 5% level of significance for factor T = 6.750 Key: H=Hybrids T = Treatments M=Mycorrhizal; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) 136

RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 21a: Carbohydrate content (%) in different hybrids of Helianthus annuas L.

Fig. 21b: Effect of RP treatments on Carbohydrate content (%) in four hybrids of Helianthus annuas L.

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Fig. 21c: Effect of mycorrhiza on Carbohydrate content (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

4.4 Mineral Composition Nutrients (N, P, K and Zn) uptake of selected hybrids of sunflower is given in tables 23-26, (Fig 22-25a,b,c). Result showed that maximum uptake of nutrient was attained by mycorrhizal plants. The result is in good agreement with work of Lester (2009) who reported that AM fungi have the capability of absorbing all essential, macro and micro nutrients which are required for plant growth Our results are also supported by the following workers (Bi et al., 2003; Xia et al., 2007; Subramanian et al., 2008; Naderi et al., 2010; Sajedi & Rejali, 2011) who investigated that uptake of nutrients was higher in mycorrhizal plants in contrast to those which are non-mycorrhizal. However, results at various rock phosphate levels varied as reported by Liu et al. (2002). In mycorrhizal plants promising results were noticed at low level of rock phosphate (RP1) while at high levels (RP2 and RP3) the non-mycorrhizal plants out performed.

4.4. I Macronutrient Phosphorus (P) Overall results showed significant increase in phosphorus concentration of seeds at all levels of RP in both inoculated and non-inoculated plants. The results of Means, ANOVA and LSD test of phosphorus concentration of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 23, table 23 and Fig.22a,b,c. The ANOVA showed that effect among treatments was significant (P 138

< 0.0420), regarding phosphorus concentration at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants, while effect among replications, hybrids and interaction between hybrids and treatments were found to be non-significant (Appendix 23,Figs. 22a,c). The present results also show that among control plants better performance was exhibited by mycorrhizal plants (0.54%) than non-mycorrhizal control plants (0.46%) (Table 23) which shows that mycorrhizal infection had positive effect on absorption of P by the host plant. Our results are supported by the work of Bai et al. (2008) who stated that maize plants infected with AMF had higher P contents than non-mycorrhizal plants. Similarly, Naderi et al. (2010) showed that sorghum plants with AMF in their roots enhanced the absorption of N, P and K than non-inoculated plants. Many other workers have reported enhancement of phosphate uptake by vesicular arbuscular mycorhizal fungi (AMF) (Kaya et al., 2003; Koide & Mosse, 2004; Henrike et al., 2007; Akhtar & Siddiqui, 2008; Giasson et al., 2008; Sawers et al., 2008; Brundrett, 2009; Hijikata et al., 2010). It is mainly due to the fact that mycorrhizal fungi discharge powerful chemicals (lytic enzymes and organic acids) in the soil that bring about dissolution of tightly bound soil nutrients like P and Fe (Lester, 2009; Jansa et al., 2011). The mycorrhiza induces antioxidant enzymes activity which may enhance phosphorus acquisition in the plants (Alguacil et al., 2003). Our results were also supported by the following workers Al-Karaki (2000) on Lycopersicon esculentum, Feng et al. (2002) on Zea mays, Tian et al. (2004) on Gossypium arboretum. Sharifi et al. (2007) on Glycine max., Amaya-Carpio et al. (2009) for Ipomoea carnea Abdel-Latef & Chaoxing (2011) for tomato and Prasad et al. (2012) for Chrysanthemum indicum L. Phosphorus as essential component of cell membrane, nucleic acids (DNA, RNA), ATP, co-enzymes, phospholipids and phosphoproteins and plays vital role in many metabolic activities like energy transfer, photosynthesis and respiration etc (Ozanne, 1980). Being essential part of nucleic acid it has important role in cell division and development of new tissues which initiate development of lateral roots and fibrous roots 139

(Brady and Weil, 2002). P is also helpful to bring increase in the leaf area and shoot dry weight of plants (Yahiya et al, 1995). It is evident from the mean data (Table 23, Fig.22b) that the effect of treatments gave significant results. With increasing rock phosphate levels phosphorus concentration of mycorrhizal plants also increases. This might be due to combined effects of fertilizer and AMF. This finding is also supported by Grant et al. (2005) that increasing level of phosphorus in the soil brings increase in the phosphorus in the plant tissues. Hybrid means elucidated that there was no significant variation among all hybrids. Phosphorus concentration was 0.52% in NKS-278 which is statictically at par with Hysun-33 (0.56%), SMH-0917 (0.57%) and SMH-0907 (0.56%) (Table 23, Fig.22a).

Table-23: Phosphorus concentration in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Values followed by different letters are significantly different (p < 0.05) Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phosphate 0917 0907 levels HxT M RP0 0.53 0.52 0.56 0.57 0.54ab RP1 0.54 0.62 0.62 0.59 0.59a RP2 0.55 0.62 0.61 0.59 0.59a RP3 0.56 0.59 0.61 0.61 0.59a

NM RP0 0.45 0.50 0.46 0.46 0.46b RP1 0.48 0.53 0.55 0.52 0.52ab RP2 0.55 0.55 0.57 0.54 0.55a RP3 0.56 0.56 0.58 0.61 0.57a Means(H) 0.52 0.56 0.57 0.56

LSD value at 5% level of significance for factor T = 0.08161

140

Key: H=Hybrids T = Treatments M=Mycorrhizal; NM= Non-mycorrhizal RP0= 0 % recommended rock phosphate (Control) RP1= 25 % recommended rock phosphate RP2= 50 % recommended rock phosphate RP3= 100 % recommended rock phosphate

Fig. 22a: Phosphorus concentration (%) in different hybrids of Helianthus annuas L.

Fig. 22b: Effect of RP treatments on Phosphorus concentration (%) in four hybrids of Helianthus annuas L.

