Solanum Lycopersicum L.) in GHANA

Home , Fusarium wilt

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PATHOGENICITY OF Meloidogyne incognita AND Fusarium oxysporum f. sp. lycopersici

ON GROWTH, YIELD AND WILT SEVERITY IN TWO VARIETIES OF TOMATO

(Solanum lycopersicum L.) IN GHANA

VIGBEDOR DELADEM HANNAH

10375148

THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN

PARTIAL FUFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER

OF PHILOSOPHY (MPHIL) CROP SCIENCE DEGREE

DEPARTMENT OF CROP SCIENCE

UNIVERSITY OF GHANA

JULY, 2019

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DECLARATION

I, HANNAH DELADEM VIGBEDOR, do hereby declare that except for references to the work of other researchers that have been duly acknowledged, this work submitted is the outcome of my original investigations and findings and that this thesis has neither in whole or in part been presented for another degree elsewhere.

…………………………… …………………..

HANNAH DELADEM VIGBEDOR DATE (STUDENT)

………………………………… …………………..

DR. S. T. NYAKU DATE (MAIN SUPERVISOR)

………………………………. …………………..

DR. E. W. CORNELIUS DATE (CO – SUPERVISOR)

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DEDICATION I dedicate this thesis to my parents, Mr. Samuel Vigbedor and Mrs. Juliana Vigbedor for their support throughout the period of this study.

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ACKNOWLEDGEMENT My deepest thanks to God Almighty for his grace and protection throughout the years. I also want to thank Dr. Seloame Tatu Nyaku, Dr. Eric Cornelius and Dr. Vincent Eziah, all lecturers at the Department of Crop Science who encouraged and directed me in the course of this study. May God bless and increase you in wisdom.

To Mr. Samuel Osabutey, Mr. Richard Otoo and Mr. Issac Bedu technicians at Plant

Pathology Laboratory I say thank you for all the technical support during the laboratory work. My deepest gratitude also goes to the teaching assistants of the Department of Crop

Science for their help on the field.

My heart felt appreciation also goes to Mr. William Asante, Mr. Nicholas Agyekum and Mr.

Stephen Atsu of University of Ghana farms for their assistance during the field experiments.

Finally, I say a big thank you to all the wonderful people who were blessings during the entire period of my study especially, Mr. Yaw Akoto Dankwa, Osei Kwadwo Mensah and

Joel Kyere Nimarko. God richly bless you all.

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Table of Content

DECLARATION ...... i

DEDICATION ...... ii

ACKNOWLEDGEMENT ...... iii

Table of Content ...... iv

List of Tables ...... vii

List of Figures ...... viii

List of Appendices ...... viii

ABSTRACT ...... xi

CHAPTER ONE ...... 1

1.0 INTRODUCTION ...... 1

CHAPTER TWO ...... 4

2.0 LITERATURE REVIEW ...... 4

2.1 Origin, Botany and Importance of Tomato ...... 4

2.2 Climatic and Soil Requirement for Tomato Growth ...... 6

2.3. Tomato Varieties in Ghana ...... 6

2.4. World Tomato Production ...... 7

2.5 Tomato Production in Ghana ...... 8

2.6 Losses in Tomato Production ...... 9

2.7 Fusarium oxysporum ...... 10

2.7.1 General Characteristics of Fusarium oxysporum ...... 10

2.7.2. Biology and Lifecycle of Fusarium oxysporum ...... 10

2.7.3. Fusarium Wilt Disease in Tomato...... 11

2.8. The Root-Knot Nematode ...... 12

2.8.1 Root- Knot Damage ...... 12

2.8.2 Biology and Life Cycle of Meloidogyne Species...... 12

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2 .8.3 Symptoms of Meloidogyne On Host Plant ...... 13

2.9. Disease Complexes and Interactions of Nematode and Fungi ...... 14

2.10. Types of Interaction Between Fungi and Nematodes ...... 17

2.10.1 Synergistic Interaction ...... 17

2.10.2. Antagonistic Interaction ...... 18

2.10.3 Neutral Interaction ...... 19

3.0 MATERIALS AND METHOD ...... 20

3.1. Isolation and Confirmation of Microorganisms Causing Root- Knot and Fusarium Wilt Disease ...... 20

3.1.1 Nematode Extraction ...... 20

3.1.2. Isolation of Fungi Causing Tomato Wilt Disease ...... 21

3.2 Determination of the Individual and Combined Effects of Fusarium oxysporum f. sp. lycopersici and Meloidogyne Spp. on the Growth and Yield of Tomato...... 22

3.2.1 Experimental Site ...... 22

3.2.2 Experimental Design ...... 22

3.2.4 Nursing of Seeds ...... 24

3.2.5 Preparation of Micro Biological Media for Isolation of Fungi ...... 24

3.2.6 Potato Dextrose Agar (PDA) Preparation ...... 24

3.2.7. Preparation of Fusarium oxysporum f.sp lycopersici Inoculum ...... 24

3.2.8 Inoculation of Tomato Plant with Fusarium oxysporum f. sp lycopersici Inoculum ...... 24

3.2.9 Re-Isolation of Fungi ...... 25

3.2.10 Data Collection ...... 25

3.3. Determination of Reproductive Ability of Meloidogyne incognita on Tomato, After Fusarium oxysporum f. sp.lycopersici Infections...... 27

3.3.1. Nematode Egg Population ...... 27

3.3.2. Root Score ...... 27

3.3.3 Determination of Nematode Reproductive Index ...... 27

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3.4 Data Analysis ...... 28

4.1 Microorganisms Causing Root- Knot and Fusarium Wilt Diseases of Tomato ...... 29

4.1.1 Meloidogyne incognita ...... 29

4.2 Individual and Combined Effects of Fusarium oxysporum f. sp. lycopersici and Meloidogyne Spp. on the Growth and Yield of Tomato...... 30

