NNAMDI AZIKIWE UNIVERSITY, AWK A

A TECHNICAL REPORT OF THE STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

Undertaken at:

INTERNATIONAL INSTITUTE OF TROPICAL AGRICULTURE (IITA), Ibadan. BY: DURU VINCENT CHIAGOZIE

REG NO: 2010584951

Submitted to:

THE DEPARTMENT OF PARASITOLOGY AND ENTOMOLOGY, FACULTY OF BIOSCIENCES.

IN PARTIAL FULFILMENT FOR THE AWARD OF Bachelor of Sciences (B.Sc.), Single Hons, DEGREE IN PARASITOLOGY AND ENTOMOLOGY

(APRIL- SEPTEMBER, 2013)

DEDICATION

This technical report is dedicated to God, the greatest scientist ( the omniscience) and the Blessed Virgin Mary, mother of God, the first to behold Gods experiment.

AND

To all lovers of research for development in the field of Agriculture.

ACKNOWLEDGEMENT

My profound gratitude goes first to God almighty, the author and finisher of my life, for His guidance and protection throughout my training days in Ibadan; to Mary, my mother and queen, for Her maternal assistance during my rainy days throughout my training days. May you be forever be glorified in my life and academic endeavours. I wish to also thank the SIWES directors and co-ordinators and all those who saw to the organisation of this program. Thank you all for this wonderful opportunity. My gratitude also goes to my benefactor and 2nd god in Ibadan, Dr. Ifeanyi A. Orjiakor, whom God used to protect and provide for me during my arrival in Ibadan and in my hazy moments. Indeed, nwanne di na mba. To my erudite scientist, the man whom I owe all these knowledge to, without whom my stay and even my training in the institute wouldnt have been possible; Dr. Stefan, Hauser, a senior scientist and a Root and Tuber systems Agronomist of high repute for his fatherly care and nuture and for giving me leave to appreciate the IITA environment and make something meaningful out of my stay in it and also for always giving me a listening ear each time without whinges. Dr. Stefan, I say a big THANK YOU. And to the host of my supervisors in the Agronomy unit;.Messrs. Adeboye Dada Johnson, Felix Friday, Joseph Olukunle, Kehinde Oyekanmi and Samuel Nsakpe and research fellows Enesi Rebecca and Benjamin Adetola, I say thank you for all your coaching and for haven given me the unmerited privilege to learn from you all throughout my stay with you. Indeed I would say I really enjoyed every bit of my stay with you all. My gratitude also goes to the research supervisor, IITA Entomology unit, Mrs. Aderanti, Rachael, and the host of the other IITA Entomology unit staff for their care and tutelage during my short stay with them. Mommy, I really appreciate your help and pray the good Lord to bless you. My unalloyed gratitude also goes to the Nematology madam, Mrs. Adewuyi Omowumi for her unalloyed care, tutelage, supervision and help she showed me during my stay with her. I pray the good Lord to also bless you for your kind heart and generosity. And to all Nematology unit staff who contributed in one way or the other to the success of my training in the unit both the research fellows, I say a very big THANK YOU to you all. My gratitude also goes to my benefactor, Dr. Duru, G.O.C, whom I would never forget in all my academic endeavours for his unalloyed support throughout my university education. Charity they say begins at home. To this I wish to acknowledge my family for haven been the pillar of support for me throughout my 6 months industrial training days. To my father, Sir Duru U.F, I say thanks Sir, to my ever-caring mother and my adviser in times of adversity, Duru, S.N, I say Daalu ezigbo nne m oma, to my brothers Chiegboka and Chinazo, I say thanks bro and to my Margaret thatcher sister, I say may God bless you for me.

Lastly, my gratitude goes to my friends on whom I fall back to for assistance when am morally down; Chukwuebuka Anuebunwa, for providing me with a conducive shelter; thanks bro na baba God go reward you sha; to Nnachor Emmanuel (chuks), Ekeoma chidi, Nnaedozie Okoli, Owie Nosah, Abisona Michael and to Nwazuruoke Jude (my choir co-ordinator, st. Cecilia students choir, Seat of Wisdom Catholic church, University of Ibadan) my pale pale I say thank you for helping me in touring IB city and in achieving something spiritually during my stay in Ibadan. Once more, thanks pals. To all whom I may have skipped tp acknowledge here, do know that you are always in my mind and my lips during prayers and I wish to say a big THANK YOU to you all.

CERTIFICATE OF SIWES TRAINING

TWIMC

SUMMARY OF WORK

This report, a four chaptered report, is aimed at explaining key areas of my training under the Student Industrial Work Experience Scheme undertaken at The INTERNATIONAL INSTITUTE OF TROPICAL AGRICULTURE (IITA), Ibadan, headquarters. It gives a brief introduction about IITA, an African-based, international research for development organization established with the aim of fostering agriculture, enhancing crop productivity and quality, reducing producer and

consumer risk and generating wealth from agriculture through her research works. This report also covers a detailed description of all the activities that were carried out during the period of the training and encapsulates each in a separate chapter. In one of the chapters, the Entomology unit, a detailed explanation of all the work undertaken at the unit during the training was given ranging from the laboratory diet preparation of different diet for different crop pests, their rearing in the adult rearing rooms/cages to the infestation of different agricultural plant cultivars with these pests either for screening purposes or for further research works. The report on Agronomy unit expanciated the various agronomic research trial works that were on-going as at the time of the training ranging from the nutrient omission trial experiments on root (cassava) and tuber (yam) crops, density trial experiments too with cassava and yam and the incorporation of maize intercropping into the research to the cultural practices in soil fertility management. The last chapter of the report which was on the Nematology unit expanciated the day-to-day activities and the different works done in the unit during the training days ranging from the nematode sampling, different extraction techniques for the nematode extraction, nematode identification and counting, nematode fishing and culturing for further research purposes to soil sterilization for nematode multiplication experiment. The work and activities enlisted in this master-piece was actually carried out by me and with my supervisors scrutiny and directives.

TABLE OF CONTENTS

Page Cover page Dedication Acknowledgement Certificate of training Summary of work Table of content i ii iii v vi vii

CHAPTER 1 INTRODUCTION 1.1 1.1.1 1.1.2 1.2 1.2.1 1.3 SIWES background and objectives SIWES background SIWES objectives Background of the establishment attached IITAs location and organization 2 1 1 2 1

List of some laboratory equipment used during the training and their usage 4

CHAPTER 2 ENTOMOLOGY UNIT 2.1 Introduction 9

2.2 Preparation of various diet compositions for insect rearing in the stem-borer laboratory for different stem-borers. 10 2.2.1 Composition and preparation of diet ingredients for the rearing of Eldana saccharina. 10 2.2.1.1 Lifecycle of Eldana saccharina. 2.2.1.2 Laboratory rearing of Eldana saccharina. 10 10 12

2.2.1.3 Composition of diet ingredients and preparation procedures for the laboratory rearing of Eldana saccharina. 2.2.1.3.1 Procedure for preparing the diet for Eldana saccharina. 12

2.2.2 Composition and preparation of diet ingredients for the rearing of Sesamia calamistis. 14 2.2.2.1 Lifecycle of Sesamia calamistis. 2.2.2.2 Laboratory rearing of Sesamia calamistis. 14 15

2.2.2.3 Composition of diet ingredients and preparation procedures for the rearing of Sesamia calamistis in the laboratory. 16 2.2.3 Composition and preparation of diet ingredients for the rearing of Maruca vitrata. 16 2.2.3.1 Lifecycle of Maruca vitrata. 2.2.3.2 Laboratory rearing of Maruca vitrata. 16 17

2.2.3.3 composition of diet ingredients and preparation procedure for the laboratory rearing of Maruca vitrata. 17 2.2.3.3.1 Procedure for preparing the diet for the laboratory rearing of Maruca vitrata.18 2.2.3.4 Preparation of an alternative diet for the laboratory rearing of Maruca vitrata.18 2.3 Field infestation of various cultivars with insect pests larvae. 19

CHAPTER 3 AGRONOMY UNIT 3.1 3.2 3.2.1 Introduction Omission trial experiments 22 21 21

Cassava omission trial experiments

3.2.1.1 field measurement for planting of cassava in cassava omission trial experiment.22 3.2.1.2 fertilizer application 3.2.1.3 field assessment of the omission trial experiment 3.2.1.4 harvesting and sample collection 3.2.2 Yam omission trial experiment 23 23 24 25

3.2.2.1 field measurement for planting of yam in yam omission trial experiment.25 3.2.2.2 yam seedling treatment for planting 3.2.2.3fertilizer application 3.2.2.4 field assessment and yam staking and vine trailing 3.3 3.3.1 Density trial experiments Cassava density trial experiment 27 27 28 29 26 26 26

3.3.1.1 cassava varieties used for the experiment 3.3.1.2 the split plot and split-split plot experimental designs 3.3.1.3 field measurement for cassava planting in cassava density trial experiment31

3.3.1.4 fertilizer application in the cassava density trial experiment 3.3.1.5 field evaluation and disease-check in the cassava density trial experiment.32 3.3.1.6 cassava-maize intercropping system 3.3.1.7 cassava-maize intercrop in the cassava density trial experiment 3.3.1.8 maize harvesting in a density trial experiment 3.3.1.9 maize post-harvest processes in the density trial experiment 3.3.2 Yam density trial experiment 35

32

33 34 34 35

3.3.2.1 field measurement for yam planting in yam density trial experiment.35 3.3.2.2 fertilizer application in the yam density trial experiment field 36

3.3.2.3 protocol for yam growth assessment in yam density trial experiment37 3.3.2.4 yam-maize intercrop in the yam density trial experiment 3.3.2.5 maize harvesting in a yam density trial experiment 3.3.2.6 maize post-harvest processes in the density trial experiment 39 3.4 3.4.1 Cultural practices in soil fertility management 40 38 39

Agronomic practices in the use of Mucuna in soil fertility management.40

3.4.2 Agronomic practices in the use of bush fallowing technique in soil fertility management 40 CHAPTER 4 NEMATOLOGY UNIT 4.1 4.2 4.2.1 4.2.2 4.3 4.3.1 Introduction Nematode sampling Soil sample collection for nematode sampling root sample collection for nematode sampling nematode extraction pie-pan/extraction tray method 45 46 46 46 47 48 48 49 41 42 42 44

4.3.1.1 extraction tray method for soil samples 4.3.1.2 extraction tray method for root samples 4.3.2 sieving method

4.3.2.1 sieving method for soil sample extraction of nematodes 4.3.2.2 sieving method for extracting sedentary cyst nematodes 4.3.3 maceration (blending) method

4.3.4 4.4 4.5 4.6 4.7

sodium hypochlorite method (hussy and barker technique) nematode identification and counting nematode fishing nematode culturing soil sterilization

50 51 53 54 55 56

PROBLEMS ENCOUNTERED AND OPINED SOLUTION

CONCLUSION AND GENERAL APPRAISAL OF THE PROGRAM

57

REFERENCES

58

APPENDIX

60

CHAPTER 1.

INTRODUCTION

1. SIWES BACKGROUND AND OBJECTIVES In the earlier stage of science and technology education in Nigeria, students were graduating from their respective institutions without any technical knowledge or working experience. It was in view of this that students undergoing science and technology related courses were mandated, for students in different institution in view of widening their horizons so as to enable them have the technical knowledge and working experience before graduating from their various institutions. It is in this vein that the Students Industrial Work Experience Scheme (SIWES) was initiated.

1. SIWES BACKGROUND The Students Industrial Work Experience Scheme (SIWES) was established by the Industrial Training Fund (ITF) in 1973 to enable students of tertiary institution, especially those in Engineering, Technology, and Sciences of tertiary institutions (universities, polytechnics, monotechnics and colleges of education) have technical knowledge of industrial work based on their course of study before the completion of their program in their respective institutions so as to smoothen their entry into industrial practices on completion of their studies and also reduce periods spent in training fresh graduates as new employees. As a result of increasing number of students enrolment in higher institutions of learning, the administration of this function of funding the scheme became enormous, hence, ITF withdrew from the scheme in 1978 and the scheme was taken over by the Federal Government and handed over to both the National Universities Commission (NUC) and the National Board for Technical Education (NBTE). By 1979, the colleges of education were not part of the scheme and later in 1984; the Federal Government reverted back to the ITF which took over the scheme officially in 1985 with funding provided by the Federal Government. 1.1.2 SIWES OBJECTIVES SIWES is strategized for skill acquisition. The major benefits accruing to students who participate conscientiously in the scheme are the skills and competences they acquire. The major reason behind the embarkment of students in SIWES was to expose them to the industrial environment and enable them develop occupational competences so that they can readily contribute their quota to national economic development and technological advancement after graduation. The specific objectives of the scheme (SIWES) as outlined in the Industrial Training Funds policy document no.1 of 1973 are as follows: To provide placements in industries for students of higher institutions of learning approved by relevant authorities (NUC, NBTE, NCCE) to acquire experience and skills relevant to their course of study. Prepare students for the real work situation they will meet after graduation. Expose students to work methods and technics in the handling of equipment and machinery that may not be available in school. Make transition from school to the labor market smooth and enhance student contact for later job placement. Provide students with the opportunity to apply their knowledge in real life work situation thereby bridging the gap between theory and practice. Strengthen employer involvement in the entire educational process and prepare students for employment in industry. Promote the desired technological know-how required for the advancement of the nation.