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Fig. 22c: Effect of mycorrhiza on Phosphorus concentration (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

Nitrogen (N) Over all result showed that there was non-significant increase in nitrogen concentration in response to rock phosphate in both AMF inoculated and non-inoculatred plants.The results of Means, ANOVA and LSD test of nitrogen concentration of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 24, table 24 (Fig.23a,b,c). The ANOVA showed that effect among replications, hybrids, treatments and interaction between hybrids and treatments were non-significant regarding seeds nitrogen concentratin at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Appendix 24, Fig. 23c). Comparison between four sunflower hybrids viz. NKS-278, Hysun-33, SMH-0917 and SMH-0907 for their nitrogen concentration at various rock phosphate levels revealed that there was no significant variation among all hybrids. Nitrogen concentration was maximum in SMH-0907 (1.45%) which is statistically at par with Hysun-33 (1.43%), SMH-0917 (1.40%) and NKS-278 (1.39%) (Table 24, Fig.23a).The present results (Table, 24) show that among control plants (RP0) mycorrhizal (M) plants performed better (1.44%) than non-mycorrhizal (NM) plants (1.35%). It has been suggested that mycorrhizal plants hypha have capacity to absorb the soil N & transfer it to plants roots (George et al., 1992; Bago & Becard, 2002). It is controlled by host plant's demand for Nitrogen (Hawkins & George, 2001). VAM plants have access to all forms of Nitrogen, unavailable to non-mycorrhizal plants (Tobar 142 et al., 1994). From the work of Johansen et al. (1994) it is revealed that external mycelium has ability to get soil inorganic nitrogen efficiently. In the AMF symbiosis arginine synthesis take place in the extra radical mycelium which is then transferred to intraradical mycelium. In the intraradical mycelium the arginine break up into simpler components and N is released which is transferred to the host plant (Tian et al., 2011). It is clear from treatment mean (Table24, Fig. 23b) data that at low level of rock phosphate (RP1) the mycorrhizal plant showed better nitrogen concentration (1.45%) as compared to non-mycorrhizal plants (1.37%), while at high levels (RP2 and RP3) the non- mycorrhizal plants out performed. Our results are in favor with the work of Chandrashekara et al. (1995) who found that AMF inoculated plants save 25-50 percent of phosphorus fertilizer as compared to non-inoculated plants. Rashid et al. (2008) also reported the same results in Solanum melongenum L.

Table-24: Nitrogen concentration (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phosphate 0917 0907 levels HxT M RP0 1.47 1.44 1.33 1.52 1.44 RP1 1.35 1.52 1.52 1.45 1.45 RP2 1.35 1.51 1.47 1.44 1.44 RP3 1.44 1.37 1.37 1.42 1.40

NM RP0 1.31 1.37 1.28 1.47 1.35 RP1 1.31 1.37 1.37 1.44 1.37 RP2 1.49 1.42 1.42 1.47 1.45 RP3 1.45 1.49 1.49 1.45 1.47 Means(H) 1.39 1.43 1.40 1.45

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Fig. 23a: Nitrogen concentration (%) in different hybrids of Helianthus annuas L.

Fig. 23b: Effect of RP treatments on Nitrogen concentration (%) in four hybrids of Helianthus annuas L.

144

Fig. 23c: Effect of mycorrhiza on Nitrogen concentration (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

Potassium (K) The results of Means, ANOVA and LSD test of potassium concentration of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 25, table 25 (Fig.24a,b,c). The ANOVA showed that effect among replications, hybrids, treatments and interaction between hybrids and treatments were non-significant regarding potassium concentration at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Appendix 25). Treatment means data (Table 25, Fig. 24b) revealed that potassium concentration is maximum (0.36 %) under mycorrhizal condition than non-mycorrhizal (0.32 %) condition. Rock phosphate fertilization showed no significant effect. Hybrid means also elucidated that there was no significant variation among all hybrids. Potassium concentration was maximum in Hysun-33 (0.36%) which is statistically at par with SMH- 0917 (0.35%), SMH-0907(0.33%) and NKS-278 (0.31%) (Table25, Fig. 24a). The tissues inhabited by mycorrhizal fungi have increased K concentration which clearly indicates an increased uptake of K by mycorrhizal plants (George et al., 1992). Higher K concentration was reported in sorghum colonized by Glomus fasciculatum (Raju et al., 1987). Alizadeh et al. (2010) reported that mycorrhiza significantly increased the absorption of nitrogen, phosphorous and potassium in shoot and root. In contrast some other results showed that in mycorrhizal plant tissues, K concentrations can be reduced (Pinochet et al., 1997). 145

Increased in potassium concentration in AMF inoculated plants have also been reported by, Sharifi et al. ( 2007) in Glycine max., Giri et al. (2007) in Acacia nilotica, Zuccarini & Okurowska (2008) in Ocimum basilicum Garg & Manchanda (2009) in Cajanus cajan, Wu et al. (2010) in Citrus tangerine, Zou & Wu (2011) in Poncirus trifoliata, Abdel Latef & Chaoxing (2011) in tomato, Evelin et al. (2012) in Trigonella foenum-graecum, Estrada et al. (2013) in maize plants, Potassium (K) does not form any organic compound with a vital role (Swan, 1971) but it activates certain enzymes to promote metabolism in the plants like protein synthesis etc (Uchida, 2000). Opening and closing of stomata is also regulated by K+ ion pump.

Table-25: Potassium concentration (%) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH-0917 SMH-0907 T Rock phosphate HxT levels M RP0 0.35 0.32 0.36 0.41 0.36 RP1 0.30 0.50 0.37 0.37 0.38 RP2 0.30 0.36 0.37 0.30 0.33 RP3 0.30 0.30 0.31 0.29 0.30

NM RP0 0.30 0.31 0.34 0.34 0.32 RP1 0.31 0.5 0.35 0.34 0.36 RP2 0.34 0.32 0.36 0.27 0.35 RP3 0.35 0.33 0.36 0.37 0.33 Means(H) 0.31 0.36 0.35 0.33

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Fig. 24a: Potassium concentration (%) in different hybrids of Helianthus annuas L.