4.2.1 Results From University Of Ghana Farms ...... 30

4.3 Reproductive Ability of Meloidogyne on Tomato After Fusarium oxysporum f. sp. lycopersici Infections ...... 47

4.3.1. Results From University Of Ghana Farm ...... 47

4.3.2 Results From National Service Farm ...... 49

CHAPTER FIVE ...... 52

5.0 DISCUSSION ...... 52

CHAPTER SIX ...... 55

6.0 CONCLUSION AND RECOMMENDATION ...... 55

6.1 CONCLUSION ...... 55

6.2 RECOMMENDATIONS ...... 55

REFERENCES……………………………………………………………………………….56

APPENDICES ...... 66

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List of Tables

Table 1: Composition Of Tomato, Value Per 100g……………………………………………5

Table 2. Top Ten Major Tomato Producing Countries In The World In 2017……………….. 7

Table 3. Tomato Production, Yield, And Area Harvested In Ghana………...………...…….8

Table 4. Climatic Data Of Experimental Area from August 2018 to January, 2019 22

Table 5. Rating Scale For Wilt Incidence……………………………………………………27

Table 6. Fresh And Dry Shoot Weights For Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The University Of Ghana Farm…………………….... 34

Table 7. Fresh And Dry Root Weights For Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The University Of Ghana Farm………………………..35

Table 8. Wilt Incidence (%) And Wilt Severity (%) In Mongal F1 And Petomech Tomato Varieties Fusarium After Inoculations On The University Of Ghana Farm………..36

Table 9. Yield For Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The University Of Ghana Farm……………………………………………………...37

Table 10. Fresh And Dry Shoot Weights For Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The National Service Farm…………………………...42

Table 11. Fresh And Dry Root Weights For Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The National Service Farm…………………………..43

Table 12. Wilt Incidence (%) And Wilt Severity (%) In Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The National Service Farm………………... 45

Table 13. Yield For Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The National Service Farm……………………………………………46

Table 14. Root Gall Score And Egg Count In Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The University Of Ghana Farm……………………… 48

Table 15. Initial Nematode Count, Final Nematode Count And Reproductive Factor Of Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The University Of Ghana Farm………………………………………………………………49

Table 16. Root Score And Egg Count In Mongal F1 And Petomech Tomato Varieties After Fusarium Inoculations On The National Service Farm………………………………… 50

Table 17. Initial Nematode Count, Final Nematode Count And Reproductive Factor Of Mongal F1 Petomech Tomato Varieties After Fusarium Inoculations On The National Service Farm……………………………………………………………………………. 51

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List of Figures

Fig.1. Micrograph Of Meloidogyne incognita, J2 (A) And Nematode Egg (B). 400 X Magnification...... 29

Fig.2. Two Weeks Old Culture Of Fusarium oxysporum f.sp. lycopersici On Potato Dextrose Agar(A) And Micrononidia Of Fusarium Oxysporum f.sp. lycopersici 400x (B)...... 30

Fig.3. Heights Of Mongal F1 And Petomech Plants Inoculated With Fusarium oxysporum f. sp.lycopersci On University Of Ghana Farm...... 31

Fig.4. Girths Of Mongal F1 And Petomech Plants Inoculated With Fusarium oxysporum f. sp. lycopersci On University Of Ghana Farm from weeks 4 to 10...... 32

Fig.5. Chlorophyll Content Of Mongal F1 And Petomech Plants Inoculated With Fusarium oxysporum f. sp. lycopersci On University Of Ghana Farm from weeks 4 to 10...... 33

Fig.6. Heights Of Mongal F1 And Petomech Plants Inoculated with Fusarium oxysporum f. sp. lycopersici on National Service Farm from weeks 4 to 10...... 39

Fig.7. Girths Of Mongal F1 And Petomech Plants Inoculated with Fusarium oxysporum f. sp. lycopersici on the National Service Farm from weeks 4 to 10...... 40

Fig.8. Chlorophyll Content Of Mongal F1 And Petomech Plants Inoculated with Fusarium oxysporum f. sp. lycopersici on the National Service Farm from weeks 4 to 10...... 41

Fig.9. Interaction In Dry Root Weight of Mongal F1 and Petomech Tomato Varieties and Pathogen Inoculation treatments…………………………………………………………………………44

List of Appendices

Appendix 1 Plant Height University Of Ghana Farm ...... 66

Appendix 2 Plant Girth University Of Ghana Farm ...... 67

Appendix 3 Chlorophyll Content University Of Ghana Farm ...... 68

Appendix 4 Wilt Incidence On University Of Ghana Farm ...... 69

Appendix 5 Wilt Severity On University Of Ghana Farm ...... 69

Appendix 6 Fresh Shoot Weight On University Of Ghana Farm ...... 70

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Appendix 7 Fresh Root Weight On University Of Ghana Farm ...... 70

Appendix 8 Dry Shoot Weight On University Of Ghana Farm ...... 70

Appendix 9 Dry Root Weight On University Of Ghana Farm ...... 70

Appendix 10 Yeild On University Of Ghana Farm ...... 71

Appendix 11 Initial Nematode Count On University Of Ghana Farm ...... 71

Appendix 12 Final Nematode Count On University Of Ghana Farm ...... 71

Appendix 13 Egg Count On University Of Ghana Farm ...... 72

Appendix 14 Root Gall Score On University Of Ghana Farm ...... 72

Appendix 15 Analysis Of Variance Of Plant Height On National Service Farm ...... 72

Appendix 16 Analysis Of Variance Of Plant Girth On National Service Farm ...... 73

Appendix 17 Analysis Of Variance Of Chlorophyll Content On National Service Farm ...... 74

Appendix 18 Analysis Of Variance Of Wilt Incidence On National Service Farm ...... 75

Appendix 19 Analysis Of Variance Of Wilt Severity On National Service Farm ...... 75

Appendix 20 Analysis Of Variance Of Fresh Shoot Weight On National Service Farm ...... 75