(Agwuna, 2012)

1.2

BACKGROUND OF THE ESTABLISHMENT

The International Institute of Tropical Agriculture, IITA, is an Africa-based, international research for development and non-profit organization established in 1967 with its headquarters located in Ibadan, Nigeria and several research stations across Africa. IITA is one of the worlds leading research partners in enhancing crop quality and productivity, reducing producer and consumer risks and generating wealth from agriculture, with the ultimate goals of reducing hunger, malnutrition and poverty. IITA is governed by an international board of trustees and is staffed by renowned scientists and other professionals from over 30 countries across the globe. IITAs mission is to enhance the food security, income and well-being of resource-poor people in sub-Saharan Africa by conducting research and related activities to increase agricultural production, improve food systems, and sustainably manage natural resources, in partnership with national and international stakeholders. Its research agenda addresses postharvest systems on the following food crops: Cassava, Cowpea, Maize, Plantain and Bananas, Soybeans and Yam; their improvement, health and resource. IITA is supported primarily by the Consultative Group for International Agricultural Research (CGIAR). It became the first African link in the worldwide network of agricultural research centres supported by the CGIAR.

1.2.1 IITAS LOCATION AND ORGANIZATION. IITA, Ibadan station and headquarters, was where I was opportune to undergo my SIWES training. It is located along old oyo road, Idi-ose, Akinyele Local Government Area, Ibadan, Oyo state, Nigeria. For efficacy and specialization, the institute is divided into several units, each headed by an erudite scientist who oversees the affairs of what goes on in the unit. There are several units based on the major research crops undertaken by the the institute viz: Cassava Breeding Unit, Yam Breeding Unit, Banana/Plantain Breeding Unit, Cowpea Unit, Maize Breeding Unit, Soybean Breeding Unit; others include those based on crop protection and improvement: Pathology Unit, Virology Unit, Entomology Unit, Nematology/Striga Unit, Soil Microbiology Unit etc. Others include Agronomy Unit, Reseach Farm Unit, Crop Transformation/Utilization Unit, Genetic Resources Center, Communication/Publishing Unit, Bio-sciences Unit, Analytical Services Unit etc. Also, there are other autonomous research centres within the institute such as The AfricaRice Centre, The International Centre for Insect Physiology and Ecology (ICIPE), HarvestPlus, International Livestock Research Institute (ILRI) which are situated within the institute and form part of the institutes research partners in achieving their vision- becoming Africas leading research partner in finding solutions for hunger and poverty.

FIG. 1. I

IITAS ORGANISATIONAL STRUCTURE.

1. LIST OF SOME LABORATORY EQUIPMENT USED DURING THE TRAINING AND THEIR USAGE.

1. Compound microscope:

used for viewing micro organisms that cannot be seen with the naked eyes. 2. Stereo microscope:

U sed for nematode egg counting and nematode fishing.

3. Microscope counting slide:

Used for nematode counting on the microscope and for egg quantification. 4. Forceps:

Used as an extension of the hand for holding lab media so as not to contaminate the media. It is also used for nematode egg fishing. 5. Petri dishes:

they are used for culturing nematodes and for holding fished nematodes or their eggs.

6. Kester cylinder:

used for arranging same media into the incubator and also for arranging equipment to be sterilized into the autoclave for sterilization. 7. Incubator:

Used to maintain the progressive development of cultured organisms by regulating viable growth factors e.g. temperature. 8. Micro flow laminar work station:

This is a chamber that is used to control particulate contamination such as dust which could affect the reliability of the work being done in the lab. 9. Carmel-hair brush:

Used for picking and infesting plants with larvae of insect pests. 10. Diet plates:

Used for preparing insect diets for their laboratory rearing.

11. Oviposition sticks:

this is placed inside a pests cage an on the pouches on it, female insect pests lay their eggs. 12. Spad :

This is used to measure the chlorophyll content in leaves of plants. 13. Area meter:

This is used for measuring the surface area of a leaf. 14. Measuring tape:

This is used for field measurement and markings. 15. Measuring cylinders:

For measuring accurately the volume of the laboratory solution/water intended to be used.

16. Beakers:

Used for holding liquid extracts and also for measuring out a particular sample volume. 17. Electric blender:

used for sample maceration in nematode extraction and for diet maceration in insect diet preparation.

18. Electric weighing balance:

used for standard weighing of samples before further activities is carried out.

19. Extraction paper:

used for nematode extraction. 20. Sieves:

Used for nematode extraction.

21. Electric mixer:

Used for mixing of the diet ingredients together. 22. Electric soil sterilizer:

sed for soil sterilization. 23. Soil auger:

Used for soil sample collection in the field.

CHAPTER 2

ENTOMOLOGY UNIT

2.1

INTRODUCTION

The crop protection and improvement division is one of the major research areas where IITA helps in bettering the lives of Africans. The entomology unit, which is one of the units under the division, takes care of the insect pests of the different crops undertaken by the institute. The unit services the institute in areas pertaining to insect pests of the major research crops undertaken by the institute and carry out researches on how their infestation can be reduced to an insignificant percentage. The unit also serves other units by providing them with certain insect pests which they ( the Entomology unit) rear in their insectaria up to the number required by the unit demanding the pests for their own researches. They also help other people from outside the institute who may have need of running a research in the institute under their unit. During my stay in the institute, I was opportuned to visit and work in this unit, and I really had nice experience during my stay there as a trainee. There, I was opportune to witness some of their research works and took part in most of them. I was also able to see some of the things I was taught in school and was able to adapt to the practical aspect at the institute without much stress. Also during my stay there, we were much involved in rearing various insects for further research works. I was much interested in rearing insect pests of maize, cassava and yam as these were the major crops my scientist, Dr. Stefan, Hauser, was experimenting on as at the time of my training in the institute. We were much involved in the pests of maize as we were also trying to screen some maize varieties for their resistivity or susceptibility to some maize stem-borers (a

PhD research work work of a research-fellow). Though there were many maize stem borers, our interest was focused on three major insect pests whose destruction has been on the high side in some African maize-producing countries: Eldana saccharina, Sesamia calamistis and Maruca vitrata. We, during the course of the research and hence too, my training , were able to rear them, culture them in their different diets prepared for each by us, inoculate them in the diets and finally infest our maize field with the pests as the research entailed. Some of this work, suffice it to say, was done by me alone or as part of a group and under my supervisors scrutiny and guidance.

2.2 PREPARATION OF VARIOUS DIET COMPOSITIONS FOR INSECT REARING IN THE STEM-BORER LABORATORY FOR DIFFERENT STEM-BORERS.

2.2.1 COMPOSITION AND PREPARATION OF DIET INGREDIENTS FOR THE REARING OF Eldana saccharina.

2.2.1.1 LIFECYCLE OF THE STEM BORER.

Eldana saccharina is an indigenous stem-borer in Africa and is widely distributed in the Sub-Saharan Africa. It attacks crops such as sugarcane, maize, sorghum and rice. The Lepidoptera belongs to the family Pyralidae and genus Eldana (Myers, P. Et.al, 2013). It attacks maize plants late in their development when it can affect grain filling which results in yield loss. It is often the most abundant borer species at the end of the maize growing season. The adult female E. saccharina begins laying eggs around flowering time of the maize plants. The eggs of the stem borer are yellow and oval and are laid in batches and in maize usually on dry dead maize leaves. For the laboratory rearing, eggs are laid on an oviposition

stick set inside the cage for the purpose. The eggs become pink just before emergence. Up to 300 eggs on the average are laid per female. The eggs hatch within five to six days into a light-brown to dark-grey coloured larvae which possesses a circular arranged crochets (Hooks) on the proleg, pinacule and tubercles along the body(Myers, P. Et.al, 2013). The larval stage is the pests destructive stage and it takes 21-35 days. Pupation occurs inside the stem or inside the tissue paper used in covering the diet( for the lab. rearing) and the pupa is covered by a co coon made of silk and plant debris. Adult E. saccharina emerges in 7-14 days and starts the cycle again.

Adult Egg mass

5-6days 7-14 days

Pupa

21-35 days

larva

FIG.2.1

LIFE CYCLE OF Eldana saccharina

2.2.1.2

LABORATORY REARING OF Eldana saccharina

Due to the destructive nature of the stem-borer, it is being reared in the laboratory for various research works on the various host plants of the pest. The pest is being reared by the Entomology unit of IITA for both the maize unit and the AfricaRice Centre as well as for individual researchers who may need the pest for one research work or another. Materials needed for the laboratory rearing include: A cage (for the adult) An infestation room (where the eggs and the larvae are infested in a diet) A microflow laminar work station Forceps Pins Oviposition sticks Scissors The eggs are collected by removing them from the oviposition stick where the female laid them into a tissue paper using a forceps. The eggs are further taken to the infestation room which is kept at a temperature of 22-25c and a relative humidity of 9%. The eggs are adhered to a pin and inserted into a diet where they stay and hatch into larval stage. The larvae on hatching, feeds on the diet and bores into it. After the estimated larval stage, (usually 21-35 days), the larvae metamorphoses into the non-motile pupa. At this stage, the pupa is removed from the diet and put into tissue papers and then taken to the adult room where there are cages for the adult pest. On emergence, the adult flies about inside the cage, mates, and starts the cycle again.

FIG 2.2 Egg removal from The oviposition stick diet

FIG 2.3 oviposition sticks

FIG 2.4 Eggs adhered to A pinhead inside the

FIG 2.5 The infested diets FIG. 2.5 Pupa emerging into adult in the arranged on the rack inside cage

The infestation room.

2.2.1.3 COMPOSITION OF DIET INGREDIENTS AND PREPARATION PROCEDURES FOR THE REARING OF Eldana saccharina

COMPONENTS FRACTION A Water for boiling Soybean flour Wheat germ Salt mixture Sugar FRACTION B Water for boiling Agar FRACTIONC Ascorbic acid Aureomicin (14.1%) appendix A Methyl-parahydrobenzoate Sorbic acid Streptomycin FRACTION D Potassium hydroxide (KOH) appendix A Propionic/phosphorus acids Choline chloride (15%) Acetic acid (25%) Formaldehyde (10%) Vitamin suspension appendix A appendix A appendix A appendix A appendix A

AMOUNT IN 16LITRES DIET.

7200ml 1024g 456g 152g 184g

7200ml 150g

100g 15.6g 26g 16g 2g

88ml 92ml 104ml 200ml 16ml 104ml

2.2.1.3.1

PROCEDURE FOR PREPARING THE DIET FOR Eldana saccharina

Materials other than the aforementioned components used for the preparation include: Two big pots Two stoves (or any other heat source) An electric blender A fork An electric weighing balance Ladle An electric mixer A measuring cylinder (1000ml) Diet plates Microflow laminar work station Using a 1000ml calibrated measuring cylinder, measure out 7200ml of water and pour into one of the big pots and place on fire. Measure out another 7200ml of water and pour into the second pot and also place it on fire. Into the second pot (i.e. pot B), weigh out 150g of Agar and pour inside, cover the pot and leave it to boil. Into the first pot, measure out all the components of Fraction A according to their stipulated measurements as stated above and pour into the pot (i.e. pot A ) also allow it to boil for about 30 minutes. Add pot B contents into pot A after 30 mins of boiling and put the pot A inside cold water and allow to cool to 60c. As the mixture is cooling off, quickly weigh out the components of Fraction C and put in a blender. Pour the already cooled concoction inside the electric mixer but not all the contents of the pot. Pour the remaining portion of the concoction left behind inside the pot ( mainly the congealed residues at the bottom of the pot) into the blender containing fraction C components and blend together. Pour the blended mixture into the ones in the electric mixer and on the machine. Measure out the components of Fraction D and pour one after the other into the mixer for optimal mixing. After about 5 minutes from the time the last component of Fraction D was added into the mixer, stop the mixer and bring down the mixing bowl and set it on the trolley and then move it nearer to the microflow laminar work station where the diet plates has been washed and arranged in readiness for the diet. Use a 500ml cup to dish out the diet and pour into each plate (usually 22 plates on the whole). Leave the diet in the plates on the microflow laminar work station to cool very well and congeal then, use a fork to draw lines (usually little perforations) in the diet (this line helps in creating holes for the newly hatched larvae to easily bore into the diet on hatching from the egg) after which, the diet is ready for infestation with eggs or 2nd instar larvae.

FIG. 2.7 measuring out of Water for the boiling.

FIG 2.8 pot A & B on fire

FIG.2.10 mixing of pot A+ B. left-over concoction.

FIG.2. 11 remnants of pot A*B Being poured into the blender.

FIG2.12 blending of Fraction C+ Of the

FIG.2.13 electric mixer Mixing the concoctions workstation.

FIG.2.14 dishing of the diet

FIG.2.15 The diets awaiting infestation on the

into the plates on the Laminar workstation.