Fig. 24b: Effect of RP treatments on Potassium concentration (%) in four hybrids of Helianthus annuas L.

147

Fig. 24c: Effect of mycorrhiza on Potassium concentration (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

4.4.ii Micronutrients Zinc (Zn) The results of Means, ANOVA and LSD test of zinc concentration of four hybrids of sunflower (Helianthus annuus L.) following different treatments are given in the Appendix 26, table 26 and Figs.25a,b,c. The ANOVA showed that effect among replications, hybrids, treatments and interaction between hybrids and treatments were non-significant regarding zinc concentration at various levels of rock phosphate in mycorrhizal and non-mycorrhizal plants (Appendix 26, Fig. 25c). Regarding the uptake of Zinc in studied species AM inoculated plants promote (0.27 mg/L) the absorption as compared to non-mycorrhizal (0.22 mg/L) control plants (Table, 26, Fig, 25b). Our findings are in agreement with the work of Sharif et al. (2011) who reported that crops (sorghum, millet and maize) inoculated with AMF enhanced micronutrient uptake considerably as compared to non-inoculated crop. There was 67% increase of Fe in millet, 186% of Zn in maize and 208% of Cu in inoculated sorghum crops. Similar results are also reported by Karaki (2000). Ramakrishnan & Selvakumar (2012) reported that AMF (Glomus fasciculatum and Glomus intraradices) inoculation in Lycopersicum esculentum Mill. significantly enhanced growth and available nutrient content (N,P, Zn and Cu) as compared to control treatment.The AM inoculated plants show enhanced acquisition of mineral nutrients with 148 low mobility, such as P, Zn, Cu and Fe by extending their fungal hyphae far from the rooting zone (Pirazzi et al., 1999; Al-Karaki, 2000). Liu et al. (2002) suggested that micronutrients uptake (Zn, Cu, Mn, and Fe) varied with P levels added to soil. It is clear from mean data (Table 26, Fig. 25 b) that at low level of rock phosphate (RP1) the mycorrhizal plant showed better (0.28 mg/L) zinc concentration as compared to non-mycorrhizal plants (0.23 mg/L), while the non- mycorrhizal plants at high level (RP2 and RP3) showed slightly better performance as compared to mycorrhizal plants (Table 26, Fig 25b). High soil phosphorus levels not only lead to low mycorrhizal colonization but also results in micronutrient defeciencies due to low uptake by the plant (Timmer & Leyden, 1980). The hybrid response showed that in inoculated sunflower hybrids there was 0.22 mg/L, 0.26 mg/L, 0.24 mg/L, and 0.23 mg/L increase of Zn concentration in NKS-278, SMH- 0907, SMH0917, Hysun-33, respectively, (Table26, Fig. 25a ). Singh et al. (2002) investigated that influence of soil P on arbuscular mycorrhizal symbiosis is governed by the host genotype. AM colonization, at various P levels improved vegetative maize plant growth, root and shoot phosphorus. However, the pattern of phosphorus and zinc uptake in mycorrhizal inoculated and non-AMF inoculated shoots was genotype specific.

149

Table-26: Zinc concentration (mg/L) in different hybrids of Helianthus annuus L. at various levels of rock phosphate (RP). Each value is a mean of three replicates. Treatment Hybrids Means Percent (%) NKS-278 Hysun-33 SMH- SMH- T Rock phosphate 0917 0907 levels HxT M RP0 0.27 0.27 0.25 0.32 0.27 RP1 0.25 0.41 0.26 0.23 0.28 RP2 0.24 0.25 0.25 0.2 0.23 RP3 0.18 0.23 0.24 0.2 0.21

NM RP0 0.2 0.19 0.24 0.25 0.22 RP1 0.21 0.23 0.25 0.23 0.23 RP2 0.23 0.28 0.26 0.22 0.24 RP3 0.25 0.27 0.24 0.2 0.24 Means(H) 0.22 0.26 0.24 0.23

Fig. 25a: Zinc concentration (%) in different hybrids of Helianthus annuas L.

Fig. 25b: Effect of RP treatments on Zinc concentration (%) in four hybrids of Helianthus annuas L.

Fig. 25c: Effect of mycorrhiza on Zinc concentration (%) under different rock phosphate levels in four hybrids of Helianthus annuas L.

151

4.5 Mycorrhizal dependency Mycorrhizal dependency is “the ratio of the dry mass of a plant exhibiting mycorrhizal association to that of non-mycorrhizal plants (Lambers et al., 2008).” Relative mycorrhizal dependency is “the difference between mass of dry shoot of plants exhibiting mycorrhizal association and those of non-mycorrhizal plants, given as percentage of mycorrhizal plant‟s dry mass (Plenchette et al., 1983).” In present study four hybrids of sunflower were analyzed for mycorrhizal dependency (MD) at various rock phosphate levels (Table, 27, Fig. 26). Overall results showed that control (RP0) plants showed the lowest mycorrhizal dependency (MD) values (26.29% in NKS-278; 10.07 in Hysun-33, 2.98% in SMH-0917 and 5.26 % in SMH-0907), which increases progressively with increasing RP levels upto recommended 50% (RP2) i.e in NKS-278 (34.34%), in Hysun-33 (29.06%), in SMH- 0917 (9.19%) and in SMH-0907 (16.94%). These results are in conformity with findings of Yao et al. (2001) who found that AMF significantly influence mycorrhizal dependency at moderate phosphorus level. However, at high RP level (RP3) the effect of mycorrhiza is found negative (Table, 27, Fig. 26). Ryan et al. (2004) and Takács et al, (2006) reported similar results that mycorrhizal dependency eliminated at high soil P concentration. According to our finding all selected sunflower hybrids differ in their mycorrhizal dependency. The comparison revealed that NKS-278 gave maximum MD values followed by Hysun-33, SMH-0907 and SMH-0917 independently of rock phosphate levels (Table 27, Fig. 26). These results are in accordance with the work of Cavalcante et al. (2001); Zangaro et al. (2007) who found that all plant species and even cultivers differ in their mycorrhizal dependency. Our results are also in agreement with the finding of Ortas & akpinar (2011) who found that different mycorrhizal species affect maize plant varieties quite differently from each other in terms of growth and dry mass of the plant. The cause for this difference in dependency is considered to be the difference in soil type, soil phosphorus and root geometry etc (Menge et al., 1983; Hatrick et al., 1993). Our results also agree with those of Chang et al. (1990), Rani & Mukerji (1991), Douds et al. (1993) and Al-Karaki & Al-Raddad (1997) who showed that mycorrhizal dependency of plant is due to higher acquisition of P, Zn, Cu and many other nutrients. 152