Appendix 21 Analysis Of Variance Of Fresh Root Weight On National Service Farm ...... 76

Appendix 22 Analysis Of Variance Of Dry Shoot Weight On National Service Farm ...... 76

Appendix 23 Analysis Of Variance Of Dry Root Weight On National Service Farm ...... 76

Appendix 24 Analysis Of Variance Of Yield On National Service Farm ...... 77

Appendix 25 Analysis Of Variance Of Initial Nematode Count On National Service Farm .. 77

Appendix 26 Analysis Of Variance Of Final Nematode Count On National Service Farm ... 77

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Appendix 27 Analysis Of Variance Of Egg Count On National Service Farm...... 78

Appendix 28 Analysis Of Variance Of Root Gall Score On National Service Farm ...... 78

Appendix 29 Root–Knot Nematode Rating Chart – Bridge And Page ...... 79

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ABSTRACT

The pathogenicity of fungus, Fusarium oxysporum f. sp. lycopersici and nematode,

Meloidogyne incognita on growth, yield and wilt severity was studied on two tomato varieties, Mongal F1 and Petomech in a randomized complete block design, factorial experiment with three replications on the University of Ghana and National service farms from July 2018 to June 2019. Each plot had an area of 10 m2 with 25 plants. Fusarium inoculum of 1.3×106 cells per 5 mL was inoculated on fields naturally infested with

Meloidogyne incognita at 7, 14 and 21 days after transplanting tomato seedlings to the fields to study the combined effect of both pathogens (NF7, NF14, NF21). To study their individual effects, some plots were inoculated with Fusarium oxysporum after transplanting but treated with nematicide (F), another plot was not inoculated with Fusarium oxysporum on field naturally infested with Meloidogyne incognita (N) and a control plot where field was not inoculated with Fusarium oxysporum and was treated with nematicide (C). Nematicide used in this study was Velum prime 400 SC with active ingredient Fluopyram. Meloidogyne incognita and Fusarium oxysporum f.sp. lycopersici were confirmed as the causal agents of root- knot and Fusarium wilt diseases respectively. Meloidogyne incognita and Fusarium oxysporum f sp. lycopersici existing either as individuals or combined did not affect Plant height, girth, fresh root weight and yield of tomato. Mongal F1 tomato variety is tolerant to

Fusarium wilt and root- knot disease caused by Fusarium oxysporum f. sp. lycopersici and

Meloidogyne incognita respectively. Fusarium oxysporum f. sp. lycopersici reduced the reproductive ability of Meloidogyne incognita on Mongal F1 and Petomech tomato varieties, with the reproductive ability of Meloidogyne incognita ranging from 0.0 to 0.3 and 0.0 to 0.2 on University of Ghana farm and National Service farm respectively. The least reproductive factor from the University of Ghana farm was (0.0) from Mongal F1 seen in the Control. The

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least reproductive factor was also 0.0 from treatment N in Mongal F1 and in treatment F in

Petomech both from the National Service farm.

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CHAPTER ONE 1.0 INTRODUCTION

Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family sometimes referred to as Nightshade family of which several plants such as Irish potato, pepper, eggplant and tobacco also of economic importance belong to (Knapp, 2002).

The Crop exists as a domesticated plant (Peralta et al., 2008) and is cultivated widely throughout the world. In Ghana, tomato is consumed in most households as fresh produce in salads, cooked as a vegetable in stews and soups or processed as tomato puree, tomato sauce, ketchup, juice and can also be dried. The fruit contains lycopene which has positive health benefits and is the second most important vegetable rich in vitamins and minerals (Bradley,

2003). It contains vitamins A, B, C and some amount of potassium, iron and phosphorus

(Vossen van der et al., 2004). Tomatoes are produced seasonally in Ghana reflecting differences in rainfall pattern and access to irrigation (Robinson & Kolavali, 2010).

Commercial production of tomatoes is intense in the Upper East, Northern, Bono and Greater

Accra regions of Ghana which supply the market at various times of the year (Adu- Dapaah

& Oppong-Konadu, 2004).

In 2011, the country produced 320,520 tonnes of tomato (FAO, 2017). Over the past decade, production has been stagnant and to some extent, declined (Bortey & Osuman, 2016).

Tomato export, in value has been declining from GH₵.1.2 million in 2008 to GH₵ 0.15 million in 2012. In the same period, its import has increased from GH₵ 50 million to GH₵

280 million (Asuming-Brempong & Boakye, 2008; Robbinson & Kolavalli, 2010). Ghana also imports 27,000 metric tonnes of processed forms in puree and paste from Europe monthly (Monney et al., 2009). Clearly, Ghana is not able to meet the demand of consumers when it comes to the production of tomatoes. The collapse of the Pwaluga tomatoes factory in

Northern Ghana which was to process tomato affected most farmers as well as stakeholders

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and contributed to import of tomatoes in Ghana (Clottey et al., 2009). Biotic and abiotic factors as well as institutional challenges contributed to the collapse of the industry (MoFA,

2009).

The excessive build-up of soil nematodes and uncontrolled spread of fungal, bacterial, viral diseases contribute to low yield during production and is widely been referred to as the

‘Tomato Disease Complex’ (Inusah, 2005; Tanzubil et al., 2004). Among these, Fusarium oxysporum and root- knot nematodes are of importance. Fusarium oxysporum f.sp lycopersici causes Fusarium wilt, one of the most economically important and wide spread disease of tomato (Reis et al., 2005).

The pathogen invades the root epidermis of the tomato plant and spreads into the vascular tissue colonizing the xylem vessels and blocking it with mycelium and conidia resulting in wilt symptoms (Beckman, 1987). Root - knot nematodes from the genus, Meloidogyne are also important agricultural pest that cause extensive damage to a wide variety of crops including tomatoes (Sasser, 1980; Sikora & Fernandez, 2005). They feed on the roots of the tomatoes, produce galls and eventually cause wilting of plants. Fusarium and root- knot nematodes are both soil borne and cause wilting in tomatoes which symptoms if not carefully examined may be mistaken as phosphorus deficiency in the soil. It is possible that, the presence of both pathogens in the soil at a time could cause interaction in their individual effects on plant which may be rather severe.