2.2.2 COMPOSITION AND PREPARATION OF DIET INGREDIENTS FOR THE REARING OF Sesamia calamistis.

2.2.2.1 LIFE CYCLE OF Sesamia calamistis This stem-borer species is found to be among the most common stalk borer of many cereal crops in West Africa. Africa pink stalk-borer, as it is also called, affects mostly cereals like rice, maize, sorghum, millet etc. the Lepidoptera belongs to the family noctuidae and genus Sesamia (Myers, P. et.al, 2013). The adult female lays the egg between the lower leaf sheaths and the stem in batches of 40 and arranged in two to four contiguous rows. On average, each female lays around 300 eggs in a period of five days (Akol, A.M et.al ). Egg laying occurs from the time the plants are two weeks old until flowering. The eggs are hemispherical, about 1.5mm in diameter. They are somewhat whitish when laid but darken as they develop. About 8 days from the day the eggs are laid, the larvae, which have variable color but are usually creamy white with a distinctive pink suffusion a brown head and a yellowish-brown dorsal abdominal segment (usually the last segment), emerges. There are 6 larval instars and the larval period lasts for about 22 days. The larva (precisely L2) is the destructive stage of the pest. They penetrate the stem shortly after they emerge which might result in dead hearts or stalk breakage. During the ear filling period, the majority of the larvae occur in the ears. Most larvae pupate within the stem or cobs (Akol, A.M et.al). There is only one pupa per stem (E.A Heinrichs). The pupal period lasts from 10 to 12 days before a new pink stalk-borer emerges and the cycle continues.

FIG.2.16 Life cycle of Sesamia calamistis

2.2.2.2 LABORATORY REARING OF Sesamia calamistis. Due to the destructive nature of the stalk-borer, it is being reared in the laboratory for various research works it would be used to undertake. The pest is being reared in IITA by the InsectRearing unit (Entomology unit) for such purposes. Materials needed for the laboratory rearing include: A cage (for the adult) An infestation room (where the eggs and the larvae are infested in a diet) A microflow laminar work station Forceps vials Oviposition sticks Scissors

The eggs are collected by removing them from the oviposition stick where the female inserted them and putting them in a tissue paper using forceps. The eggs are further sterilized in 10% formaldehyde for about 20 minutes and then put in the microflow laminar work station for 10 minutes before they are infested in their diet in the infestation room.the eggs are separated into 60 eggs per diet and put inside vials, ready for infestation. The vials are placed inside the diet, mouth open and slanty so that the emerging larvae can easily crawl down and out of it into the diet. The larvae on hatching feeds on the diet. The larvae is being transferred to new diets as they molt from one stage to another. After the estimated larval period (22 days), the larvae metamorphose into the non-motile pupa and can be seen attached to the tissue papers used in covering the diet. At this stage, they are being removed from the diet and taken to the adult room where there are cages for the adult pest. On emergence, the adult flies about inside the cage, mates and starts a new cycle.

2.2.2.3 composition of diet ingredients and preparation procedures for the rearing of Sesamia calamistis in the laboratory. The composition of diet ingredients and preparation procedures for the rearing of Sesamia calamistis in the laboratory is the same as that of Eldana saccharina save that streptomycin, one of the components of Fraction C is not added in S. calamistis diet.

2.2.3 COMPOSITION AND PREPARATION OF DIET INGREDIENTS FOR THE REARING OF Maruca vitrata. 2.2.3.1 LIFE CYCLE OF Maruca vitrata The legume stem-borer, M. vitrata (Lepidoptera: crambidae) is a serious pest of grain legumes in the tropics and subtropics. M. vitrata female normally lay eggs on floral buds and flowers, although oviposition on leaves, leaf axils, terminal shoots and pods has also been recorded (Taylor, 1963). A female may lay up to 400 eggs in batches of 2 to 16. The eggs are light yellow, translucent and have faint reticulate sculpturing on the delicate chorion. The eggs hatch in about 5 days into a 17-20mm larvae. The larvae pass through 5 instars. The young larvae of M. vitrata (1st,2nd and 3rd instars) especially injure the terminal shoots and the flower buds whereas the old larvae (4th and 5th instars) particularly damage the open flowers and the pods. Pupation takes place in a silken cocoon amongst webbed leaves/pods or in soil after about 8-14 days of the larval development and lasts for about 5-10 days before the emergence of the adult insect. The life cycle is completed in 18-35 days.

adult

Egg mass

18-35 days

5-10days

8-14 days Pupa Larva

FIG. 2.17 Life cycle of Maruca vitrata

2.2.3.2 LABORATORY REARING OF M. vitrata

M. vitrata, like other pests of crops, is being reared in the laboratory for further research works it would be used to undergo. Materials needed for the lab. rearing include:

A cage (for the adult) An infestation room (where the eggs and the larvae are infested in a diet) A microflow laminar work station Forceps vials Oviposition sticks The eggs are collected by removing them from the oviposition stick where the female laid them and putting them in a tissue paper using forceps. The eggs are further taken to the maruca infestation/larvae holding room which is kept at a temperature range of 20-24c. the eggs are separated into 45 eggs per diet and put inside vials, ready for infestation. The vials are placed inside the diet, mouth open and slant so that the emerging larvae can easily crawl down and out of the vial into the diet. The larvae on hatching, feeds on the diet. The larvae are being transferred to new diets as they molt from one stage to another. After the estimated larval period (8-14 days), the larvae metamorphose into the pupal stage and could be seen attached to the tissue paper used in covering the diet container. At this stage, they are removed from the diet and taken to the adult room where they are placed in cages for the adult pests. On emergence, the adult flies about in the cage, mates and starts the cycle again.

2.2.3.3 COMPOSITION OF DIET INGREDIENTS AND PREPARATION PROCEDURES FOR THE LABORATORY REARING OF Maruca vitrata

COMPONENTS FRACTION A Cowpea flour Maize flour Wesson salt mix Ascorbic acid Chloramphenicol/Aureomycin Sugar Methyl-parahydrobenzoate

AMOUNTS IN 4 LITRES DIET

400g 127.2g 44.4g 25g 3.9g 60g 3.6g

Sorbic acid Water for blending FRACTION B KOH (4m) Choline chloride (15%) Acetic acid (25%) Formaldehyde (10%) Vitamin suspension FRACTION C Water for boiling agar Agar (appendix A)

6.8g 2000ml

22ml 29.6ml 50ml 26ml 30ml

2000ml 59.2g

2.2.3.3.1

PROCEDURE FOR PREPARING THE DIET FOR Maruca vitrata

Materials other than the aforementioned components used for the diet preparation include: 1 medium-sized pot 1 heat source (hot plate) Electric blender Fork Weighing balance Microflow laminar station Laddle Measuring cylinder (1000ml) Diet plates

Using a 1000ml calibrated measuring cylinder, measure out 2000ml of water and pour into the pot and place on fire. Measure out the specified quantity of agar and pour into the pot on fire and leave to boil. Measure out all the components of Fraction A according to their stipulated measurements and pour inside the blender, also add 2000ml of water for blending and blend for 3 minutes. Measure out also components of Fraction B and pour into the blender too and blend for another 3 mins. and then set aside the blender. Wait for the Fraction C on fire to boil then bring it down and cool it down to 60c. Pour the agar into the mix in the blender (i.e. mixture of Frac. A + Frac. B blended together) and blend the whole ingredients together for 5 minutes and then set aside. Wash the diet plates and place in the microflow laminar work station to dry. Dish the diet using a 500ml cup into 12 diet plates on the microflow laminar work station and then leave to solidify. Use the fork to make lines on the diet.

2.2.3.4 PREPARATION OF AN ALTERNATIVE (LESS COSTLY) DIET MEDIUM FOR REARING Maruca vitrata IN THE LABORATORY. Asides using the aforementioned components in the preparation of a media for rearing Maruca vitrata for further research work, a less costly, money conserving media was fashioned out by the unit and so far, its been efficient. The procedure for the preparation of the diet is easy and less material consuming. It is prepared thus: Pick cowpea (Vigna unguiculata) that is susceptible to Maruca vitrata, about 10 cups. Put in a bowl and wash very well. After washing, take out all the bad ones that floats on the water and throw them away. Add 45% sodium hypochloride (NaOCl) i.e. JIK and leave for 10 minutes. This is to sterilize the cowpea. After 10 mins, wash away the NaOCl with clean tap water twice and

then add another clean tap water. Add 2g of any fungicide of your choice into it. This is to avoid fungal contamination of the diet. Leave for up to 8 hrs and then drain the water out of the cowpea. Then leave the cowpea inside the bowl to germinate under room temperature (it takes at least 2 to 3 days under the lab. Room temp.) Once the cowpea has germinated enough as to contain the number of the pests to be added, infest the medium with M. vitrata larvae in their 1st instar stage although eggs can also be used for infestation. Place the medium on a shelf in the Maruca rearing room under a temperature range of 20-24c. A routine check of the media should be observed at least once in a week. During this check, the tissue paper at the base of the container (upon which the cowpea was placed) should be changed and the hatched, more matured larvae should be transferred to new media to avoid over-crowding and competition hence which may result in losses. When the larvae starts pupating on the tissue papers, take them to the Maruca cage where the adults are kept to await the emergence of new adults.

FIG 2.18 picture of the alternative maruca Cowpea diet 2.3 INFESTATION OF MAIZE CULTIVARS WITH INSECT PEST LARVAE

AIM: To screen two different maize cultivars, S1301 and S1303 for their resistivity or susceptibility to S. calamistis attack. MATERIALS USED: a diet plate/ Petri dish, an infestation Carmel-hair brush PROTOCOL: two maize cultivars were planted on a field (WBII) for screening. The field was mapped into four blocks (22m9m) with a 2m pathway surrounding and separating one block from another. Each block was further divided into 6 plots and the two maize varieties were planted randomly in separate plots on a block. Hence there were 2 treatments (the two maize cultivars), 6 plots with randomized treatment and four replicates (4 blocks). In each block, 6 rows of each maize cultivar and six columns were planted. Of the six columns, 2 columns at the both sides of the plot were side boarders whereas the two rows at the front and back of the plots served as the front and back boarders respectively hence leaving only 4 rows and 4 columns (i.e. 16 plants per plot) as the main plants. The pest, S. calamistis, in its 1st larva instars was used as the inoculants and was inoculated into the 21-days old maize cultivars. To each maize plant in each block and subsequently in each plot, 3 larvae inoculants were inoculated in-between the newly sprout leaves of the maize plant using the infestation brush to pick the larvae. Care was taken to ensure that the pests larvae were rightly inoculated into the hub of the leaves to ensure efficacy in the experiment. The maize cultivars were fertilized with urea fertilizer (150g per plot) and watered using over-

head irrigation system. FIG 2.19 field maize cultivar infestation on-going

FIG 2.20 the maize infested field. CHAPTER 3

AGRONOMY UNIT

3.1

INTRODUCTION

Agronomy, according to the Merriam-Webster Unabridged Dictionary, is a branch of agriculture dealing with field crop production and soil management. It is a science that deals with the methods used by farmers to raise crops and care for the soil including irrigation and the use of herbicides, pesticides and fertilizers. IITA being an agriculture-oriented institute has the Agronomy unit as one of its bedrocks in achieving its speculated goals. The agronomy unit of the IITA undertakes research works pertaining to the field production of the mandate crops of the institute (especially roots and tuber crops) and soil management especially in the agronomic management practices involving the use of fertilizers in boosting crop yields and land conservation to obtain maximum yield. During my stay in the unit, I was opportuned to work in the field where various research works were on-going ranging from the omission trial, density trials to cultural practices in soil fertility management. During the course of my training in the unit, I took part in various agronomic operations, experimental evaluations, data collection, etc. I also was able to learn to use and operate some of the machines used during the course of the research works. These include the Area meter, the Electric weighing balance, spring balances and nitrogen extractors amongst others.

3.2

OMISSION TRIAL EXPERIMENTS

The omission trial experiment was one of the research works undertaken by the Agronomy unit during my stay there. The experiment focused on the nutrient (fertilizer) omission to ascertain the varied effect each particular nutrient had on the yield of the crops and what the reaction would be on the yield if the nutrient was omitted. The experiment involved the use of seven (7) different nutrients viz: Nitrogen Phosphorus Potassium Magnesium Sulphur Zinc. Boron

However, Nitrogen was added using urea fertilizer, while Phosphorus was added using Triple Super Phosphate (TSP) fertilizer, potassium was added using potassium sulphate (KSO4) or potassium chloride (KCl) fertilizers; magnesium was added by using magnesium sulphate (MgSO4) or Magnesium chloride (MgCl) fertilizers; zinc was added by using zinc sulphate (ZnSO4) or zinc chloride (ZnCl) fertilizers while boron was added using boric acid (H3BO3) fertilizer. Sulphur however was not added through any particular fertilizer but through all these other ones that contained sulphate (ZnSO4, MgSO4, KSO4). Hence, in a plot where Zn and S should be added, ZnSO4 fertilizer was added to take care of the two whereas where only Zn needed to be added, ZnCl fertilizer was applied to take care of the nutrient. This also applied to the other ones. This experiment was sponsored by the International Fertilizer Development Corporation (IFDC) and it was also aimed at testing their fertilizers which they developed for root and tuber crops. The experiment was carried out in two different fields BS20 and part of WBIII. Two crops yam (Dioscorea rotundata) and TME419 cassava (IITA landrace accession) were used for the experiment and the Randomized Block Design (RBD) experimental design was used in the course of the experiment for the statistical analyses.