The results of our investigation have showed that sunflowers hybrids were more responsive to mycorrhizal association but degree of dependency also varies according to rock phosphate levels as reported by Cardoso et al. (2008), in Mangaba tree. Mycorrhizal dependency increases with increasing P levels (Habte & Manjunath, 1991) as compared with the treatment without rock phosphate. Although increased phosphorus levels reduce AM colonization of plant roots, the dependency of plants on AM symbiosis is also decreased. Root branching determines plant dependence on the symbiosis (Smith & Read, 1997; Barakh & Heggo, 1998). Oliveira et al. (2006) reported that for good mycorrhizal dependency between AMF and the host plant well developed extra radical mycelium (ERM) are needed than those with low mycorrhizal dependency having small sized ERM..

Table-27: Effects of rock phosphate (RP) fertilizers on Mycorrhizal Dependency (MD) % of four hybrids of sunflower MD % MD % MD % MD % RP0 (No RP) RP1 (25% RP) RP2 (50% RP) RP3 (100 % RP) NKS-278 26.29 % 30.85 % 34.34 % -12.75 % HYSUN-33 10.07 % 11.78 % 29.06 % -51.84 % SMH-0917 2.98% 5.41 % 9.19% -6.20 % SMH-0907 5.26 % 13.36 % 16.94 % -3.40 %

Fig. 26: Effects of rock phosphate (RP) fertilizers on Mycorrhizal Dependency (MD) % in four hybrids of Helianthus annuas L. 153

4.6 Effect of indigenous AMF and Rockphosphate levels on Spore Density and root colonization 4.6.i AMF Spore Density The results given in (Table 28, Fig. 27) showed the effect of various rock phosphate levels on the AMF spore density in the rhizospheric soil of selected sunflower hybrids. Mycorrhizal enhancement regarding AMF spore density followed RP0>RP1>RP2>RP3 trend in all hybrids (Fig. 27). It has been observed that spore density was higher in the soil of control (RP0) plants, which decreases progressively with increasing fertility level, less number of spores was found at RP3 level in all hybrids. Generally the population of AMF spores and soil phosphorus are inversely related to each other (Hao et al., 1991). Chandrasekara et al. (2005) and Panwar & Tarafdar (2006) also found that interaction of mycorrhiza and phosphorus fertilizer had no significant effect on AMF spore density. The spore density got declined sharply at high P level is also reported by Guillemin et al. (1995); Antunes et al. (2007) and Arumugam et al. (2010) but these results negate the findings of Sharathbabu & Manoharachary, (2006) who reported that dual inoculation of AMF (Glomus fasciculatum) and rock-phosphate significantly enhanced percentage of mycorrhizal colonization than in single inoculation or in control Tylophora indica plants. Average number of spores counted per 100 gm. of soil was different from hybrid to hybrid at various levels of treatments as showed in tables and figures (28, Fig. 27). The AMF spore densities ranged from 56-260 spores/ 100 gm. soil in selected sunflower hybrids. It was found that among control plants (RP0) the highest number of spores was recorded for Hysun-33 (260/100 grams of soil) followed by SMH-0917 (251/100 grams of soil), SMH-0907 (246/100 grams of soil) and NKS-278 (202/100 grams of soil). The combined effect of AMF+ RP results showed that AMF spore density followed Hysun- 33>SMH-0917, SMH-0907> NKS-278 trend at all RP levels (Table 28). AMF spores are ubiquitous in most ecosystems (Marleau et al. 2011) and are essential component of soil micro biota (Hindumathi & Reddy, 2011). AMF exists in soil as spores, hyphae, as vegetative propagules or infected root pieces for infecting plants, but mostly inoculation of plants is brought about by extraradical mycelium (Sylvia & 154

Jarstfer, 1992). Occurrence or distribution of AMF varies with host ranges (Sarwade et al., 2011).

Table28: Effect of mycorrhiza on AMF spores in the roots of sunflower hybrids at various levels of rock phosphate (RP) Spore Density/ 100 gm Soil RP levels NKS-278 Hysun-33 SMH-0917 SMH-0907 RP0 202 260 251 246 RP1 171 202 182 171 RP2 103 172 145 122 RP3 56 102 88 79

Key: RP0 (0% recommended rock phosphate); RP1 (25% recommended rockphosphate); RP2 (50% recommended rock phosphate); RP3 (100% recommended rockphosphate).

Fig. 27: Effects of rock phosphate (RP) fertilizers on spore density/100 gm. soil of four hybrids of Helianthus annuas L.

4.6.ii AMF SPECIES Table 29 showed that the following species were identified in rhizospheric soil of selected sunflower hybrids at different rock phosphate levels (Pl. 7a-f).