Studies on interactions between root-knot nematode and Fusarium oxysporum are well observed on other crops such as banana (Jonathan & Rajendran 1998), cotton (Jeffers &

Roberts, 2003), vine (Harris & Ferris, 1991) and Beans (France & Abawi, 1994). Disease complex caused by interaction of nematodes and Fungus has been known to cause changes on mineral absorption, physiological and biochemical changes in infected plants (Lobna et al.,

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2017) to reduce vigour of the infected plants and finally cause death (Masse et al., 2002). For a disease complex to develop, a synergistic interaction must occur between the organisms involved (Back et al., 2002). Interactions may be synergistic which could be either be positive or negative. Positive interaction between the pathogens produce a much higher damage compared to the sum of individual damage by each pathogen existing individually. In an antagonistic interaction between nematode and fungus, less plant damage results compared to the sum of the individual organismal parasitism on the host plant. A third form of interaction is neutral where nematode- fungus interactions produce the same damage to plants as the individual organisms (Back et al., 2002).

Not much work has been done on the interactive effect of Meloidogyne incognita - Fusarium oxysporum f. sp. Lycopersici interaction on crop varieties in Ghana. It was therefore important that the associations between these pathogens were studied to inform decision on their management for improved tomato yield and livelihood of tomato farmers.

The main objective of this study was to evaluate the pathogenicity of Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita on tomato in two field experiments.

The specific objectives were to:

• Isolate and confirm microorganisms causing root- knot and Fusarium wilt

diseases of tomato.

• Determine the individual and combined effects of Fusarium oxysporum f. sp.

lycopersici and Meloidgyne spp. on the growth and yield of tomato.

• Determine the reproductive ability of Meloidogyne on tomato after Fusarium

oxysporum f. sp. lycopersici infections.

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Origin, Botany and Importance of Tomato

Tomato, Solanum lycopercium L. was formally named Lycopersicon esculentum Mill until further research into its molecular sequences considered it to be included in the genus,

Solanum L. (Perltra et al., 2008) and renamed as Solanum lycoperscium. Wild tomato was known from pre- Columbian American history to have originated from Western South

America. Its domestication, however, is unclear since it there two hypotheses. Some believe it to be domesticated by the Peruvian while others say it has Mexican origin (Peralta et al.,

2008). The crop was later introduced in Europe in the sixteenth century where it was believed to be poisonous (Preedy, 2008) so it was not until two hundred years before it was consumed.

Even though it was consumed in Italy (Vergani, 2002), botanists and Consumers mistrusted the fruit (Saavedra et al., 2017) and was considered a perennial crop from the Mediterranean area which killed people who ate alkaloids (Gomez et al., 2010). Tomato was then considered an ornamental plant from the mandrake family (Vergani, 2002).

Tomato is a self-fertilizing variety and highly homozygote. It is mostly cultivated as an annual but is a perennial plant. The leaves of the tomato plant is bipinnate with hairy stem and its flowers usually has five petals. It is however not unusual to find seven or more petals

(Blanca et al., 2012). Solanum lycopercium L depending on the variety could be determinate of indeterminate with an average height of about three metres (OECD, 2017). The tomato fruit botanically has characteristics of berries, a fleshy fruit which encloses the seed in a pulp.

The outer skin has the fruit wall and the placenta (OECD, 2008). The tomato crop does well in temperatures between 26°C and 32°C during seed germination and between 17°C to 27°C for cultivation (Dumas et al.,2003). At higher temperatures, flowers may drop. The plant

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requires a controlled amount of water, excess irrigation at flowering stage increases flower drop whiles less water reduces yield and plant growth (Gautier et al., 2008) .Tomato is one of the most versatile vegetable fruits which can be eaten raw in salads or processed into several other forms including tomato paste, ketch up or even cooked as stews. The preference of consumers to tomato products is usually influenced by quality characteristics, including texture, flavour and colour. Tomato also contributes beneficially to human health and is known to reduce the risk of cardiovascular diseases (Willcox et al., 2003) as well as prostate cancer (Giovannucci, 1999; Khachik et al., 2002). These benefits are as a result of its high carotenoids’ content such as lycopene; which is most abundant, Carotene, phytofluene, phytoene and neurosporene (Gomez et al., 2010) (Table.1).

Table 1: Composition of Tomato, Value per 100g Nutrient (Unit) Amount Nutrient (Unit) Amount

Water (%) 94.50 Sodium (mg) 5

Energy (Kcal) 18 Zinc (mg) 0.17

Carbohydrate (g) 3.92 Thiamin (mg) 0.014

Protein (g) 0.88 Riboflavin (mg) 0.019

Ash (g) 0.50 Manganese (mg) 0.114

Lipid (g) 0.20 Vitamin C (mg) 12.7

Fibre (g) 1.20 Niacin (mg) 0.594

Potassium (mg) 237 Lycopene (µg) 2573

Phosphorous (mg) 24 Alpha – Carotene (µg) 101

Copper (mg) 0.059 Lutein+ Zeaxanthcin (µg) 123

Beta- Carotene (µg) 449

Source: USDA food composition data (2007).

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2.2 Climatic and Soil Requirement for Tomato Growth

Tomato is a warm-season crop with a fairly cool, dry climate to obtain high yields. The optimum temperature for most varieties range between 21°C and 24°C (Green et al., 1989).

Fruit set colour and colour of leaves are affected by light intensity. If the plant is water stressed for a long period, flowers and buds may drop and fruit may also split. In too heavy rains, fruit rot occur as a result of favourable conditions for fungal infection and growth

(Shankara et al., 2005). Plant tissue get damaged at temperatures below 10°C and above 38°C

(Rice et al., 1993).