3.2.1

CASSAVA OMISSION TRIAL EXPERIMENT

The IFDC cassava nutrient omission trial experiment was carried out in field BS20 and the aim, just as afore stated was to test the effects of the IFDC fertilizers on cassava yield and the effect of the lack of each nutrient on the yield also. The cassava cultivar used for the experiment was TME 419 (Tropical Manihot esculentum 419) which is tolerant to most cassava diseases. It is a land race accession from Togo (Dixon, A.G.O, et al, 2010). One of the physical characteristics is that it grows very tall (at least up to 3m height) before it begins to produce branches and the stem color is light brown. The experiment involved processes spanning from field measurement mapping, pegging, replication, plot numbering, border demarcation and cassava planting to fertilizer application, field evaluation, harvesting and sample collection, oven-drying and drying matter content determination etc.

3.2.1.1 FIELD MEASUREMENT FOR PLANTING OF CASSAVA IN CASSAVA OMISSION TRIAL EXPERIMENT.

FIELD MEASUREMENT AND PEGGING: The field BS20 was first of all cleared using a tractor. The field was measured and marked into 4blocks using bamboo pegs. Each block measured 100m x 80m. Each block was further marked into 16plots each making a total of 64plots (i.e. 16plots x 4blocks = 64plots) in the whole field. A plot, suffice it to say, is always on the right of the peg (i.e. pegs indicating the plot number is on the left hand side while the plot numbers is on the right hand). After the measurement, the tape was once again tensioned at both ends and placed vertically on each block and then, 1m intervals were pegged at both sides of the plot. The tape was also used to peg an 80cm horizontal gap interval in the plots using small pegs to indicate the exact place where the cassava stems would be planted on the plate. There were usually a row and a column of plant (usually the 1st column) set aside in each plot to serve as the boarder plants these plants are not included in the result analysis during harvesting or any other evaluation. The main cassava stem planting was usually done after all the markings and pegging has been concluded. There are 49 cassava plants in a plot but 24 out of the 49 serves as the boarder to the 25 main plot plants.

3.2.1.2

FERTILIZER APPLICATION

Fertilizer application, the opium of the research work, commenced some weeks after the main cassava planting was done. The 7 nutrients were applied to each plot in each block in a randomized manner and one plot was always left out as the control plot in each block. The fertilizers were prior to the application weighed out into small nylon bags according to the proportion needed to be applied in each plot. In a plot where Nitrogen (N) for instance was to be applied, the urea fertilizer was used; in a plot where N, P & K were to be applied, NPK fertilizer was used, in a plot where Sulphur and potassium should be applied, KSO4 fertilizer was used. However, if the plot required only Sulphur but no potassium, MgSO4 or ZnSO4 fertilizer was used instead. Also, in a plot where only Zn was needed, ZnCl fertilizer was applied instead of ZnSO4 to avoid the addition of Sulphur and since chlorine (Cl) is not among the test-nutrients. Care was taken not to let the fertilizers touch the cassava plants. The procedure of randomized application was effected through the use of field plans.

3.2.1.3 FIELD ASSESSMENT OF THE CASSAVA OMISSION TRIAL

EXPERIMENT

Field assessment involves the general evaluation of an experimental field to ascertain the general well-being of the experiment and check for external influence that may disrupt the unassailability of the experiment and other development/progress achieved in the experiment. For the cassava omission trial experiment field assessment, it involved evaluating the plant to check for stems (i.e. number of main stems per cassava plant), the branches, the nodes and number of leaves on a plant. Some of the plants, suffice it to say, had 1, 2, or 3 main stems, 1 or 2 branches and several leaves. Some nodes, however, were noticed to be closely packed together indicating some form of poor growth during the dry season. Some nodes, during the assessment too, were observed to have been greatly attacked by the cassava green mite (Mononychellus tanajoa) and so could not produce branches.

FIG 3.1 cassava plant attacked by the cassava grren mite Some leaves too were noticed to have been also attacked by leaf blight bacteria which caused small dark patches on the leaves while at the back of the leaves, tiny speckles of yellow coloration showed the presence of the green mite. In other plants, the leaves were found to have also died and fallen off as a result of wilting. During the assessment, some pegs which had been removed or fallen were replaced again. Also, the main stems of some cassava plants were noticed to have bent sideways and the area exposed to sunlight had started germinating all over again. All these observations suffice it to say, were all being noted. Usually after fertilization, the number of main stems, branches and the height of each cassava plant were taken.

FIG 3.2 Cassava plant assessments in a cassava omission trial field.

3.2.1.4

HARVESTING AND SAMPLE COLLECTION

Harvesting, which usually marks the end of every planting season, started on the cassava omission trial field few weeks after the last batch of fertilizer application. At this juncture, the TME419 cultivar had gotten to heights ranging from 2.5m to about 4m and actually due for harvesting. The harvesting, unlike the normal way of harvesting cassava, was done in a systematic way so as not to incur errors that are drastic to the research and hence mar the aim of the research. The leaves, the stems and the roots (both good and bad) were all harvested separately and packed together according to the plots where they were harvested from. After the harvesting in each plot, the mass of the whole cassava leaves were taken by plucking and packing the leaves inside sacks and then weighing them using spring balances. After the mass weighing and recording, samples of the leaves from each plot were taken from the leaf mass in each plot and weighed separately and put in an envelope, ready to be taken to the oven. This leaf sample taken would be used to determine the dry-matter content of the cassava leaves in that plot (the initial sample weighing was to determine the fresh-leaves mass). The same procedure was also used in the stem harvesting save that the stems were broken into tiny pieces for easy package in the envelopes. During the root harvesting, the number of good roots and bad roots were noted, the mass of the total good roots (in kg), mass of the sub-samples of the good roots (in g) and the mass (in g) of the bad roots were also recorded but however, the bad roots were thrown away as they were of no use any longer. The root sub-samples weight that was taken was used as the mass of the fresh O.K roots and these sub-samples were later taken to the lab for drying in the oven. The samples that were taken to the oven were kept at a temperature of 65 c and for 7days before they were brought out and grounded into powders. Prior to the grinding, the dry weight of the samples (leaves, stems and roots) for each plot was ascertained. These tasks and procedures, although they seemed rigorous, were systematically and tactfully executed with high precision and accuracy as watchwords. The grounded samples would further be shifted to the Analytical Services Unit of the institute for further laboratory analyses and research on the samples.

3.2.2

YAM OMISSION TRIAL EXPERIMENT

The yam omission trial experiment, just like the cassava omission trial, was carried out to ascertain the effect of different nutrients (fertilizers) on the yield of yam. The experiment involved processes spanning from field measurement and pegging, replication, plot numbering and border demarcation, yam seedling treatment and yam planting proper to fertilizer application, field evaluation, staking and trailing.

3.2.2.1 FIELD MEASUREMENT FOR PLANTING YAM IN YAM OMISSION EXPERIMENT

TRIAL

FIELD MEASUREMENT AND PEGGING: Some part of WBIII field was cleared using a tractor. The field was divide into two and one half was ridged while the other half was left unridged i.e. flat. The refuse gotten was burnt on the field in a randomized manner after which the measurement and the markings commenced. The field was marked into 4 blocks (4 replicates) (2 for ridged and 2 for flat land) using bamboo pegs. Each block was further divided into 8 plots making a total of 32 plots (i.e. 4 blocks 8plots= 32 plots) in the whole field. A plot as usual started on the right-hand side of the peg. The pegs were numbered according to plot numbers

with OY signifying the trial experiment that was going on in the plot (i.e. OY RN or RF) for omission yam ridge nil or omission yam ridge fertilize or (OY FF or FN) for omission yam flat fertilize or omission yam flat nil. After the measurement, the tape was once again tensioned at both vertical ends and placed on each block and then, 1m gap interval were pegged at both end of the blocks. The tape was also tensioned at the horizontal ends of the block and 80cm gap intervals were pegged using small pegs (this was to indicate the exact place where the yam seedlings would be planted on the plots). There were usually a row and a column of plant (as in cassava trial experiment) set aside in each plot to serve as the border plants. These plants are not included in the result analysis during harvesting or any other evaluation. The main yam planting was usually done after all the markings and pegging had been concluded. There were 32 yam plants in a plot out of which 16 are border plants and the rest 16 are the main plot plants.

3.2.2.2 YAM SEEDLING TREATMENT FOR PLANTING After the field measurements and marking was completed and prior to the yam planting proper, the seed yams to be planted were treated with insecticides and other chemicals to prevent pest and disease attacks on the seedlings Yam tubers were cut into specific sizes and were weighed using a weighing balance (for a viable yam seedling, the standard weight was between 100g-200g). these yam seedlings were arranged into different baskets in hundreds. 50 liters of water was poured each into two big buckets and into them were added 10% insecticide (cypermethrin 10% EC) and a fungicide (Zforce) of which the active ingredients were cypermethrin and mancozab 80% WP respectively. (mancozab 80% WP is of the family Ethylene Bisdithiocarbonate EBOC). 300g of Z-force was measured out with a saucer on a weighing balance (as it was in powdered form) and 100ml of cypermethrin was also measured out using a measuring cylinder (as it was an emulsifiable solvent). These two chemicals were then mixed in each of the big buckets. Yam seedlings from each basket were placed inside nets so as to ensure uniformity in the treatment. The yam seedlings inside the nets were dipped into the solution and left to stay for 3 minutes. They were further taken to the glasshouse for drying. The glasshouse allows for maximum sunlight on the yam seedlings. After about 1 week in the glasshouse, the seedlings were taken to the field for planting.

3.2.2.3 FERTILIZER APPLICATION Fertilizer application, which was the core of the research commenced weeks after the main planting was done. However, the fertilization actually commenced practically when most of the yams had germinated and started producing tendrils. The fertilizers used for the yam omission trial was not different from that that was used in the cassava save that three more nutrients, charcoal, woodchips and poultry manure, were inculcated into the yam experiment. The nutrients were applied to each plot in each block in a randomized manner and one plot was always left out as the control and another plot receiving the whole treatments (i.e. the 10 nutrients). Care was

taken not to let the fertilizers touch the yams hence, they were applied some 15cm away from the yam tendrils in a ring form. The procedure of randomized application was effected in all the blocks and much care was taken to ensure that the randomization was accurate through the use of field plans. The fertilizers were applied as follows: P as TSP, Mg an S as MgSO4 or as MgCl2, K as KCl or K2SO4, B as H3BO3, Zn as ZnSO4, N as urea, in some cases the Chlorite version needed to be used to avoid combined application. The P, Mg S, Zn and B were applied shortly after yam emergence as a single dressing. N and K were split into three dressings applied shortly after yam emergence, 3 and 5 months after planting.

3.2.2.4 FIELD ASSESSMENT AND YAM STAKING AND VINE TRAILING General field assessment in the yam omission trial experiment started when the yams started germinating and producing tendrils. The evaluation involved germination count to ascertain how many seedlings germinated in a plot and then replacement of the ones that had not germinated. The yam tendrils were staked with bamboos and ropes as soon as they started trailing on the ground. For efficiency and uniformity, the stakes were done using different colors of rope. One particular color was used to stake the borders while another color was used for the main plots. The control had another color of its own too. Vine trailing was also one of the field assessment exercise carried out in the yam omission trial field. The work on the yam omission trial experiment, though seemingly tedious, was tactfully carried out. The harvesting and post harvest experimental works would come later as soon as the yams were due for harvesting and as soon as the necessary treatments has been duly carried out on the different blocks.

3.3

DENSITY TRIAL EXPERIMENTS

The density trial experiment was one of the research works that were on-going during my stay in the Agronomy unit. The experiment sought to find out which planting density that would be most favorable for planting and on which land tillage (i.e. flat land or ridged) would this density be most efficacious. The experiment also went further to check the effect of cropping systems (i.e. monocropping and intercropping) on yield in the different planting densities. The trial crops were Cassava and Yam. However, maize was used in the intercropping. This experiment was carried out by my scientist, Dr. Stefan Hauser and with an MSc. research fellow (who was also doing her research on Yam Density Trial Experiment). Some of the justifications for his carrying out the research were due to the low output (in tonnes) of cassava at the end of the planting season in Sub-Saharan Africa although cassava is a staple crop here and also to seek ways of helping farmers in making maximum use of their lands in getting what they want- maximum profit and low cost of production. The scopes of the experiment spanned from field measurement and markings, pegging, planting, field assessments and data collection to harvesting and post-harvest analysis. I, suffice it to say, partook actively in all the proceedings of the experiment, and got quality explanations for questions that seemed hazy to me from my scientist, Dr. Stefan (a senior scientist in Root and Tuber Systems Agronomy of the institute). The experiment was carried out in four different fields: D23, D19A &B, D15 and WBIII. The experiment was divided into cassava density trial experiment and yam density trial experiment with different statistical experimental plot designs for each.