 Acalospora mellae (Spain & N.C. Schenck)  Acaulospora laevis Gerd. & Trappe (1974) 155

 Glomus fasiculatum (Thaxt. Gerd. & Trappe)  Glomus mosseae (T.H. Nicolson & Gerd)  Glomus aggregatum (N.C. Schenck & G.S. Sm.)  Sclerocystis pakistanica (S.H. Iqbal & Perveen)  Gigaspora gigantea (Gerd. & Trappe) Nasim et al. (1998) showed that spores are the means of identification of these fungi. In the present work soil was collected from different pots with plants at reproductive stages of growth. Four genera of endogonaceous spores were identified which were Acaulospora (2 spp. i.e. A. mellae, A. laevis), Glomus (3 species i.e. G. mosseae, G. aggregatum and G. fasiculatum), Sclerocystis 1 specie i.e. (S. pakistanica) and Gigaspora (1 spp. G. gigantean) (Pl.7a-f). In this research we found that the species of Acaulospora were most common and predominant followed by Glomus, Sclerocystis, and Gigaspora. Our findings are further supported by the work of other researchers (Lovelock et al., 2003; Wongmo, 2008; Tchabi et al., 2008; Charoenpakdee et al., 2010; Gao & Guo, 2010; Songachan & Kayang, 2011) who investigated that there is higher number of Acaulospora in the soil followed by Glomus species. The predominance of Acaulospora species might be due to their adaptation to wide variety of soil types, host species and Ph and nutrient availability etc (Jefwa et al., 2006; Straker et al., 2010). It suggests that AMF strains are biological specific for the host plant as reported by Bever et al. (1996). The large number of AMF spores may be attributed to the deficiency of low phosphorus in the soil. Generally the population of VAM spores and soil phosphorus are inversely related to each other (Hao et al., 1991). The presence of small number of Gigasporaceae might be due to the fact that they are usually found in sandy dunes (Lee & Koske, 1994) and are usually large sized, which requires long period for their development than the small sized spores species (Hepper, 1984). Moreover, Gigaspora are very common in wild plants than field crops (Gai et al., 2006).

156

Table29: Effect of mycorrhiza on AMF spores species in sunflower hybrids.

Percent Rock AMF Spore Spore Spore Spore density in phosphate levels spores density density density SMH-0907 in NKS- in Hysun- in SMH- 278 33 0917 Acaulospora +++ +++ +++ +++ RP0 Sclerocystis ++ ++ ++ ++

Glomus +++ +++ +++ +++

Gigaspora - + - -

Acaulospora ++ +++ +++ +++ RP1 Sclerocystis - ++ + -

Glomus + ++ ++ +

Gigaspora - - + -

Acaulospora + +++ ++ + RP2 Sclerocystis - + + -

Glomus - + + +

Gigaspora - - + -

Acaulospora - + - - RP3 Sclerocystis - - - -

Glomus - + - -

Gigaspora - - - -

+ = Present - = Absent +++ = 100 %; ++ = 75 %; + = 25 %

157

PLATES 7

7a: Spore of Sclerocystis pakistanica

7b: Spore of Acaulospora mellae

158

7c: Spore of Gigaspora gigantea

7d: Spore of Glomus aggregatum

159

7e: Spore of Glomus fasciculatum

7f: Spore of Glomus mosseae

160

4.6.iii AMF Colonization in Roots The results given in (Table 30a,b, Fig. 28) showed the effect of various rock phosphate levels on the percent root colonization in the rhizospheric soil of selected sunflower hybrids. Mycorrhizal enhancement regarding percent root colonization followed RP0>RP1>RP2>RP3 trend in all hybrids (Fig. 28). AMF root colonization was determined by the presence of external hyphae, internal hyphae, vesicles and arbuscules. The general AMF infection in sunflower hybrids at various rock phosphate levels was low (Table 30a) as compared to control (RP0) viz 100%. According to the results of the present study the vesicular infection was common and maximum at all RP levels in all hybrids (Pl. 8). These results agree with the results of Iqbal and Bareen, (1986) and Burni et al. (1993). As found in our results the high vesicular infection occurs at the reproductive stage of the plant. Our results are in accordance with the findings of Nasim et al. (1996) and Iqbal et al. (1988) who also found high vesicular infection at the reproductive stage of the plant. Similarly, Fattah & Razak (1996) and Zhao et al. (1997) reported that there exist relationship between the AMF development and the age of the host plant. Moreover, Al-Raddad (1995) observed that the type of crop and harvesting greatly affect the root colonization. The comparison revealed that highest number of vesicles was recorded in Hysun-33 followed by SMH-0917, SMH-0907 and NKS-278 at all RP levels shown in Table (30b), as reported by Linderman & Davis (2004) in marigold, Janouskova et al. (2007) for tobacco and Sensoy et al.(2007) for Capsicum annuum L. The results (Table 30a,b) showed that AMF inoculated plants had significant positive effects on AMF root colonization. However, this positive effect of AMF inoculated plants decreased with increasing RP level; lowest root colonization was found at RP3 level in all hybrids. Redecker (2005) found that high concentration of phosphate seems to induce low fungal colonization level by the plants. It was found that AMF colonization was higher in the control plants of all hybrids whereas it decreased to a minimum 32% in NKS-278, 35% in SMH-0907, 42% in SMH-0917 and 46% in Hysun-33 at high P levels (RP3) (Table 30a, Fig. 28, Pl. 8). These results agreed the results of Soleimanzadeh (2012) who showed that positive effect of AMF colonization decreases with increasing P levels. Similar results were reported by Chandrashekara et al. (1995), Mohammad et al. (2003) and Pragatheswari 161 et al. (2004). This might be attributed to the fact that low phosphorus result in exudation of certain chemicals from the root which enhances AMF colonization and spore germination but such exudations does not take place when phosphorus level is high (Juniper & Abbott, 2006; Murkute et al., 2009). Sharif et al. (2011) and Manske (1990) showed that low availability of soil phosphorus increases AMF colonization. But the Satpal & Kapoor (2000) results showed that dual inoculation of Vigna radiata plants with rock phosphate and AMF stimulated root colonization as compared to those without rock phosphate. The results (Table 30b) also showed that at control and low level of rock phosphate the internal hyphae and arbuscular infection were moderately frequent and scattered throughout the cortex which is actually the sites of nutrients exchange, (Powell & Bagyaraj 1984; Parniske, 2000). The external hyphae and arbuscules were not seen in any of the studied root segments at high RP level (RP3). High soil phosphate level has direct effect on reduction of hyphal growth and spore production.