Generally, tomatoes grow well in sandy loam soils (Shankara et al., 2005) but will also grow in a variety of soils if the soils are warm, not saline, have good aeration and have a high- water holding capacity. The soil should also have a pH of 5.5 to 6.8 and contain easily available nutrients (Rice et al., 1993).

2.3. Tomato Varieties in Ghana

Tomato varieties all over the world are classified based on colour and shape of fruit, days to maturity, height of plant and other growth parameters which include whether it is determinate or not (Norman, 1992). Most varieties in Ghana used for commercial purposes are introduced varieties which are well adapted to the local environment. The local varieties cultivated are

‘Caterpillar’, ‘Cocoba’, ‘Burkina’, ‘Ashanti’, ‘Power’, ‘Ankoma’ and ‘Rando’ (Khor, 2006).

Recommended varieties of tomato include Roma VF, Pectomech, Pectomech VF, Wosowoso,

Rio Grande, Cac J and Lauranno 70 (MOFA, 2009) with Pectomech being the most preferred variety by consumers and for processing (Robinson and Kolavalli, 2010). In the Upper East

Region of Ghana (Vea), Pectomech and Roma are mostly grown (Clottey et al., 2009).

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The main sources of seeds for Ghanaian farmers are reputable seed dealers from recognised seed companies. There are also new and improved varieties that are developed by plant breeders to enhance the characteristics of the local varieties. Some of these varieties are

Petomech, Mongal F1, Pantha 17, Kiara F1, Tropimech and Sumo F1.

2.4. World Tomato Production

Even though tomato is believed to have originated from the Southern part of America, its cultivation has now been adopted around the world and is much consumed compared to other vegetables (Eric et al., 2015). Countries all over the world have mastered its production with

China being the world’s leading producer of tomatoes (Table 2). In 2016, China cultivated

110,000 hectares of land and harvested 56.8 million tonnes of tomato with the least farmer earning about $21,800 annually (FAO, 2017).

Table 2. Top Ten Major Tomato Producing Countries in the World in 2017 Rank Country Value (tonnes) per annum

1 China 59,514,773

2 India 20,708,000

3 Turkey 12,750,000

4 United States of America 10,910,990

5 Egypt 7,297,108

6 Iran 6,177,290

7 Italy 6,015,868

8 Spain 5,163,466

9 Mexico 4,243,058

10 Brazil 4,243,058

Source: USDA (2017)

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2.5 Tomato Production in Ghana

Ghana is a country with over 25 million people as its population and has a land area of

238,500 square km with cultivated land being about 79 million hectares out of which 50,000 hectares is suitable for tomato production (Addo et al., 2015). In Ghana, tomato is a very important vegetable because almost all Ghanaian dishes require tomato as an ingredient

(Tambo & Gbemu, 2010). It is not surprising that Ghana was cited as second largest consumer of tomato paste in the world (Baba et al., 2013). The National Tomato Produces

Federation, Ghana, state that the country imports 7,000 metric tonnes of fresh tomatoes monthly from neighbouring countries, 27,000 of processed tomato from Europe yearly and produces about 510,000 metric tonnes every year. Tomato produced from the areas cultivated has not been sufficient to meet the countries demands over the years (Table 3). The country is unable to produce enough tomato to meet consumption. Commercial production of tomato occurs in the Greater Accra, Northern, Upper East and Bono Regions in Ghana. These regions make supply throughout the year in the country (Adu- Dapaah & Oppong-Konadu,

2004).

Table 3. Tomato Production, Yield, and Area Harvested in Ghana. Year Production (tons) Yield (hg/ha) Area harvested (ha)

2008 284,000 60,426 47,000

2009 317,520 72,000 44,100

2010 315,520 72,063 44,200

2011 320,500 72,103 44,450

2012 321,000 71,732 44,450

2013 340,218 73,800 46,100

2014 366,772 78,037 47,000

2015 366,772 78,037 47,000

2016 366,772 78,037 47,000

2017 371,811 77,570 47,932

Source: FAOSTAT (2017)

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2.6 Losses in Tomato Production

Pre harvest, harvest and post-harvest practices affect the quality of tomato (Sablani et al.,

2006). Post harvest loses are highest in developing countries (FAO, 2017). Aworth &

Olorunda (1981) reported that 50% of fresh tomato is lost between harvesting, transportation and consumption of the tomato in Nigeria. Tomato is one of the plants which is highly susceptible to damage and is infected by almost all plant pathogens. The fruit is relatively soft and can easily be damaged during handling and transportation. A major cause of loss in tomato production is caused by parasitic pathogens (Horna et al., 2006) thus bacterial, fungal, viral and nematode diseases which cause considerable losses to farmers (Shankara et al.,

2005).

Some of the diseases of tomato worldwide include, bacterial canker (Clavibacter michiganensis subsp. michiganensis), Tomato mosaic virus, tomato leaf curl, bacterial wilt caused by Ralstonia solanacearum, tomato spotted wilt (Tomato spotted wilt virus) and bunchy top (Tomato bunchy top virus). Important diseases are Fusarium wilt caused by

(Fusarium oxysporum f.sp. lycopersici) grey leaf spot (Stemphyllium solani), early blight

(Alternaria solani), Pythium damping off (Pythium debaryanum) and late blight

(Phytophthora infestans).

Important species of nematodes infecting tomato include the root- knot nematode

(Meloidogyne spp.), stubby-root nematode (Trichodorus spp. and Paratrichodorus spp.), lesion nematode (Pratylenchus sp.) and sting nematode (Belonolaimus longicaudatus).

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2.7 Fusarium oxysporum

2.7.1 General Characteristics of Fusarium oxysporum

Fusarium species cause a wide range of diseases in different host plants (Irom et al., 2011).

