KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY, KUMASI, GHANA. COLLEGE OF AGRICULTURE AND NATURAL RESOURCES DEPARTMENT OF CROP AND SOIL SCIENCES ORGANIC AND INORGANIC FERTILIZERS APPLICATION ON THE GROWTH, YIELD AND ARTEMISININ CONTENT OF ARTEMISIA ANNUA L. IN THE HUMID TROPICS OF GHANA. STEPHEN YEBOAH JUNE, 2010 ORGANIC AND INORGANIC FERTILIZERS APPLICATION ON THE GROWTH, YIELD AND ARTEMISININ CONTENT OF ARTEMISIA ANNUA L. IN THE HUMID TROPICS OF GHANA. A THESIS SUBMITTED TO THE DEPARTMENT OF CROP AND SOIL SCIENCES, COLLEGE OF AGRICULTURE AND NATURAL RESOURCES, KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY, KUMASI, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN AGRONOMY (CROP PHYSIOLOGY). STEPHEN YEBOAH B.ED (HONS) AGRICULTURE JUNE, 2010 DECLARATION I hereby declare that the research work presented in this thesis is my own original research and that, to the best of my knowledge; this thesis has not been presented at any occasion by another person for the award of a degree. References to other people’s work have been duly acknowledged. Stephen Yeboah …………………… …………………… (Student) Signature Date I declare that I have supervised the student in undertaking the research submitted herein and confirm that the student has my permission to present it for assessment. Certified by: Prof. R. Akromah ……………………… …………………….. (Supervisor) Signature Date Prof. R. Akromah …………………… …………………….. (Head of Department) Signature Date i i ACKNOWLEDGEMENT I wish to express my sincere gratitude to the Almighty God for his strength and divine guidance throughout this programme. My first appreciation goes to my supervisor, Professor Richard Akromah, for giving me this wonderful opportunity to work on such a novel project. I am thankful for his invaluable suggestions, constructive criticisms, encouragement and generosity in my course of studies. I am indebted to Professor Charles Quansah without whom I would not have been able to learn as much as I have during my time here. I extend my heartfelt gratitude to Dr. Adu-Amankwaah and Mr. Ayittey of the Chemical Engineering Department for their support during the crude extraction. Words cannot express my appreciation to Dr. Joseph Sarkodie – Addo and Dr. Alfred Arthur, for their services during the data analyses. My greatest appreciation goes to my family: to my father who taught me the value of working hard and with honour; to my mother who taught me to never let go of a goal and to persevere in the face of adversity; and to my wife who supported me in all my endeavours. I am grateful to the staff of the Department of Crop and Soil Sciences for their pieces of advice during my period of study. Mr. Paul Abaidoo of the Department deserves special mention for the assistance in data collection. Finally, to all and sundry whose prayers and support have made this work a reality; I say may God bless you all. ii i DEDICATION To God as my provider. iv ABSTRACT A study was conducted at the Agriculture Research Station, Anwomaso during the 2008 major growing season to evaluate the effect of organic (poultry manure) and inorganic (chemical) fertilizers on the growth, yield and artemisinin content of Artemisia annua. The experiment also examined the effects of variation in the stage of harvest on the plant growth and yield. The experiment was arranged in a randomized complete block design with three replications and six treatments. The treatments were 0, 45 and 90 kg N/ha compound fertilizer and poultry manure at 2, 4 and 6 t/ha. During the study, data were collected on: plant height, plant canopy spread, stem width, internodes distance, number of branches per plant, fresh and dry leaf yield (kg/ha), fresh and dry shoot weight (g), crude extract weight (g), artemisinin content (%) and artemisinin yield (kg/ha). The results showed that 4 t/ha poultry manure treatment was effective and gave the highest fresh and dry leaf yield at both pre-flowering and full bloom harvests when compared to other treatments. Growth, development and yield response to organic and inorganic fertilization by Artemisia annua largely followed the trend 4 t/ha PM> 90 kg N/ha > 6 t/ha PM > 45 kg N/ha > 2 t/ha PM > 0 kg N/ha. The results indicated that application of the treatments significantly affected most of the parameters when compared to the control. There was significant differences (p<0.05) between the treatments for plant height, plant canopy spread, stem width, number of branches, fresh and dry leaf yield at pre-flowering, dry leaf yield, fresh and dry shoot weight, artemisinin content and artemisinin yield at full bloom stage. The results of the study also indicated that while the highest crude extract weight was obtained from 4 t/ha v poultry manure at pre-flowering, the highest crude extract weight was obtained from 90 kg N/ha compound fertilizer at full bloom. However, no significant differences (p>0.05) among the different fertilizer rates were observed. Application of 4 t/ha poultry manure gave the highest artemisinin yield of 9.57 kg/ha and 37.24 kg/ha at both pre-fowering and full bloom stages, respectively. The results also showed considerable increase in crude extract weight and artemisinin content at pre-flowering compared to values recorded at full bloom. The results showed that the artemisinin yield was positively correlated with leaf yield and stage of harvests. From the results of this study, it is recommended that application of 4 t/ha poultry manure can be used as an alternative to compound fertilizers which are expensive. This will reduce the cost of Artemisia annua production. v i TABLE OF CONTENTS CONTENT PAGE DECLARATION ............................................................................................................ ii ACKNOWLEDGEMENT ............................................................................................ iii DEDICATION ............................................................................................................... iv ABSTRACT .................................................................................................................... v TABLE OF CONTENTS ............................................................................................. vii LIST OF TABLES ......................................................................................................... xi LIST OF FIGURES ...................................................................................................... xii LIST OF PLATES ....................................................................................................... xiii CHAPTER ONE ............................................................................................................. 1 1.0 INTRODUCTION .............................................................................................. 1 CHAPTER TWO ............................................................................................................ 6 2.0 LITERATURE REVIEW ................................................................................... 6 2.1 ORIGIN AND BOTANY OF ARTEMISIA ANNUA .......................................... 6 2.1.1 Distribution and Geographical Location of Artemisia annua ....................... 