162

Table30a: Effects of various levels of RP fertilizers on REC index in the roots of sunflower hybrids. RP levels NKS-278 Hysun-33 SMH-0917 SMH-0907

% age infection % age infection % age infection % age infection

RP0 100% 100% 100% 100%

RP1 72% 96% 91% 88%

RP2 66% 88% 84% 72%

RP3 32% 46% 42% 35%

Fig. 28: Effects of rock phosphate (RP) fertilizers on % infection of 4 hybrids of sunflower

163

Table30b: Effects of various levels of RP fertilizers on AM infection Morphologies, external hyphae, internal hyphae, Arbuscules, vesicles, (%) in roots of mycorrhizal Sunflower hybrids.

Percent AMF infection (%) Hybrids Treatments External Internal hyphae Vesicles Arbuscules Hyphae (%) (%) (%) (%) NKS-278 RP0 24±5.29 24.33±6.65 24±5.29 7.33±6.42 RP1 20±10 23.33±5.77 23.33±5.77 4±6.08 RP2 6.66±5.77 13.33±5.77 13.33±5.77 * RP3 * 3.33±5.77 6.66±11.54 *

Hysun-33 RP0 40.66±9.01 44±6.92 50.66±1.15 14±6.37 RP1 33.33±5.77 33.33±11.54 36.66±5.77 6.66±5.77 RP2 13.33±11.54 20±0 20±0 3.33±0 RP3 * 10±0 13.33±5.77 *

SMH- RP1 40±10 43.33±5.77 50±0 7.33±6.42 0917 RP0 23.33±5.77 23.33±5.77 23.33±5.77 3.33±6.35 RP2 10±10 16.66±5.77 20±0 * RP3 * 6.66±5.77 10±10 *

SMH- RP0 36.66±1.54 37.33±5.77 40±0 13.33±5.94 0907 RP1 20.66±10.06 24±6.92 24±5.29 4±6.92 RP2 3.33±5.77 10±0 10±0 * RP3 * 3.33±5.77 3.33±5.77 *

± standard error *(absent) RP0 (0% rock phosphate); RP1 (25% rockphosphate); RP2 (50% rock phosphate); RP3 (100% rockphosphate)

164

Plate 8

A B

C D

E F Plate A-F: AM infection in roots of sunflower hybrids. A: arbuscular infection; b-e: vesicular infection; f: internal hyaphe

165

CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

5.1 CONCLUSIONS

1. The present study strongly suggests that the AMF-rock phosphate combination produces better results in the enhancement of the productivity of sunflower hybrids even in P-deficient soils. 2. The results of the experimental work revealed that AMF is beneficial in promoting agronomic parameters (Plant height, root length, number of leaves/plant, leaf size, seed yield, head diameter, dry weight), oil yield, unsaturated fatty acid profile, proximate and nutrient composition of the hybrids. Moreover, spore density and root colonization of the studied plants were also increased over non-inoculated plants in nutrient deficient soil. 3. The use of biofertilizer is not only eco-friendly but also economical as it reduces our dependence on expensive chemical fertilizers. 4. AMF-rock phosphate combination plays an important role in bringing increase in the fertility of soil and availability of phosphorus to the plants. 5. The use of low–moderate doses of rock phosphate with AMF gave better results; while high levels of rock phosphate is antagonistic to AMF. 6. Dual use of AMF and low-moderate dose of rock phosphate has profound effect on different vegetative and reproductive parameters like plant height, root length, number of leaves/plant, leaf size, seed yield, head diameter, dry weight , seed yield, head diameter, dry weight, proximate and nutrient composition and oil content of the sunflower as compared to control. However the response varied in different hybrids. 7. This research has revealed that the use of AMF along with low doses of rock phosphate promote mono unsaturated (oleic acid) and polyunsaturated fatty acids (linoleic acid), while the use of AMF along with high doses of rock phosphate bring increase in the production of saturated fatty acids like Palmitic acid and stearic acid in sunflower seeds. 166

8. This research work has substantial health significance as unsaturated fatty acids are more beneficial for health as they are responsible for lowering “bad” LDL cholesterol (low density lipoproteins) and simultaneously increasing the "good", HDL cholesterol (high density lipoprotein). On contrary, the saturated fatty acids behave otherwise. 9. AMF inoculated plants save 25-50 percent of phosphorus fertilizer as compared to non-inoculated plants for production of same amount of total biomass and oil contents. AMF inoculation compensate for high dose of phosphorus fertilizer. 10. Out of four hybrids used in experiment Hysun-33 is more suitable to agronomical conditions of Peshawar climate due to its high oil yield.

167

5.2 Recommendations 1) The aim of this chapter is to introduce new techniques of the use of biofertilizer such as AMF to achieve adequate production level with least utilization of synthetic fertilizers for sustainable agriculture practice. 2) The role of AMF as a potential biofertilizer is well recognized but not well exploited due to the agronomic practices in vogue. This needs further exploitation so that this technology should become available to the farmers at local level at cheaper rates to overcome economic constraints. 3) Pakistan soil, being mostly phosphorus (P) deficient. The unbalanced and injudicious use of phosphorus fertilizers by the farmers causes upheaval and increase in phosphorus level of the soil which becomes one of the limiting factors for crop productivity. Proper P-fertilizer management is important to find out the optimum fertilizer rate for the crop under cultivation. Moreover, diversified farm management is required which is location specific test for finding out the potentials of the biofertilizer. 4) AMF inoculants strains and plants varieties should be selected in such a combination which is highly compatible and competitive for achievement of better yield. 5) Hybrids are genetically good but Farmers should select such hybrids which are suitable to the environment in which they are placed e.g Hysun-33 is more suitable to agronomical conditions of Peshawar climate. It is therefore recommended that the farmers should use these AMF inoculated hybrid along with low doses of rock phosphate for getting better yield of seeds rich in unsaturated oils which are more beneficial for health. 6) Keeping in view the importance of AMF for the increase of yield in sunflower, prudent monitoring of the soil is required for identification and isolation of some effective AM endophytes associated with oil seed crops. Moreover, its preservation in bulk is needed for future supply to the farmers. 7) The farmers should also be educated accordingly about the importance of AMF biotechnology through media for proper use of AMF strains along with recommended doses of rock phosphate, to avoid costly synthetic agrochemicals. 168

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Web 1: http://www.dawn.com/news/214932/phosphate-rock-deposits-remain-unexploited Web2: http://old.parc.gov.pk/1SubDivisions/NARCCSI/CSI/oil.html.