Fusarium oxysporum is soil dwelling with a large number of strains which could be pathogenic or non-pathogenic. The pathogen can survive long periods in the soil as a saprophyte feeding on dead decaying organic matter. In the rhizosphere, Fusarium can survive as mycelium and in different spore types but usually as chlamydospores (Schippers & van Eck, 1981). The pathogen can be airborne, soil borne or can be carried within plant residue. It can also be recovered or isolated from any part of the plant (Summeral et al.,

2003). In culture, Fusarium produces a wool to cotton like, flat spreading colony with a front colour ranging from cream, white to yellow and underneath colour ranging from colourless, dark brown, brown to tan (De Hoog et al., 2000).

2.7.2. Biology and Lifecycle of Fusarium oxysporum

Fusarium oxysporum produce three types of asexual spores (chlamydospore, microconidia and macroconidia) and do not produce sexual spores (Booth, 1971; Nelson et al., 1981).

Chlamydospore are produced from modified vegetative hyphal segments and usually formed on senescent host tissues or old cultures. They are also formed either in pairs or singly even though they can be formed in chains or clusters. They have huge numbers in hyphae and conidia with thick walls that are either rough or smooth. Their shape is globuse to sub- globuse. (Agrios, 2005).

Microconidia on the other hand are the most abundant spores produced mostly on the aerial mycelia. They have an oval to kidney shape, uninucleate and germinate poorly (Ebbole &

Sachs, 1990).

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Macroconidia have three to five cells which are curved at the edges. They are sub cylindrical, equally and gradually tapering in both ends with a pointed apical cell (Nelson et al., 1981).

They are also abundant and found on plants infested with Fusarium wilt.

2.7.3. Fusarium Wilt Disease in Tomato

The disease occurs in temperate to tropical regions. The main diseases caused by the pathogen are wilt and rot. Fusarium oxysporum is the most common fungi that causes losses in agricultural crops (Lesile et al., 2005). They produce secondary metabolites and mycotoxins such as fusaric acid, beauvericin, moniliformin and gibberellic acid (Lesile et al.,

2005). This disease is most important among fungal diseases of tomato (Sokhi et al., 1990).

Fusarium wilt in tomato is caused by Fusarium oxysporum f, sp lycopersci which is soil borne. The pathogen is ubiquitous with a high ecological as well as genetic diversity

(Mohammed et al., 2016). The fungus when introduced into the soil remains for a long a period. Temperatures between 21°C and 32° C coupled with wet weather favours the growth and multiplication of the fungus. According to Mark & Brooke, (2006), well drained soils compared to poorly drained soils are less susceptible to infection of Fusarium oxysporum f.sp lycopersci. The pathogen can affect tomato plant at all stages of growth, from nursery to fruiting stage. It causes pre-emergence damping-off where the germinating seed rots and post-emergence damping-off at the collar region and death of the young plant (Verma ,1954;

El- Helaly et al., 1962).

The fungi start by invading the root epidermis, makes it way to the vascular tissue, producing mycelium and conidia in the xylem tissue clogging and preventing water from reaching the upper parts of the tomato plant (Lucas & Knights,1987).

Initial symptoms of Fusarium wilt occur as bright yellowing of older lower leaves, usually at one side only. In mature plants, symptoms are noticed from the blooming to fruit maturation

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stage. Infected plants become stunned, older leaves droop curved downwards, plant wilts and dies eventually (Momol et al., 2004). Signs appearing as mycelial layers can even be seen on dead plants usually during wet weather (Singh, 1985).

2.8. The Root-Knot Nematode

2.8.1 Root- Knot Damage

Among nematode species, the root-knot nematode is the most dominant species causing about 15% to 60 % loss in vegetable production (Ravichandra, 2008) of which 35% of the losses has been reported on tomato (Jonathan et al., 2001). In Ghana, tomato production is limited by root-knot nematode (Meloidogyne incognita), causing over 70% losses to farmer fields (Nyaku et al., 2018). Their distribution is worldwide especially in warm climates with short mid- winter (Karssen et al., 2013). Over 100 species have been identified in the genera

Meloidogyne, (Onkendi et al., 2014) six of them are recognized to cause considerable damage

(Adam et al., 2007). The Meloidogyne species is also difficult to control (Bird & Kaloshian,

2003; Hussain et al., 2012). Measures to control them are either less efficient, too costly or harmful to the environment. They attack over 2,000 plant species which include cultivated crops and can cause up to 5% and more damage on individual fields (Agrios, 2005). In tomato fields, incognita, arenaria, javanica and hapla are most important (Jacquet et al.,

2005). Meloidogyne incognita is the most abundant species in terms of tomato attack (Kayani et al.,2017).

2.8.2 Biology and Life Cycle of Meloidogyne Species.

The Meloidogyne species has a short life cycle of about six to eight weeks allowing it the advantage to mature as the host plant does (Shurtleff & Averve, 2000). The cycle starts with

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the egg stage with the second juvenile (J2) stage developing inside the egg. After hatching, the second juvenile stage comes out of the egg into the soil to locate a suitable entry point on the host plant using their stylet to thrust through the plant (Hussey, 1985). This point is usually behind the root tip where the tissue is soft (Davis et al., 2004). The Second Juvenile stage can survive for about four weeks in the soil before penetrating the host plant (Riga,

2004). In the root, a permanent feeding site is established by injecting secretory proteins produced from the oesophageal glands through the stylet forming giant cells (Abad et al.,

2003). These cells provide nutrients for the developing second juvenile stage. The tissues surrounding the feeding site, undergoes hypertrophy and hyperplasia resulting in the formation of root-knots or galls (Moens et al., 2009). The galls or root-knots are what makes the Meloidogyne species to be easily identified (Caillaud et al., 2008). After the feeding site is established, the second Juvenile stage develops to the third and later, the fourth juvenile stage and finally into an adult male or female nematode. The female also produces egg mass in a gelatinous matrix known as an egg mass on the outer surface of the galled root using mitotic parthenogenesis (Moens et al., 2009). The gelatinous matrix protects the eggs from dehydration (Pattison, 2007). A female nematode is capable of producing over a thousand eggs (Mai and Abawi, 1987).