7 2.1.2 Genotypes...................................................................................................... 8 2.2 CONSTRAINTS TO ARTEMISIA PRODUCTION ....................................... 10 2.2.1 Growth Requirements for Artemisia Production ........................................ 11 2.3 ARTEMISININ PRODUCTION ..................................................................... 13 2.3.1 Glandular Trichomes as Sites of Artemisinin Accumulation ..................... 16 2.3.2 Mechanism of Action of Artemisinin ......................................................... 18 2.4 METHODS OF EXTRACTION OF ARTEMISIA ANNUA ............................. 19 2.4.1 Extraction with Petroleum Ether/Hexane Ethyl Acetate Mixture ............... 20 2.4.2 Extraction of Artemisinin by Supercritical CO ......................................... 21 2 2.4.3 Extraction of Artemisinin Using Hydrofluorocarbon HFC-134a ............... 22 2.4.4 Extraction Using Organic Ionic Liquids ..................................................... 23 2.4.5 Extraction of Artemisinin Using Ethanol.................................................... 24 vi i 2.5 PURIFICATION OF ARTEMISININ ............................................................. 25 2.6 MODE OF HARVESTING OF ARTEMISIA ANNUA .................................... 26 2.7 BIOTECHNOLOGICAL PRODUCTION OF ARTEMISININ ...................... 28 2.8 RESPONSE OF ARTEMISIA ANNUA TO NITROGEN FERTILIZER .......... 29 2.9 RESPONSE OF ARTEMISIA ANNUA TO ORGANIC MANURE ................. 32 CHAPTER THREE ...................................................................................................... 36 3.0 MATERIALS AND METHODS ..................................................................... 36 3.1 EXPERIMENTAL SITE .................................................................................. 36 3.2 EXPERIMENTAL MATERIALS .................................................................... 36 3.3 SOIL STERILIZATION .................................................................................. 36 3.4 NURSING OF SEEDS AND SEEDLINGS ..................................................... 37 3.5 LAND PREPERATION AND FIELD LAYOUT ........................................... 37 3.5.1 Transplanting .............................................................................................. 37 3.6 INITIAL SOIL SAMPLING AND ANALYSIS .............................................. 38 3.6.1 Soil Physical Analyses ................................................................................ 38 3.6.1.1 Particle size analysis ............................................................................. 38 3.6.1.2 Soil bulk density (ℓb) ............................................................................ 39 3.6.2 Soil Chemical Analyses .............................................................................. 40 3.6.2.1 Soil pH .................................................................................................. 40 3.6.2.2 Soil organic carbon ............................................................................... 41 3.6.2.3 Total nitrogen ....................................................................................... 41 3.6.2.4 Available phosphorus ........................................................................... 42 3.6.3 Exchangeable Cations Determination ........................................................... 43 3.6.3.1 Extraction of the exchangeable bases ................................................... 44 3.6.3.2 Determination of calcium ..................................................................... 44 3.6.3.3 Determination of calcium and magnesium ........................................... 44 3.6.3.4 Determination of exchangeable potassium and sodium ....................... 45 3.7 TREATMENT APPLICATION ....................................................................... 46 3.8 CULTURAL PRACTICES .............................................................................. 46 3.8.1 Watering ...................................................................................................... 46 vi ii 3.8.2 Weeding ...................................................................................................... 47 3.9 PARAMETERS MEASURED ......................................................................... 47 3.10 DATA COLLECTION ..................................................................................... 48 3.11 HARVESTING FOR EXTRACTION ............................................................. 49 3.12 EXTRACTION ................................................................................................. 49 3.12.1 Purification .................................................................................................. 52 3.12.2 Detection of Artemisinin in Crude Extract by Liquid Chromatography-Mass Spectromatry ............................................................................................... 52 3.13 STATISTICAL ANALYSIS ............................................................................ 52 CHAPTER FOUR ........................................................................................................ 53 RESULTS AND DISCUSSION ................................................................................... 53 4.1 INITIAL SOIL PROPERTIES AND TOTAL NUTRIENT CONTENT OF POULTRY MANURE ..................................................................................... 53 4.1.1 Results ......................................................................................................... 53 4.1.1.1 Soil characteristics ................................................................................ 53 4.1.1.2 Nutrient content of poultry manure ...................................................... 53 4.1.2 DISCUSSION ................................................................................................... 55 4.2 GROWTH AND DEVELOPMENT OF ARTEMISIA ANNUA ....................... 56 4.2.1 Results ......................................................................................................... 56 4.2.1.1 Plant height (cm) .................................................................................. 56 4.2.1.2 Plant canopy spread (cm) ..................................................................... 56 4.2.1.3 Stem width (cm) ................................................................................... 57 4.2.1.4 Internodes distance (cm) ....................................................................... 58 4.2.1.5 Number of branches per plant .............................................................. 59 4.2.2 Discussion ................................................................................................... 60 4.3 YIELD OF ARTEMISIA ANNUA ..................................................................... 65 4.3.1 Results ......................................................................................................... 65 4.3.1.1 Fresh and dry leaf yield of Artemisia annua ........................................ 65 4.3.1.2 Fresh and dry shoot weight (g) ............................................................. 66 4.3.1.3 Crude Extract content of Artemisia annua (g) ..................................... 67 ix