3.3.1

CASSAVA DENSITY TRIAL EXPERIMENT

Cassava, being one of the mandate crops of the institute, was used in most of the research works that were undertaken in the institute. This is due to the fact that cassava is one of the staple crops that are commonly grown by most African farmers. Cassava was also used in most of the researches that were undertaken by the Agronomy unit of which the cassava density trial was among them. The cassava density trial experiment was carried out to check the different planting densities that would be most efficacious and cost minimizing and the effect of planting cassava and another crop together at such density on the yield. It also tried to analyze the tillage type on

which the efficacious density was most efficient and the effect of fertilizers on the yield of the cassava. The experiment involved systematic processes spanning from the field clearing and measurement, pegging and plot numbering, border demarcation and cassava planting proper to fertilizer application, field evaluation and viral infection assessment of the cassava cultivars, maize planting and harvesting together with the post-harvest processes. Also worthy of note are the Split-split plot experimental design adopted in the course of the experiment and the cassava varieties used for the research. 3.3.1.1 CASSAVA VARIETIES USED FOR THE EXPERIMENT Parameters PEDIGREE TME 419 TMS 97/2205 During the course of the cassava density trial experiment, two cassava cultivars gotten from the Cassava Breeding unit of the institute were used. The two cultivars were TMS 97/2205 and TME419. Some of the noticeable characteristics that differentiate the two varieties are tabulated as follows:

Landrace from 30572TME6 Togo Green purple Moderate Zig-zag Dark green Dark brown white

1ST FULLY EXPANDED Bright green LEAF COLOR PUBESCENCE OF YOUNG absent LEAF GROWTH HABIT OF STEM straight STEM COLOR Light brown OUTER ROOT SKIN COLOR Light brown ROOT FLESH COLOR White/cream

Table 3.1 differences between the two cassava cultivars. Source: A.G.O Dixon et.al, 2010

3.3.1.2 DESIGNS.

THE SPLIT PLOT AND SPLIT-SPLIT PLOT EXPERIMENTAL

The split plot design is specifically suited for a two-factor experiment that has more treatments than can be accommodated by a complete block design. In a split plot design, one of the factors is assigned to the main plot. The assigned factor is called the main-plot factor. The main plot is divided into sub-plots to which the second factor, called the subplot factor is assigned. Thus, each main plot becomes a block for the subplot treatments. With a split-plot design, the precision for the measurement of the effects of the main plot factor is sacrificed to improve that of the sub-plot factor. Sometimes in experiments, subjects are assigned at random to a set of treatments. Then those are subdivided into sub-units to which another set of treatments are applied. And then, those in turn are subdivided again and a third set of treatments are applied. This type of experimental design is what is called THE SPLIT-SPLIT PLOT EXPERIMENTAL DESIGN. It is an experimental design that is uniquely suited for a three-factor experiment where three different levels of precision are desired for the various effects. Each level of precision is assigned to the effects associated with each of the three factors. This design is characterized by two important features (Gomez, K.A et.al, 1984) There are 3 plot sizes corresponding to the 3 factors, namely, the largest plot (the main plot) for the main plot factor, the intermediate plot (sub plot) for the sub plot factor and the smallest plot (sub-sub plot) for the sub-sub plot factor. There are 3 levels of precision, with the main plot factor receiving the highest degree of precision. It can also be imposed on a completely randomized design (CRD). FIELD MARKS: Main experimental subjects of a RCB are divided further into additional independent units (sub plots) to which another set of treatments are randomly assigned. These subplots are additionally split into sub units to assigned randomly to yet another set of treatments. Main treatments are assigned at random within blocks of adjacent subjects, each treatment once per block. The number of blocks is the number of replications.

Any main treatment can be adjacent to any other treatment, but not to the same treatment within the block. ANOVA table format: Source of variation Degrees of Sums of squares Mean square (MS) F

variation Blocks (B) Treatments (Tr) Error-main plots (Em) Subplots (S) Subplots X Treatments (SxT) Error-subplots (Es) Split-subplots (U )

freedoma b-1 t-1 (t-1)*(b-1) s-1

(SSQ) SSQB SSQTr SSQEm SSQS

square (MS) SSQB/(b-1) SSQTr/(t-1) SSQEm/((t-1)*(b-1)) SSQS/(s-1) MSB/MSEm MSTr/MSEm MSS/MSEs

SSQSxT/(t-1)*(s-1) MSSxT/MSEs (t-1)*(s-1) SSQSxT t*(b-1)*(s-1) SSQEs SSQEs /(t*(b-1)*(s-1)) SSQU SSQU/(u-1) MSU/MSEu u-1 SSQUxT/(t-1)*(u-1) MSUxT/MSEu Split-subplots X Treatments (UxT) (t-1)*(u-1) SSQUxT Split-subplots X Subplots (UxS) (s-1)*(u-1) SSQUxS SSQUxS/(s-1)*(u-1) MSUxS/MSEu SSQUxSxT/((t-1)(sSplit-subplots X Subplots X (t-1)*(s- SSQ MSUxSxT/MSEu UxSxT Treatments (UxSxT) 1)*(u-1) 1)*(u-1)) SSQEu/(t*s*(b-1)*(ut*s*(bSSQEu Error-split-subplots (Eu) 1)*(u-1) 1)) SSQTot Total (Tot) t*b*s*u-1
awhere

t=number of main treatments, b=number of blocks and s=number of subplots. anova table for statistical analysis of a split-split plot design.

Table 3.2

Source:www.tfrec.wsu.edu/anova/rcbspsp.html

3.3.1.3 FIELD MEASUREMENT FOR CASSAVA PLANTING IN CASSAVA DENSITY TRIAL EXPERIMENT. FIELD MEASUREMENT AND PEGGING: The different fields (i.e. D23,D19b,D15 &WBIII) were cleared using a tractor and measured using measuring tapes for accuracy and precision. The cassava field was divided into two halves and one half was ridged while the other half was kept unridged (i.e. flat land) this was due to the fact that while some farmers preferred planting on ridges others preferred on flatland. On the ridged part, the field (which served as block) was marked into plots using big bamboo pegs. The pegs, as usual, were on the left side of the plot. The ridged part was divided into two sub plots one sub plot being marked by for one cassava variety and the other sub plot for the other variety. The big bamboo pegs that were used in mapping in each sub plot divided the subplots into four sub-sub plot hence making a total of 8 sub-sub plot on the ridged part of the field. The same procedures were also carried out on the flat land side of the field. These were done in all the cassava density fields used for the experiment. Small bamboo pegs were made and used in marking vertically on the sub plots (i.e. the ridge and flat lands) the exact points on the field where the cassava stems would be planted to ease the stress in measuring again during the planting proper. The tapes were stretched from one point on each row in the field to the other extreme end where it ended and tensioned for accuracy. On each row, the numbers below were pegged on the ridge and flat land at the exact point where it was on the tape touching the ground. Two sub plots were pegged for planting at the same time because they were just lying side-by-side each other.

FIRST PLOT (in m): low to high 1.00 2.00 3.00 3.90 4.80 5.70 6.60 7.40 8.20 9.00 9.80 10.50 11.20 11.90 12.60 13.20 13.80 14.40 15.00 15.50 16.00 16.50 17.00 17.40 17.80 18.20 18.60 19.00 19.30 SECOND PLOT (in m): high to low 19.90 20.00 20.40 20.80 21.20 21.60 22.10 22.60 23.10 23.60 24.20 24.80 25.40 26.00 26.70 27.40 28.10 28.80 29.60 30.40 31.20 32.00 32.90 33.80 34.70 35.60 36.60 37.60 38.60 39.60 It was from these spacing that the density trial was effected. Some pegs had 1m spacing while others had 90cm and others 80cm, 70cm, 60cm, 50cm, 40cm, and 30cm respectively. The plot numberings were done by assigning ordinary numbers to each and then using big bamboo pegs to mark where a new plot started. On the pegs also were alphabets like (RIF), (FMN) etc which denotes Ridge Intercrop Fertilize and Flat Monocrop Nil. These connote that the plot was on ridge/flat land and it was a mono/inter cropping system and it was to be fertilized or not during the fertilizer application. CASSAVA PLANTING PROPER : The stems of the two cassava cultivars (TME 419 and TMS 97/2205) were cut into small pieces of about 15cm. each cultivars stem were planted at the spots where the small bamboo pegs that were used earlier during the measurement were placed. The cassava stems were planted in such a way that two-third (2/3) of the whole stem were below the ground only one-third (1/3) of the stem were above the ground. Care, however, was taken to make sure that the nodes on the stems were facing the sky as those were where the emerging young leaves would emanate from when they start germinating. 3.3.1.4 FERTILIZER APPLICATION IN THE CASSAVA DENSITY TRIAL EXPERIMENT Fertilizer application was one of the procedures that were undergone during the course of the experiment. The 10 Weeks old cassava plants were fertilized with different kinds of IFDC trial fertilizers as and when due. Worthy of note, was however, that not all the sub-sub plots were fertilized during the periods of any fertilizer application. This was to incorporate into the research the aspect of testing for the effect of fertilizers on the yield. Hence, pegs with numbers like CD/RMN/02 or CD/FIN/12 means that that was a cassava density experiment, ridge/flat, monocrop/intercrop, no fertilizers then the serial number. This helped during the fertilizer application to know which plots were to be fertilized and which ought not to be fertilized. The fertilizers applied were NPK 15:15:15 at 200 kg / ha at 2 weeks after planting, followed by a dressing of urea equivalent to 30 kg/ha of N. A third application of KCl was conducted at 4 months after planting at an equivalent of 45 kg/ha K. Care was taken not to let the fertilizers be in very close contact with the cassava stems. 3.3.1.5 FIELD EVALUATION AND DISEASE CHECK IN THE CASSAVA DENSITY TRIAL EXPERIMENT FIELD. For the cassava density trial experiment field evaluation, it involved routine evaluation of the cassava plants to check for the number that germinated in each plot, if the pegs were still in place and if the ridges were still as they should etc. necessary actions were taken to restore and amend these abnormalies in the field. The weeds in the field were also taken care of monthly throughout the course of the experiment. As part of the experiment, the heights (in cm), number of main stems on each cassava plant, number of branches on each plant too was checked and recorded fortnightly. (Appendix **** record sheet for the data collection). From the evaluation, it was noticed that although TME419 surpassed TMS 97/2205 in terms of heights of the main stems, the later had more main stems and branches per plant than the former. Also worthy of note was the virus-assessment done in the two cassava variety fields. The two

cassava cultivars were assessed and graded based on the gravity of the viral symptoms and spots found on the leaves. Although the two cultivars showed some sign of infection by the virus, the symptoms were however more conspicuous in TME419 cultivars than in TMS 97/2205.

CGM= Cassava Green Mite

. FIG 3.3 Cassava field assessment and data taking

3.3.1.6 CASSAVA-MAIZE INTERCROPPING SYSTEM Intercropping is a type of mixed cropping which involves the agricultural practice of cultivating two or more crops in the same space at the same time with the aim of increasing productivity per unit of the land. Cassava-maize intercrop is carried out on a piece of agricultural land with the aim of intensifying the productivity per unit of the land. This intercropping is made effective due to the fact that cassava takes a longer time before it is harvested whereas maize doesnt and hence the reason for their intercropping. 3.3.1.7 CASSAVA-MAIZE INTERCROP IN THE CASSAVA DENSITY TRIAL EXPERIMENT. The cassava-maize intercrop was carried out in the cassava density experiment fields also. This

was to check the effect on yield of the cassava and the maize at various planting densities and also to check for the differences in yield between the monocrop (i.e. cassava alone and maize alone) fields and the intercrop fields. The maize was planted on both ridges and flat land with a vertical spacing of 20 cm between each maize plant and a horizontal distance of 75cm. The intercrop fields had the same field layout as the monocrop save for the two crops. As in the monocrop, pegs with inscriptions like RIF** or FIN** means that the field is ridged/flat, the cropping system is intercrop and it would receive fertilizer treatments or none. Field evaluation and plant assessments were also carried out in the intercrop fields as well. The heights (in cm), number of main stems on each cassava plant, number of branches on each plant etc were also checked and recorded fortnightly as in all cassava density trial fields. The maize variety used, TZL comp.3 DT F2, was gotten from the Maize Breeding unit of the institute. The maize cultivar was treated with CIBAPLUS, an insecticide cum fungicide which protects the seed from most pests and fungal diseases attacks. 3.3.1.8 MAIZE HARVESTING IN A DENSITY TRIAL EXPERIMENT. The maize plants used in the density trial experiment for cassava were harvested four months after the time they were planted. The harvesting was done in a systematic and careful manner so as not to alter the research results or make it erroneous. The harvesting involved the use of ropes tied to pegs to demarcate the different densities on each field in the rows. That is to say that those with 1m gap were demarcated from the border using a rope tied to two pegs that had 1m inscribed on it. This applied to those with 90cm gap (i.e.0.9m), 80cm (0.8m)30cm (0.3m). The maize in each batch were harvested separately row by row (there were 6 rows in each plot). The results required and recorded for the experiment include the number of maize stands in each row in a batch, the number of fresh o.k. cobs and the number of bad cobs. After each harvest in a batch, the total mass (in kg) of the fresh o.k. cobs were weighed and also recorded same as that of the bad cobs in each batch too. Sub-samples of the fresh o.k. cobs from each batch were taken and put inside paper bags and weighed. This weight (in g) would serve as the fresh cob weight of the batch during the result analyses.