2 .8.3 Symptoms of Meloidogyne On Host Plant

The damage on the roots of the plant by the nematode restricts the movement of nutrients and water in the affected plant preventing the plant from growing healthy. This also creates an avenue for other pathogens to infect the plant (Nicol & Van Heeswijick, 1997).

Above ground symptoms are usually mistaken as water stress or nutritional deficiency. There is stunting, wilting during the day and chlorosis of the leaves of infected plants (Pattison,

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2007). A major underground symptom is the galling of the roots which is obvious in the name root-knot nematode (Sasser & Freckman, 1987). Infected roots produce excessive secondary roots, the root surfaces become rough appearing like a club which may be small with necrosis and show injuries (Pandey & Kalra, 2003). In tomato plants infected by

Meloidogyne, the extent of galling may be dependant on the number of nematodes present, the variety of tomato grown and the nematode species (Mai & Abawi, 1987).

2.9. Disease Complexes and Interactions of Nematode and Fungi

In the rhizosphere, pathogenic and non- pathogenic micro-organisms are in constant competition at a microscopic scale for resources (Sikora & Reimann, 2004). Microbial groups of fungi, nematodes, bacteria, protozoa, algae and micro-arthropods (Raaijmakers, 2001) continue to interact. Disease development is known to be as a result of the complex interaction between host plants, pathogens and the prevailing conditions of the environment.

According to Back et al (2002), there is further interaction in soil borne pathogens between other microorganisms as they occupy the same ecological niche. Naturally, plants are not exposed only to a single pathogen. In reality, ‘nature does not work with pure culture’ and plants diseases are as a result of associated organisms (Fawcett, 1931). Nematodes’ primary component of disease complexes causing and interactions with other diseases causing organisms may be their main economic importance (Powell, 1971). It is also known that nematodes predispose plants to secondary invasion by fungal pathogens (Mai and Abawi,

1987).

The first report of nematode interaction was recorded in cotton between Meloidogyne incognita and Fusarium oxysporum (Atkinson, 1892). After this report, other nematode-

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fungus disease complexes have also been studied over the years (Sasser, 1989; Khan &

Reddy, 1993).

In a study conducted by Singh et al (1994) on french beans, simultaneously inoculating nematodes alone with fungus and ten days before fungus, reduced fresh shoot weight and height of French beans whiles maximum wilt was also expressed. Inoculation of only fungus and different combinations of both, decreased significantly the fresh root weight. Moderate wilting and maximum root-knot index was observed when both nematode and fungus were inoculated either in different combinations or individually. Maximum percentage wilt was observed after simultaneously inoculating both pathogens and nematode inoculation prior to fungal inoculations while moderate wilts occurred when fungus was inoculated alone and fungus inoculation before inoculation of nematode.

A study by Patel et al (2000) also revealed that Fusarium oxysporum f. sp. cicei with

Meloidogyne incognita on Chicken pea cv. Dahwood Yellow, either combined or individually reduced fresh shoot and root weights and plant height drastically. This reduction however was made by Meloidogyne incognita as compared to Fusarium oxysporum f. sp. cicei. Nematode population and root gall index on chicken pea were at its maximum when only nematode was inoculated but reduced in the presence of fungus. Severity of disease increased in the presence of fungus and root-knot nematode was present. Simultaneous inoculation of both organisms produced maximum wilt.

Interaction on blackgram cv.T-9 between Fusarium oxysporum and Meloidogyne incognita was studied (Mahapatra & Swain, 2001). There was less effect when fungus was inoculated prior to the nematode inoculation and galling and nematode population decreased. Combined inoculation caused an adverse effect on plant growth as well as rhizobia inoculation.

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Simultaneous inoculation of both organisms and nematode preceeding the fungus, synergistically reduced nodulation and plant growth.

There was a reduction in nematode multiplication when fungus was inoculated prior to nematode. Simultaneous inoculation of nematode and fungus, also of nematode followed by fungus 15 days after, caused 100% root-knot disease and significant reduction compared to inoculation of fungus alone or fungus inoculation prior to nematode (Senthamaria et al.,

2006).

Interaction between Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici on tomato CV.CO3 under glasshouse conditions showed the suppression of plant growth in combination of both pathogens than with fungus alone. There was a synergistic effect on the reduced growth of plant. A maximum reduction in plant height (33.1 cm) occurred when nematode and fungus were simultaneously inoculated (Samuthiravalli & Sivakumar, 2008).

Disease complexes of nematodes with organisms interrupt the uptake of nutrients and water by plants from the soil eventually reducing plant vigour and causing the death of plants

(Masse et al., 2002). Fungus and nematode cause changes in the physiology of the host plant.

There is reduction in the photosynthetic rate of the plant (Meena et al., 2016). Some researchers have suggested that nematodes may affect fungal resistance in plants and make plants susceptible to fungi which are innocuous without nematodes present (Batten & Powell,

1971). This means that nematodes induce factors which can change the susceptibility of plants to fungal diseases and increase the fungus ability to penetrate and develop within the host plant tissues.

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2.10. Types of Interaction Between Fungi and Nematodes

As the relationship between fungi and nematode has gained attention over the years, the type of interaction that exists between these organisms have gained interest over the years.

Naturally, the interaction that occurs between organisms could be synergistic, antagonistic or may even remain neutral (Khan, 2008). Ravichandra (2014) suggested that there may be mechanical wound, host modifiers, fungal pathogen, rhizosphere modifiers and resistance breakers for a successful interaction to occur between organisms.