Web 3: http://www.grainsa.co.za/use-of-sunflower-oilcake-in-dairy-cattle-rations. Web 4: https://en.wikipedia.org/wiki/Peshawar web. 5: http://www.gmo-compass.org/eng/database/plants/68.sunflower.html web:6 https://en.wikipedia.org/wiki/Peshawar#Climate

226

APPENDICES Appendix1: Composition of hoagland's solution used in this experiment

Stock solutions:

1. Ca (NO3)2.4H2O 236.1 g/l

2. KNO3 101.1 g/l

4. MgSO4.7H2O 246.5 g/l 5. Trace elements (make up to 1 L)

H3BO3 2.8 g

MnCl2.4H2O 1.8 g

ZnSO4.7H2O 0.2 g

CuSO4.5H2O 0.1 g

NaMoO4 0.025 g 6. FeEDTA - 10.4 g EDTA.2Na

7.8 g FeSO4.7H2O 56.1 g KOH

Make up 1 L of KOH, adjust pH to ~5.5 using H2SO4. Then add EDTA.2Na and

FeSO4.7H2O. To make 1 L Hoagland's solution (-P) from these stocks, add

7 ml Ca(NO3)2 stock

5 ml KNO3

2 ml MgSO4 1 ml Trace elements 1 ml FeEDTA to 1 L water

227

Appendix 2: ANOVA for plant height of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 255.610 127.805 1.0467 0.3572 2 Hybrid(H) 3 10512.132 3504.044 28.6965 0.0000 4 Treat(T) 7 9838.499 1405.500 11.5104 0.0000 6 H*T 21 1956.106 93.148 0.7628 -7 Error 62 7570.638 122.107 ------Total 95 30132.985 ------

Coefficient of Variation: 11.61%

Appendix 3: ANOVA for root length of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 8.591 4.295 4.5367 0.0145 2 Hybrid(H) 3 57.141 19.047 20.1169 0.0000 4 Treat(T) 7 206.843 29.549 31.2089 0.0000 6 H*T 21 274.661 13.079 13.8138 0.0000 -7 Error 62 58.702 0.947 ------Total 95 605.938 ------Coefficient of Variation: 8.50%

228

Appendix 4: ANOVA for number of fresh leaves of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 1.646 0.823 0.1714 2 Hybrid(H) 3 395.792 131.931 27.4775 0.0000 4 Treat(T) 7 230.458 32.923 6.8569 0.0000 6 H*T 21 181.375 8.637 1.7988 0.0388 -7 Error 62 297.688 4.801 ------Total 95 1106.958 ------

Coefficient of Variation: 18.10%

Appendix 5: ANOVA for number of wilted leaves of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 4.938 2.469 0.8212 2 Hybrid(H) 3 135.115 45.038 14.9809 0.0000 4 Treat(T) 7 85.073 12.153 4.0425 0.0010 6 H*T 21 121.635 5.792 1.9266 0.0243 -7 Error 62 186.396 3.006 ------Total 95 533.156 ------

Coefficient of Variation: 27.07%

229

Appendix 6: ANOVA for leaf length of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.470 0.235 1.1991 0.3084 2 Hybrid(H) 3 0.414 0.138 0.7046 4 Treat(T) 7 5.527 0.790 4.0266 0.0011 6 H*T 21 4.845 0.231 1.1766 0.3025 -7 Error 62 12.156 0.196 ------Total 95 23.412 ------

Coefficient of Variation: 12.37%

Appendix 7: ANOVA for leaf width of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.069 0.035 0.2884 2 Hybrid(H) 3 0.491 0.164 1.3611 0.2630 4 Treat(T) 7 2.376 0.339 2.8217 0.0128 6 H*T 21 2.008 0.096 0.7950 -7 Error 62 7.457 0.120 ------Total 95 12.402 ------

Coefficient of Variation: 15.70%

230

Appendix 8: ANOVA for dry weight of plant of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 8.804 4.402 4.6540 0.0131 2 Hybrid(H) 3 6430.609 2143.536 2266.2531 0.0000 4 Treat(T) 7 10192.930 1456.133 1539.4959 0.0000 6 H*T 21 7623.188 363.009 383.7911 0.0000 -7 Error 62 58.643 0.946 ------Total 95 24314.174 ------

Coefficient of Variation: 0.98%

Appendix 9: ANOVA for head diamter of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 4.463 2.232 2.2721 0.1116 2 Hybrid(H) 3 29.721 9.907 10.0864 0.0000 4 Treat(T) 7 47.405 6.772 6.8948 0.0000 6 H*T 21 109.468 5.213 5.3072 0.0000 -7 Error 62 60.897 0.982 ------Total 95 251.953 ------

Coefficient of Variation: 18.55%

231

Appendix 10: ANOVA for number of seeds/head of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 54.333 27.167 1.0048 0.3720 2 Hybrid(H) 3 80165.531 26721.844 988.3203 0.0000 4 Treat.(T) 7 50927.073 7275.296 269.0804 0.0000 6 H*T 21 141662.219 6745.820 249.4974 0.0000 -7 Error 62 1676.333 27.038 ------Total 95 274485.490 ------Coefficient of Variation: 3.28%

Appendix 11: ANOVA for weight of seeds/head of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.544 0.272 0.2945 2 Hybrid(H) 3 194.172 64.724 70.0787 0.0000 4 Treat.(T) 7 143.537 20.505 22.2016 0.0000 6 H*T 21 171.182 8.152 8.8259 0.0000 -7 Error 62 57.263 0.924 ------Total 95 566.698 ------