3.3.1.9 MAIZE POST-HARVEST PROCESSES IN THE DENSITY TRIAL EXPERIMENT The post harvest processes involved in the density trial experiment as pertained to maize include the oven-drying, the shelling, grinding, weighing, laboratory analyses etc. after the harvesting of the maize in each of the density fields, the sub-samples from each batch were paced in the oven at a 65c centigrade temperature for about 2-3 days to dry. The weight of the dry o.k. cobs were recorded after the drying. The maize cobs were shelled (i.e. the act of removing the grains from the cobs) and the dry weight of the grains were also taken. The dry grins were grounded into powdery form and further wrapped in nylons according to batches and plots in preparation for the laboratory analyses that would be run on them. In general, the cassava density trial experiment was more rigorous and demanding than the omission trial experiment in cassava but however, I learnt a lot from the works which I did during the course of my training in the unit.

3.3.2

YAM DENSITY TRIAL EXPERIMENT

Yam, also one of the mandate crops of IITA, was used by the Agronomy unit of the institute in their density trial experiment. The yam density trial experiment was carried out to check the different planting densities that would be most efficacious and cost minimizing and the effect of intercropping at such density on the yield of yam. It also tried to analyze the tillage type on which the efficacious density was most efficient and the effect of fertilizers on the yield of the yam The experiment involved systematic processes spanning from the field clearing and measurement, pegging and plot numbering, border demarcation and yam planting proper to fertilizer application, field evaluation and disease-check (scoring), maize planting and harvesting together with the post-harvest processes. Also worthy of note is the Factorial in a Split-plot experimental design adopted in the course of the experiment.

3.3.2.1 FIELD MEASUREMENT FOR YAM PLANTING IN YAM DENSITY TRIAL EXPERIMENT. Field measurement and pegging: The different fields (i.e. D23, D19a and b, and D15) were cleared using a tractor and measured using measuring tapes for accuracy and precision. The yam field was divided into two halves and one half was ridged while the other half wasnt (flat land). This was to accommodate the fact that some farmers preferred flatland while others go for ridged lands. On the ridged part, the field (which serves as block) was marked into plots using big bamboo pegs. The pegs, as usual, were on the left side of the plot. The ridged part was divided into 4 sub-plots and on each were planted the same species of yam (Dioscorea rotundata). The same procedure was also carried out on the flat land side of the field. Small bamboo pegs were made and used in marking vertically on the sub-plots (i.e. the ridge and the flat land) the exact points on the field where the yam seedlings would be planted after treatment to ease the stress in measuring again during the planting proper. The tapes were stretched from one point on each row in the field to the other extreme end where it ended and tensioned for accuracy. On each row, the numbers below were pegged on the field at the exact place where it was on the tape. Two sub-plots were being pegged for planting at the same time because they were adjacent each other. FIRST PLOT (in m): low to high 1.33 2.66 3.99 4.99 5.99 6.99 7.89. 8.79 9.69 10.49 11.29

12.09 12.79 13.49 14.19 14.89 15.49 16.09 16.69 17.29 17.79 18.29 18.79 19.29 20.09 20.49 20.89 21.29 21.59 21.89 SECOND PLOT (in m): high to low 22.19 22.49 22.89 23.29 23.69 24.09 24.49 24.99 25.49 25.99 26.49 27.09 27.69 28.29 28.89 29.59 30.29 30.99 31.69 32.49 33.29 34.09 34.99 35.89 36.79 38.79 39.79 41.12 42.45 43.78 45.11 It was from this spacing that the density trial was effected. Some pegs had 133cm spacing while others had 100cm and others 90cm, 80cm, 70cm, 60cm, 50cm, 40cm, and 30cm spacing respectively. The plot numberings were done by assigning ordinary numbers to each and then

using a big bamboo peg on which the numbering was inscribed to mark where a new plot started. On the pegs also were alphabets like RIF, FMN etc which denote Ridge Intercrop Fertilize and Flat Monocrop Nil. These connote that the plot was on ridge/flat land and it was a mono/inter cropping system and it was to be fertilized or not during the fertilizer application. YAM PLANTING PROPER: The seedlings of the yam species D. rotundata were cut into pieces of 80-100g and treated with cypermethrin and z-force. The yam seedlings were later taken to the field for planting and were planted at the spot where the small bamboo pegs that were used earlier during the measurement were placed. 3.3.2.2 FERTILIZER APPLICATION IN THE YAM DENSITY TRIAL EXPERIMENT. Fertilizer application was one of the procedures that were undergone during the course of the experiment. Theweeks old yam were fertilized with different kinds of fertilizer as and when due. Worthy of note was however, the fact that not all the subplots were fertilized during the periods of any fertilizer application. This was to incorporate into the research the aspect of testing for the effect of fertilizers on the yield. Hence, pegs with numbers like YD/RMN/02 or YD/FIN/12 means that that was a yam density experiment, ridge/flat, monocrop/intercrop, no fertilizers then the serial number. This helped during the fertilizer application to know which plots were to be fertilized and which ought not to be fertilized.

3.3.2.3 PROTOCOL FOR YAM GROWTH ASSESSMENT IN YAM DENSITY TRIALS EXPERIMENT.

Variables 1 Height 2 Branching 3 Number of leaves (canopy status)

Keys: Height Absent = 0 0 - 60 cm= 1 61 120 cm = 2 121 180 cm = 3 181 300 cm = 4 301 and above = 5

Branching Absent = 0 2 - 10 = 1 22 20 = 2 22 30 = 3 32 40 = 4 >40 Branches and reaching on top of the stake = 5

Number of leaves No fully expanded leaves = 0 Few leaves close to the ground ranging from 0 - 20 = 1 Few leaves stretched along vine up to 120 cm = 1.5 Leaves ranging from 21 - 40 = 2 Leaves (21 - 40 ) stretched along vine up to 180 cm = 2.5 Leaves from ground to 180 cm height but > 40 = 3 Leaves ( > 40 ) stretched along vine up to 300 cm = 3.5 Leaves up to 300 cm height but > 60 = 4 Leaves on top of stakes but > 80 = 5 Also in the assessment was the use of spad (pic) to measure the chlorophyll contents in the leaves of the yam plants. The surface area of the yam leaves were also measured using a leaf area meter (pic)

3.3.2.4 YAM-MAIZE INTERCROP IN THE YAM DENSITY TRIAL EXPERIMENT The yam-maize intercrop was carried out in the yam density experiment fields also. This was to check the effect in yield of yam and the maize at various planting densities and also to check for the differences in yield between the monocrop (i.e. yam alone and maize alone) fields and the intercrop fields. The maize was planted on both ridges and flat land with a vertical distance of 20 cm between each maize plant and a horizontal distance of 75cm. The intercrop fields had the same field layout as the monocrop save for the two crops. As in the monocrop, pegs with inscriptions like RIF** or FIN** were meant for intercrops. Field evaluations and plant assessment were also carried out in the intercrop fields as well. The

results gotten from the evaluation was recorded fortnightly as in all yam density trial fields. The maize variety used, TZL comp3 DT F2, was gotten from the Maize Breeding unit of the institute. The maize cultivar was treated with CIBAPLUS, an insecticide cum fungicide which protects the seed from most pests and fungal diseases attacks.

3.3.2.5 MAIZE HARVESTING IN A DENSITY TRIAL EXPERIMENT. The maize plants used in the density trial experiment for yam were harvested four months after the time they were planted. The harvesting was done in a systematic and careful manner so as not to alter the research results or make it erroneous. The harvesting involved the use of ropes tied to pegs to demarcate the different densities on each field in the rows. That is to say that those with 1m gap were demarcated from the border using a rope tied to two pegs that had 1m inscribed on it. This applied to those with 90cm gap (i.e.0.9m), 80cm (0.8m)30cm (0.3m). The maize in each batch were harvested separately row by row (there were 5 rows in each plot). The results required and recorded for the experiment include the number of maize stands in each row in a batch, the number of fresh o.k. cobs and the number of bad cobs. After each harvest in a batch, the total mass (in kg) of the fresh o.k. cobs were weighed and also recorded same as that of the bad cobs in each batch too. Sub-samples of the fresh o.k. cobs from each batch were taken and put inside paper bags and weighed. This weight (in g) would serve as the fresh cob weight of the batch during the result analyses.

3.3.2.6 MAIZE POST-HARVEST PROCESSES IN THE DENSITY TRIAL EXPERIMENT The post-harvest processes involved in the density trial experiment as pertained to maize include the oven-drying, the shelling, grinding, weighing, laboratory analyses etc. after the harvesting of the maize in each of the density fields, the sub-samples from each batch were paced in the oven at a centigrade temperature for about 2-3 days to dry. The weight of the dry o.k. cobs were recorded after the drying. The maize cobs were shelled (i.e. the act of removing the grains from the cobs) and the dry weight of the grains ere also taken. The dry grins were grounded into powdery form and further wrapped in nylons according to batches and plots in preparation for the laboratory analyses that would be run on them. In general, the yam density trial experiment was more rigorous and demanding than the omission trial experiment in yam but however, I learnt a lot from the works which I did during the course of my training in the unit.

3.4

CULTURAL PRACTICES IN SOIL FERTILITY MANAGEMENT

Agronomy, as already defined, is a branch of agriculture that not only involves crop production

but also the soil and its management. The Agronomy unit of IITA being headed by a seasoned agronomist made sure that their activities spanned through all the scopes of the field starting from crop production to soil management. Two techniques were adopted by the unit in restoring the fertility of an agricultural land viz: Bush fallowing and Cover cropping (using Mucuna pruriens). My scientist, Dr. Stefan Hauser, was trying to evaluate between the two techniques which would be more efficacious. He, though not as a full-time or result-oriented research, wanted to explore other potentials of the plant as it was said to suppress nematode population (J.A. Adediran et.al, 2005), help reduce labor involved in weeding and control of most noxious weeds like Imperata cylindrica (Akobundu and Udensi, 1995) and its ability to restore up to about 70% of soil fertility. 3.4.1 AGRONOMIC PRACTICES IN THE USE OF Mucuna pruriens IN THE CULTURAL PRACTICE OF SOIL FERTILITY MANAGEMENT. Mucuna seeds were planted on a field (F23) that had been used previously for intensive farming. The land was measured and marked into 8 blocks (plots). 4 plots taken systematically (i.e. every 2nd plot) was used for the planting. On each plot, 20 seeds were planted on each column and 60 on each row. The plants were staked with bamboo stakes as soon as their tendrils were able to trail them. The mucuna plants were to be left in the field to grow, mature and produce seeds which would be later be harvested and stored and the vines left to wither in the field.

3.4.2 AGRONOMIC PRACTICES IN THE USE OF BUSH FALLOWING TECHNIQUE AS A CULTURAL PRACTICE IN SOIL FERTILITY MANAGEMENT On the same field (F23) where mucuna seeds were planted (i.e. the remaining 4 plots), bush fallowing as a soil fertility management technique was employed. The fallow was left to grow alongside the mucuna plants too.

CHAPTER 4 NEMATOLOGY UNIT 4.1 INTRODUCTION

Nematodes are a diverse group of worm-like animals found in almost every environment both as parasites and as free-living organisms. They are mostly found in wet surroundings which aid their movement and other activities. They move in the film of water that surrounds soil particles and the moisture contained in plant tissues. Most nematodes inhabit the soil and are microbial feeders or predators on other soil organisms but some parasitize plants and are known as Plant Parasitic Nematodes (PPN). Plant nematodes are divided into parasitic and non-parasitic (K.P.N, Kleynhans, 1999). The parasitic ones are differentiated from the non-parasitic in the possession of a movable, needle-like structure called stylet in the mouth cavity which they use to puncture plant cells to inject digestive enzymes and

draw the modified cell contents into their esophagus. Because nematodes are difficult or impossible to see in the field, and their symptoms are often non-specific, the damage they inflict is often attributed to other, more visible causes. The Nematology/Striga unit of the IITA is the section charged with the duty of curtailing the menace caused by these worms in the field. Since damage by these worms is one of the most resounding constraints to sustainable crop production that farmers in Africa have to contend with and IITA, being a research institute that seeks to alleviate agriculture in Africa through its research for development projects, has taken it upon itself to fight these cankerworms to the barest minimum where their activities would be most insignificant to affect crop yield. The activities of the unit include research for crop protection and improvement, sample analyses and data evaluation, healthy practices in crop planting etc. They also assist research fellows (M Sc, PhD, etc) in their researches and service other units within the institute that needs professional guidelines on nematode control with their unalloyed services and guidelines. During my training in the unit, I was opportuned to witness, partake and learn some of the procedures and practices in the field of Nematology ranging from the sample collection, extraction techniques, nematode identification, fishing of nematodes and culturing of nematode to soil sterilization for nematode multiplication. I was also privileged to operate some equipment and machines which, had I not been here, may not have seen or operated. My stay in the unit, suffice it to say, was really a worthwhile adventure as I grabbed some practical knowledge (that are really career-changing) in phytonematology which would enable me digest the theoretical aspect I got from the lecture hall. 4.2 NEMATODE SAMPLING

Due to the destructive nature of these worms and the need for agricultural development and yield improvement, plant parasitic nematodes are being sampled in both the soil and the plant tissues. Nematode sampling is much needed to diagnose nematode-related problems. Usually, roots of plants, soil and above-ground plant tissues , are sampled for nematodes. When only soil is sampled, the samples are generally taken to a depth of 15-20cm. nematodes are not uniformly distributed in the soil (David J. Hooper et.al, 2005). Areas of nematode damage may be circular to oval or rectangular in outline. Sampling for stem and foliar nematodes should be from symptomatic plants. Most migratory plant parasitic nematodes are found around plant roots and hence the need to also take root samples. Soil samples and plant materials to be examined for nematodes should be kept moist. Also, labeling of samples is also a very important step in nematode sampling. Nematode sampling equipment include: spade, a hand trowel, soil auger, knives, scissors, polythene bags, envelope bags, tags, cool box, writing material (permanent marker).

FIG 4.1 Nematode sampling equipment Absence of nematodes in a sample may indicate their absence in the sampled field, but may also indicate that the populations are too low to be detected by sampling. 4.2.1 SOIL SAMPLE COLLECTION FOR NEMATODE SAMPLING

Soil samples are collected from fields suspected for having nematode population built up in it. This could be for either research purposes, to assess the nematode population in the given field or to collect the nematodes for identification, culturing or multiplication for further research works. Nematodes are usually, as stated before, not evenly distributed in a field and soil sampling procedures need to take this into account to obtain a true representative sample. Taking enough samples, should however be a good admonition, to ensure they are a true representative of the situation in the field. The greater the number of sub-samples/cores combined for each field sample, the more accurate the assessment will be. A balance between available time for the sample collection and resource is, however, necessary. For plots up to 100sq.m in area, at least 20-30 sub-samples (cores) were taken per sample. For uniform areas up to 1hectare, at least 50 sub-samples were taken. In an experimental/research field, sub-samples that would incorporate the various treatments applied in the field were taken. However, sampling very wet or very dry soil was avoided except in cases where they must if samples from such area should be needed. Usually few nematodes occur in the top 5cm of soil which can be discarded from samples. Nematodes are most abundant within the root zone and for shallow-rooted crops, samples were taken to a depth of about 20-30cm. this was made possible because of the use of soil auger. Random or systematic (i.e. W or zig-zag pattern) sampling patterns are two sampling patterns used for soil sampling for nematodes. After the collection, the soil auger was placed in a plastic plate and with a strong blunt stick, all the conents of the auger were scraped into the plate. Care was taken to ensure that all the soil sample in the auger were removed before taking another sample. The samples wer later put in polythene bags with a tie at the top well labeled with permanent marker. The label bore the following information: The location The sampling date Plot number (if it were within an experimental field) Treatments applied to the field/crops grown After collection and labeling, the samples were placed in a cool box and later taken to the lab for further research works.

FIG 4.2 soil sample collection packaged sample

FIG 4.3 scraping of FIG 4.4 well the sample into a plate

and labeled soil

FIG 4.5 The soil sample stored in a box to be taken to the lab. 4.2.2 ROOT SAMPLE COLLECTION FOR NEMATODE SAMPLING

cool

Nematodes are often found in highest numbers in the root zone of the crops towards the end of the growing season. Badly stunted plants may have too small a root system to support many nematodes, and samples from nearby, less affected; plants may yield more specimens (D.J, Hooper et.al, 2005). Root samples can be collected at the same time and from the same locations as for soil. Generally, 25-100g of roots per total sample is sufficient, but a lower weight may be collected for finer roots such as from rice and a higher weight for thick roots such as from plantain. Dead plants or those in advanced stages of senescence are usually not sampled as nematodes would have migrated from these to other food sources (roots). Lift the plants and their roots from the soil using a hand trowel (spades can also be used), so that a sizeable proportion of the root system is unearthed intact, and taking care not to break off the roots and leave them in the ground. After tapping soil free, randomly remove roots with a knife or scissors. A visual examination can be made of roots e.g to detect the presence of galls or nematode egg masses, females or cysts.

nematode infested carrot

nematode in fested cassava root

nematode infested yam tuber FIG. 4.6 PICTURE OF NEMATODE INFESTED ROOTS.

nematode infested lettuce root

Place the root samples inside polythene bags and tie the mouth with a tag carrying the labels or put in a paper envelope and boldly label on the envelope. The samples are either taken to the lab immediately or kept in a cool place away from the heat of the sun.

4.3

NEMATODE EXTRACTION

After sample collection for nematode sampling, the next stage was to extract nematodes from the samples. This was usually done as soon after the sampling as possible to avoid sample deterioration. Various methods and techniques to extract nematodes from soil and plant tissues have been developed and were employed by the unit for her extractions. There are 5 extraction methods employed by the unit for extraction of different kinds of nematodes from different types of samples viz: 1. 2. 3. 4. 5. Pie-pan/Extraction tray method Sieving method Maceration (blending) method Sodium hypochlorite method (Hussey and Barker technique) Centrifuge method

The choice of which method to use depends on the conditions and materials available, the sample type and also the kind of nematodes available.

TYPE OF SAMPLE KIND OF NEMATODE Extraction tray method Sieving method Maceration (blending) method NaOCl method Centrifuge method

Soil sample Sedentary nematodes

Migratory nematodes

Root/foliar sample Sedentary Migratory nematodes nematodes

Table4.1 the suitability of extraction methods for different kinds of nematode and types of samples

Before the extraction proper begins, the samples would be prepared for the exercise by mixing the soil samples thoroughly, breaking up clumps and removing stones, roots and debris. Roots are separated from the soil by tapping gently or rinsing gently under a tap water, drying and then chopping into desired sizes. Care, however, was taken not to mix up the labels or their containers. 4.3.1 PIE-PAN/EXTRACTION TRAY METHOD

This is the modification of the Baermann-funnel technique. It extracts only live, active nematodes from the soil and plant materials. It is a very simple technique. Equipment needed include: A sieve with coarse mesh

A tray /plate, slightly larger than the sieve Extraction paper Beakers or cups to wash the extract into Wash bottle Permanent marker Knife/scissors Weighing balance

4.3.1.1

PIE-PAN/EXTRACTION TRAY METHOD FOR SOIL SAMPLES

I. Use a coarse sieve to remove stones and debris from soil and break up soil lumps. II. Using a calibrated beaker to remove a measure of soil (e.g. 100ml, 200ml etc.). III. Place the extraction paper in the plastic sieve that had been already placed on a plate/tray making sure that the extraction paper covers the base of the sieve very well. IV. Place the soil measure on the extraction paper in the sieve. Care should be taken to ensure that the soil was on the extraction paper else, it would result to a dirty extraction. V. Add water to the extraction plates. Care should also be taken in pouring the water gently into the plate and not onto the extraction paper or soil through the gap between the edge of the sieve and the side of the tray. A set volume of water should be added to each dish to wet but not cover the soil, however ensuring there was sufficient not to dry out. VI. The apparatus would be left undisturbed for 48 hours so that the nematodes can easily swim out of the soil and into the water. VII. After the extraction period, the extraction paper and the soil sample are discarded. VIII. Next, the water from the tray/plate would be washed into a labeled beaker, using a water bottle to rinse the plate. The extract suspension was then, after the transfer, ready for further research works.

II

IV &V

VI Extraction tray method for soil samples

VII

VIII FIG 4.7

4.3.1.2 EXTRACTION TRAY METHOD FOR ROOT/PLANT TISSUE SAMPLES

I. Gently tap off the soils on the roots/tubers or rinse under a tap and then leave to dry. Peel tubers carefully with a knife to just below the surface. II. Chop the roots (or tuber peels) finely with a knife and place in a labeled dish. Mix all chopped root material thoroughly. III. Remove and weigh a sub-sample of chopped root material using weighing balance. IV. Place weighed sub-sample on the extraction paper in the labeled sieve. V. Add water to the extraction plate (as in V above). VI. Leave the apparatus for at least 24 hours so that the nematodes can swim out of the samples. VII. After the extraction period, discard the extraction paper and the root samples in it. VIII. Water suspension from the tray should be poured into a labeled cup and kept ready for further usage. This method is more efficient to extract nematodes from soil samples than from root samples.

4.3.2 SIEVING METHOD This method is good for all kinds of nematodes and useful for cyst extraction from soil samples. Equipment needed include (for soil nematodes): Beakers and bucket Permanent marker (for labeling) Sieves of variable meshes: 2mm, 90m and 38m Extraction tray apparatus

Equipment needed for sedentary cyst extraction: Sieves of variable meshes: 2mm, 250m,150m Funnels Extraction papers/filter paper

4.3.2.1

SIEVING METHOD FOR SOIL MOTILE NEMATODES

I. Fill a bucket with six liters of water. Mark a water line on the inside of the bucket with a permanent marker for consistent water volume between samples gotten for the same purpose. II. Place a sub-sample of sieved and mixed dry soil measured by displacement of water in a beaker (i.e. if a 300ml beaker is filled with water to the 200ml mark and then soil samples were added and it rose up to 300ml then it meant that 100ml of soil samples was added to the beaker) into the bucket. III. Stir the mixture thoroughly using hand. Allow larger particles to settle for 30 secs. IV. Slowly pour off the upper of the water through the nested sieves: use the 2mm to catch the debris for disposal and 90m and 38m to capture the nematodes. Care should be taken when sieving proper is done so as not to lose the nematodes. V. Refill the bucket to the marked line and repeat the process once or twice. VI. Wash off the debris from the 90 and 38m sieves into a well labeled cup, ensuring that the sieves are properly cleared by washing them from behind.

VII. Leave the cup for 2-3 hours for nematodes to settle at the bottom. Gently decant excess water and set ready for further usage.

4.3.2.2 SIEVING METHOD FOR EXTRACTING SEDENTARY CYST NEMATODES I. Air-dry the soil samples before the extraction. II. Fill a bucket with 6 liters of water with a water line marked inside the bucket. III. Pour the sample and mix the water thoroughly using hand, then allow soil particles to settle for 60 secs. Cysts should float. IV. Slowly pour off the top of water through the sieves: 2mm to catch debris for disposal, and 250 and 150m to trap the cysts. V. Wash off the debris from the 250 and 150m sieves into a labeled beaker. VI. Refill the bucket to the marked line and repeat the process as much as necessary until one is satisfied that no cysts remain in the bucket. VII. Prepare and label a paper lining (using an extraction paper/filter paper) for a funnel held in a stand or in a beaker. VIII. Pour the wash-off suspension in the beaker through the filter in the funnel. allow water to drain through. IX. Carefully remove the extraction papers from the funnel and place in a moistened tray to await observation under the microscope. Viewing can be done under a stereomicroscope.

ii

iii

vii

viii

ix FIG 4.8 sieving method for cyst extraction

4.3.3

MACERATION (BLENDING) METHOD

This is a quick and useful method of examining roots for the presence of some nematodes. Soil

samples cannot be extracted using this technique. Equipment needed for the extraction include: Beakers Scissors/knife Wash bottle Permanent marker Procedure: 1. Wash the root samples gently to remove soil particles. 2. Cut the roots or tuber peels into tiny pieces. 3. Weigh a sub-sample. Place it in an electric blender with just enough water to cover the blades. 4. Run the motor for 10-30 secs intermittently for 3 times. The time and frequency varied according to the amount and kind of materials being processed. 5. Pour the blended suspension of roots and water into a beaker, rinsing out the blender container of all debris, using a wash bottle. 6. Set up an extraction tray apparatus and gently pour the suspension into the extraction paper. 7. Then the next procedures follow that of the extraction tray method. Weighing balance Domestic blender

ii

iii

iv

v FIG 4.9 blending method of extraction 4.3.4

vi

SODIUM HYPOCHLORITE (NaOCl) TECHNIQUE (HUSSEY AND BARKER METHOD)

This extraction technique was designed by Hussey R.S and Barker, K.R in 1973 basically for the extraction of meloidogyne eggs in roots to be used for inoculation. Materials needed for the extraction include: A conical flask Sieves of variable appertures: 75m and 26m Wash bottle Beakers

Procedures: 1. 2. 3. 4. 5. 6. 7. 8. 9. Wash the root samples gently to remove soil particles. Cut the roots of the samples into tiny pieces of about 1-2cm. Prepare a 10% NaOCl solution by using 10ml of jik in 90ml of water. Weigh a sub-sample. Place it inside the conical flask. Pour the NaOCl solution and shake vigorously for 2-4 mins. Quickly pass the mixture through a 200 mesh (i.e 75m) sieve nested over a 500mesh (26 m) sieve to collet freed eggs. Quickly place the 500 mesh sieve with eggs under a strem of cold water from the tap in order to remove the residuals of the NaOCl. Rinse for several minutes. Rinse the remaining roots in the flask with water. Using a wash bottle, rinse the extract on the 500-mesh sieve into a beaker of known water capacity(say 200ml) and use clean water to bring the volume up to the200ml mark. Then using a Pasteur pipette, take a sub-sample of the extract and view for counting under the stereomicroscope.