Coefficient of Variation: 21.99%

232

Appendix 12: ANOVA for oil of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.763 0.382 0.3717 2 Hybrid(H) 3 166.013 55.338 53.8918 0.0000 4 Treat.(T) 7 238.851 34.122 33.2300 0.0000 6 H*T 21 296.360 14.112 13.7437 0.0000 -7 Error 62 63.663 1.027 ------Total 95 765.650 ------Coefficient of Variation: 3.08%

Appendix 13: ANOVA for linoleic acid of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.250 0.125 0.1216 2 Hybrid(H) 3 48.018 16.006 15.5666 0.0000 4 Treat.(T) 7 309.794 44.256 43.0414 0.0000 6 H*T 21 187.085 8.909 8.6642 0.0000 -7 Error 62 63.750 1.028 ------Total 95 608.897 ------

Coefficient of Variation: 1.40%

233

Appendix 14: ANOVA for oleic acid of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.438 0.219 0.1950 2 Hybrid(H) 3 8.875 2.958 2.6368 0.0575 4 Treat.(T) 7 32.789 4.684 4.1749 0.0008 6 H*T 21 69.252 3.298 2.9392 0.0005 -7 Error 62 69.563 1.122 ------Total 95 180.917 ------

Coefficient of Variation: 6.72%

Appendix 15: ANOVA for palmitic acid of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 2.215 1.108 1.1351 0.3280 2 Hybrid(H) 3 0.177 0.059 0.0604 4 Treat.(T) 7 11.015 1.574 1.6126 0.1486 6 H*T 21 25.148 1.198 1.2273 0.2617 -7 Error 62 60.498 0.976 ------Total 95 99.053 ------

Coefficient of Variation: 25.77%

234

Appendix 16: ANOVA for steric acid of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 1.273 0.637 0.6407 2 Hybrid(H) 3 5.344 1.781 1.7928 0.1578 4 Treat.(T) 7 28.958 4.137 4.1637 0.0008 6 H*T 21 50.554 2.407 2.4229 0.0037 -7 Error 62 61.600 0.994 ------Total 95 147.729 ------

Coefficient of Variation: 18.11%

Appendix 17: ANOVA for protein of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.813 0.406 0.3986 2 Hybrid(H) 3 64.713 21.571 21.1655 0.0000 4 Treat.(T) 7 80.527 11.504 11.2876 0.0000 6 H*T 21 106.685 5.080 4.9847 0.0000 -7 Error 62 63.188 1.019 ------Total 95 315.924 ------

Coefficient of Variation: 5.16%

235

Appendix 18: ANOVA for ash content of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 9.060 4.530 5.3669 0.0071 2 Hybrid(H) 3 2.788 0.929 1.1011 0.3555 4 Treat.(T) 7 80.118 11.445 13.5596 0.0000 6 H*T 21 50.839 2.421 2.8681 0.0007 -7 Error 62 52.333 0.844 ------Total 95 195.139 ------

Coefficient of Variation: 29.59%

Appendix19: ANOVA for moisture of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.438 0.219 0.2134 2 Hybrid(H) 3 2.080 0.693 0.6762 4 Treat.(T) 7 58.803 8.400 8.1939 0.0000 6 H*T 21 56.880 2.709 2.6420 0.0016 -7 Error 62 63.562 1.025 ------Total 95 181.763 ------

Coefficient of Variation: 17.36%

236

Appendix 20: ANOVA for fats of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 2356.937 1178.469 19.0023 0.0000 2 Hybrid(H) 3 25.733 8.578 0.1383 4 Treat.(T) 7 300.604 42.943 0.6924 6 H*T 21 152.969 7.284 0.1175 -7 Error 62 3845.062 62.017 ------Total 95 6681.306 ------

Coefficient of Variation: 22.47%

Appendix 21: ANOVA for fibers of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 6.812 3.406 0.8341 2 Hybrid(H) 3 68.118 22.706 5.5602 0.0019 4 Treat.(T) 7 94.334 13.476 3.3000 0.0048 6 H*T 21 75.080 3.575 0.8755 -7 Error 62 253.187 4.084 ------Total 95 497.532 ------

Coefficient of Variation: 20.96%

237

Appendix 22: ANOVA for carbohydrates of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 1564.937 782.469 11.4389 0.0001 2 Hybrid(H) 3 291.947 97.316 1.4227 0.2447 4 Treat.(T) 7 3527.516 503.931 7.3670 0.0000 6 H*T 21 1652.284 78.680 1.1502 0.3254 -7 Error 62 4241.063 68.404 ------Total 95 11277.748 ------

Coefficient of Variation: 29.96%

Appendix 23: ANOVA for phosphorus of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.008 0.004 0.3986 2 Hybrid(H) 3 0.025 0.008 0.8107 4 Treat.(T) 7 0.160 0.023 2.2471 0.0420 6 H*T 21 0.023 0.001 0.1078 -7 Error 62 0.632 0.010 ------Total 95 0.848 ------

Coefficient of Variation: 18.21%

238

Appendix 24: ANOVA for nitrogen of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.250 0.125 0.1216 2 Hybrid(H) 3 0.063 0.021 0.0205 4 Treat.(T) 7 0.189 0.027 0.0262 6 H*T 21 0.186 0.009 0.0086 -7 Error 62 63.750 1.028 ------Total 95 64.437 ------

Coefficient of Variation: 71.11%

Appendix 25: ANOVA for potassium of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 0.317 0.158 1.0107 0.3699 2 Hybrid(H) 3 0.347 0.116 0.7388 4 Treat.(T) 7 0.933 0.133 0.8513 6 H*T 21 2.808 0.134 0.8538 -7 Error 62 9.711 0.157 ------Total 95 14.116

Coefficient of Variation: 106.96%

239

Appendix 26: ANOVA for zinc of Helianthus annuas L. A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 3.729 1.865 1.2765 0.2862 2 Hybrid(H) 3 3.764 1.255 0.8589 4 Treat.(T) 7 9.649 1.378 0.9437 6 H*T 21 33.359 1.589 1.0875 0.3844 -7 Error 62 90.565 1.461 ------Total 95 141.066 ------

Coefficient of Variation: 305.25%