4.4

IDENTIFICATION AND COUNTING OF NEMATODES

Once nematodes have been extracted from the samples; soil or plant materials, they must first be identified and then quantified. Nematode identification requires special techniques to extract nematodes from soil or plant materials, high-powered microscopes to observe minute morphological features and special training in identification procedures. A brief taxonomical classification of the plant parasitic nematodes would be relevant in the identification process. The phylum Nematoda is divided into two classes: Adenophorea and Secernentea. The class Adenophorea is divided into two sub-classes Enoplia and Chromadoria. The subclass Enoplia contains an order of PPN- the Dorylaimids found in soil and freshwater and ectoparasites of plants. The genus Longidorous and Xiphinema are two notable genera under the order. The class Sercernentea contains about 9 important orders of plant and animal parasitic nematodes. The order Aphelenchida and Tylenchida are two notable PPN orders under the class. The order Aphelenchida contains PPN of the genera Aphelenchus, Megadorous etc. while the order Tylenchida contain PPN of the families Tylenchidae (e.g. of genus under this family is Tylenchulus), Anguinidae (e.g. Ditylenchus sp.), Hoplolaimidae (e.g. Hoplolaimus sp., Helicotylenchus sp., Rotylenchus sp., Scutelonema sp.), Pratylenchidae (e.g. Pratylenchus sp, Hirschmaniella sp), Heteroderidae (e.g. Heterodera sp, Globodera sp, Meloidogyne sp.), Criconematidae (e.g. Criconema sp, Criconemella sp), Tylenchulidae (Tylenchulus, Trophonema, Paratylenchus, Tylenchocriconema) etc. (Myers, P et.al, 2013)

Hence the three Plant Parasitic Nematode (PPN) orders include: Order Dorylaimida Order Tylenchida Order Aphelenchida The three orders can be differentiated morphologically using some parameters:

PARAMETERS Stylet type Esophagus Phasmids Basal knob

Dorylaimida Odontostylet Two-part without a valvulated region Absent Absent

Tylenchida Stomatostylet 3-part esophagus with a valvulated metarcorpus Present Present and conspicuous present

Aphelenchida Onchiostylet 2-part with valvulated basal region Absent Present Absent

Cephalic framework Absent Table 4.2 F. Mai et.al,

some morphological differences btw the 3 main orders of PPNs. Source: William

However, individual nematode species in the same order were identified using other morphological features like the presence/absence of a vulval region/spicules and their position (in %) on the body length, the type of esophageal overlap (posterior or anterior) etc.

FIG 4.10 TYPICAL NEMATODE STRUCTURE. Source: D.L, Coyne, et.al,2012

The tedious task of counting large numbers of nematodes can be eased by extracting several fixed-volume sub-samples of nematodes from a suspension and counting them in graduated microscope counting slide (pic.). Usually, Pasteur pipettes are used to take a sub-sample from the suspension extract and viewed under the microscope, the different nematodes seen in the sample are identified to generic level and the number of species in each genus counted using a handheld counter (pic) and the figure derived, recorded in the nematode counting sheet. After the first count, the sub-sample is usually returned to the suspension and the counting procedure repeated two or more times and then estimate of the standardized number of eggs/nematodes per ml and inversely the whole suspension is made.

FIG. 4.11 nematode egg counting using a stereo microscope

4.5

NEMATODE FISHING

After the identification and assessment, the nematode genus that would be used for a further research work or for culturing were usually fished out from the suspension and placed on a glass petri dish. This is usually a no-easy task but gets easier with practice. Various instruments can be used for the fishing e.g. a fine insect pin, a bamboo splinter,an eyelash, a sharpened toothpick. The procedure for the fishing is thus:

1. Pour or use a pipette to place some of the nematode suspension into a petridish/counting dish. 2. Place on a stereomicroscope using the lowest convenient magnification 3. Locate a nematode and gently lift the nematode to the surface of the water with the fishing tool. Adjust microscope focus to keep the nematode in view whilst fishing the nematode out of the water solution. 4. Holding the fishing instrument under the nematode, lift the nematode out of the water. The nematode should be hanging on the tip of the picking instrument. 5. Gently place the tip of the pick into a drop of water on a slide. 6. Cover the slide with the coverslip and view under compound microscope.

FIG 4.12 Nematode fishing

4.6

NEMATODE CULTURING

PROTOCOL FOR NEMATODE CULTURING FURTHER EXPERIMENTAL RESEARCH Aim: To culture and multiply a named nematode for further research works under a laboratory condition. Materials used: Knife/peeler Spatula Culturing media (carrots) Micro flow laminar workstation 10ml measuring cylinder Carmel hair brush tissue paper heat source forceps kanisters Bulb pipette Petri-dishes

Protocol: A named nematode genus was extracred and fished out for culturing for further research work (inoculation). The metal equipment were sterilized in an auto clave (120c) for 15 mins. The equipment were placed inside the laminar workstation with the carrot (the culturing media) washed and wrapped in tissue paper. Using an ethanol spray to sterilize the hands too to avoid contaminating the medium, the forceps were picked and used to hold the carrots. The carrots were cut into sizeable rings and each ring placed in a petri-dish. 0.06g of streptomycin was measured and poured into a 10ml of water in a measuring cylinder. Then, the fished nematode species were put inside the solution. Using a Pasteur pipette, the nematode solution were inoculated (about 3-4 drops per dish) into each petri-dish containing the culturing media and on top of the media. The petri-dishes were covered and sealed with parafilm and then arranged inside a canister box and placed inside the

incubator.

4.7

SOIL STERILIZATION

For screen house research experiments, the cultivars/ inoculants are not just planted on any-how soil as such carelessness may make or in most cases mar the aim of the research. Soil sterilization simply refers to making a quantity of research soil free from microbial contaminations. In IITA, soil for planting of cultivars in the screen house were sterilized before they were used. There are 3 soil sterilization machines or ways of sterilizing a soil for research plantings: Use of electric sterilizer Use of steam soil sterilizer Use of the crude/ traditional method (i.e. cooking of the soil on fire for 2 hours).

PROBLEMS ENCOUNTERED DURING THE TRAINING AND OPINED SOLUTIONS. The International Institute of Tropical Agriculture (IITA) was like a home away from home for me during my training days there. I was so keen to learn and they were also eager to impact knowledge. In fact I found it a bit difficult to write this section of my report about the institute. But however how good a system is, it must have its short comings. IITA being a human-run institution has its short-comings pertaining to SIWES training. One of the institutes short comings was their inability to provide accommodation for us. Due to the nature of the job we do; research for development, the work area ought not to be left far from the workers abode so as to easily be near your research works and see to its timely conclusion. Another short coming of the institute is their un-organized SIWES training unit. The unit, apart from the students unit supervision, does not do any supervisory work to check on the students progress in his/her attached unit. In my opinion, I would suggest that the institute build a residential area for her workers to enable them to be closer to their research works inside the institute. Also, for the training units case, I would suggest the management braces up the unit and sensitize and mobilize them on how to cater for the general well-being of the trainee students they admit for training. In all, the institute, in my own view, is the best in terms of human management and training in areas of agricultural research for development.

CONCLUSION AND GENERAL APPRAISAL OF THE PROGRAM. The SIWES has positively contributed to my training as a future researcher. At the SIWES workplace (i.e. IITA), I was able to reconcile theoretical principles learnt in school with real parasitological and entomological practices especially in the agricultural aspects. I also learnt various techniques and research procedures that are relevant to my course of study and would help nurture me into my future career as a researcher, the biostatistics I learnt and employed in some of the experiments carried out in some of the units I was attached helped in broadening my view and in applying all that I was taught in Bio303 (Biometry) and STAT202 (Biostatistics for Agric and Biosciences ) respectively in school. Furthermore, I received extensive training on the basic laboratory ethics required of a research scientist. I was also, thanks to this SIWES program, able to go for agric-extension works outside my SIWES work place and in most remote farmerareas for sample collections and or for any other research purposes as may be required. In general, SIWES gave me the opportunity to learn about good work ethics, good interpersonal and communication skills necessary to prepare me for the real work situation I will meet after graduation and expose me to work methods and technics in the handling of research equipment and machinery that I havent seen before and finally make my transition from school to the labour market smooth and enhance my contacts for later job placement.

REFRENCES

Adediran, J.A , A.A. Adegbite, T.A. Akinlosotu, G.O. Agbaje, L.B. Taiwo, O.F. Owolade and G.A. Oluwatosin.(2005) Evaluation of fallow and cover crops for nematode suppression in 3 agroecologies of south-western Nigeria. African journal of Biotechnology. Vol.4 (10) pp 10341039; October,2005.

Akobundu, I.O. and Udensi, U.E.(1995) Effects of Mucuna species and fertilizer levels on the control of Imperata cylindrical. Abstract in Weed Science Society of Nigeria, Proceedings of the 22nd Annual conference of the Weed Science Society of Nigeria. IITA Ibadan, Nigeria, 6th10th November, 1995.

Agwuna, R.N. (2012) Detailed manual on SIWES guidelines and operations for tertiary institutions. Rex Charles and Patrick limited.

Akol, A.M., Maneno, Y. Chidege, Herbert A.L. Talwana, and John R. Mauremootoo. Sesamia calamistis Hampson, 1910-African pink stalkborer.(2011) BioNET-EAFRINET keys and fact sheets. http://www.eafrinet@africaonline.co.ke. Date accessed: 3rd October, 2013.

Coyne, D.L., Nicol, J.M. and Claudius-Cole,B (2007) Practical Plant Nematology: A field and laboratory guide. SP-IPM secretariat, IITA, Cotonou, Benin.

Dixon, A.G.O. et.al (2012). Improved CassavaVariety Handbook. http://www.iita.org/pastpublication. Date accessed: 21st October,2013.

Heinrichs, E.A. (2004). Rice-feeding insects and selected natural enemies in west Africa: biology, ecology and identification. http://www.riceweb.org/publications/awards/articles/Ricefeedinginsects2.pdf. Date accessed: 3rd October, 2013.

Hill, Dennis S. (2008) Pests of crops in warmer climates and their control. Pg 470-471. Springers.

Hooper, D.J., Johannes Hallmann and Sergei Subbotin. Methods for extraction, processing and detection of plant and soil nematodes. In Michael, Luc, R.A. Sikora and J. Bridge(2005). Plant Parasitic Nematodes of Sub-tropical and Tropical Agriculture. Pp 53-86. CABI publishing.

Manyong, V.M., V.A. Houndekon, P.C. Sanginga, P. Vissoh and A.N. Honlonkou. (1999) Mucuna fallow diffusion in southern Benin. IITA publication and Meg-comm Network. Myers, P., R. Espinosa, C. S. Parr, T. Jones, G. S. Hammond, and T.A.Dewey.(2013). The Animal Diversity Web (online). Accessed at http://animaldiversity.org. Date accessed: 21st

October, 2013

Sharma, H.C. et.al.1999. The legume podborer, Maruca vitrata: Binomics and Management. Information bulletin no 55 (InEn. Summaries in En,Fr.) Pg 42. International Crops Research Institute for Semi-arid Tropics.

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APPENDIX APPENDIX A

PREPARATION OF VARIOUS STOCK SOLUTIONS FOR INSECT REARING IN THE BORER LABORATORY 1. Preparation of 1% Sodium Hypochlorite solution: Add 400ml water to 100ml of sodium hypochlorite solution to obtain 1% solution. The stock sodium hypochlorite solution is 5%. 2. Preparation of 10% formaldehyde: Add 270ml of water to 100ml of the 37% stock solution of formaldehyde to obtain a 10% solution. Or 10ml of water to 100ml of water to 10ml of formaldehyde. 3. Preparation of 15% choline chloride solution: Add 100ml of water to 15g of choline chloride to obtain 15% solution. 4. Preparation of 25% acetic acid solution: Add 75ml of water to each 25ml of acetic acid to obtain 25% solution. 5. Preparation of 4ml potassium hydroxide solution: Add 224g of KOH pellets to 1000ml of water to obtain a 4ml solution of KOH 6. Sorbic acid: 40ml of a stock solution (100g sorbic acid in 500ml of 95% ethyl alcohol) 7. Methyl parahydrobenzoate: 75ml of a stock solution (200g of methyl parahydroxybenzoate in 1000ml of 95% ethyl alcohol) 8. Mixture of phosphoric acid and propionic acid: 86ml of a stock solution (836ml of propionic+84ml of phosphoric+ 1000ml of distilled water)

9. Vitamin suspension per 100ml of suspension in water contain: Calcium pentaonate 2.4g; Niacin 0.6g;Riboflavin 0.3g; Folic acid 0.3g; Thymine HCl 0.15g; Pyridoxine HCl 0.15g, Biotin 0.012g; B12 0.0006g.