2.10.1 Synergistic Interaction

There is a synergistic interaction between fungi and nematode when a higher damage is caused by the combination of both organisms compared to the cumulative effect of one pathogen. In the United States of America, experiments showed 100% wilt occurred in tomato plants when the plants were infected with Fusarium-Meloidogyne incognita complex as compared to 60% wilt when Meloidogyne hapla alone was present (Powell 1971; Sayre &

Walter 1991). These tests proved that the effects of the nematodes on the physiology of tomato was dependent on the type of nematode present and was evident in other combinations of species (Ragozzino & D’Errico, 2011). In the case of Pratylenchus spp-

Verticillum spp complex on tomato, the nematode –fungus combination enhanced nematode development as a result of CO2 produced by the damaged roots of the plants. This gas is known to attract nematodes (Zuckerman & Jansson, 1984). Also, with the above nematode- fungi combination, Verticillum promoted and increased the reproduction of Pratylenchus spp in tomato and eggplant. Nematodes are known to break down genetic resistances in wilt fungi. When varieties of plants, usually resistant to fungi are previously attacked by nematodes, the plant show symptoms of wilt. This could be as a result of the physiological

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and morphological changes that occured in plants due to the nematode attack (Stirling, 1991;

Sikora, 1992) even though mechanical wounds by nematodes might also induced some host response that lowers natural resistance.

2.10.2. Antagonistic Interaction

Belonolaimus longicaudatus, the sting nematode is an aggressive pathogen in cotton, it delays wilting considerably and may not express wilting at all. This also happens on cotton when

Meloidogyne spp. are involved. Meanwhile, past studies have shown that galled roots are highly susceptible to fungal invasion than non- galled roots. Hypertonic plant tissues enhance the growth of several fungi (Ragozzino & D’Errico, 2011). A histological study on tobacco reported root- knot nematodes, make plants susceptible to root rot by common soil inhabitant including Curvularia, Penicillium and Trichoderma. These fungi when inoculated to healthy tobacco roots in a greenhouse experiment, did not express necrosis or tissue invasion in the tobacco plant (Ragozzino & D’Errico, 2011). It implies that galled tissues are easily invaded by fungi which are antagonistic to phytopathogenic fungi. In the interaction between

Globodera rostochiensis and Colletotrichum atramentarium, the fungus causing the black-dot of some solanaceous plants including potato, and tomato, the fungi exudes certain chemicals which decreases hatching of both cysts and eggs of the nematode, and reduces root invasion by nematode juveniles. The ratio of males to females also increase resulting in the decrease in cysts numbers. (Powell, 1971).

The opposite occurs between other sedentary endoparasitic nematodes such as Globodera spp., Meloidogyne spp., Heterodera spp., and some phytopathogenic fungi. These nematodes prevent root invasion by fungi on tomato. There seems to be antibiotic-like substances involved (Ragozzino & D’Errico, 2011).

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The above relationship where the presence of both nematode and fungi, negatively affect the normal processes of the other such that less damage occurs on the plant, is described as an antagonistic interaction. In such an interaction, less damage is recorded on the plant when both pathogens are present than the sum of individual damage.

2.10.3 Neutral Interaction

In neutral interaction, the damage caused by both nematode and fungi is the same as the damage caused from the sum of individual pathogens. It is difficult to observe such an interaction because the damage observed is similar to when the two organisms do not interact

(Back et al., 2002).

In an experiment by Bowman & Bloom (1966), using the split root technique, Fusarium wilt was observed in both areas were root was exposed to nematode and where root was not exposed to nematode. This proved that predisposition to fungal invasion is not restricted to galled areas or sites of nematode activity.

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CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1. Isolation and Confirmation of Microorganisms Causing Root- Knot and Fusarium

Wilt Disease

3.1.1 Nematode Extraction

Soil samples were taken initially from individual plots before transplanting of tomato seedlings and also the end of the experiment. The soil samples were taken at a depth of 0 cm to 20 cm below the ground from each of the 36 plots randomly. They were labelled and taken to the laboratory for extraction. The extraction was done using the sieving and sucrose- centrifugation method. The sucrose solution was prepared by adding distilled water to 454 g of sugar to a volume of one liter and the mixture stirred until the sugar completely dissolved.

Two hundred (200) cm³ of soil samples from each of the experimental plots were poured into containers and mixed thoroughly with water twice the volume of the soil using the hand. The mixture settled for three minutes and the supernatant poured into a 36 µm sieve. The sieve was gently tapped to make the drainage faster. With the help of a wash bottle, the nematodes were washed to a section of the sieve and later transferred into well labelled centrifuge tubes to a volume of 20 mL for a uniform weight before centrifugation. The centrifuge (Thermo scientific, Germany) tubes were placed in balanced pairs, six at a time in the centrifuge. The first spin was at 1700 rpm for five minutes. The tubes were removed and allowed to settle for five minutes and the suspension was decanted 1cm above the pellet. The prepared sucrose solution was added to the pellet and shaken vigorously to disperse the pellet. The addition of sucrose was done such that all samples had equal volumes. A second spinning was done, this time at 1000 rpm for one minute after which, the nematodes and clay were suspended in the sucrose solution and clear suspension sieved using the 36 µm sieve. It was then rinsed gently into well labelled vials using a wash bottle to five ml. The vials were then stored in a

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refrigerator at 4 ºC for counting. A compound microscope (Optical technology, Germany) was used to view nematodes and a tally counter used to count them. Identification of the nematodes was based on the length, presence of stylet, shape and length of body and tail (Fig.

1, Page 31).

3.1.2 Extraction of Nematode Eggs from Plant Root

Roots from sample plants of each plot were washed under running water and cut to pieces.

These were weighed using an electronic balance (Kern, Balingen, Germany) to obtain one gram per treatment. The roots were placed in well labelled bottles containing 10% NaClO for five minutes and shaken vigorously and poured into a 36 µm sieve. It was then washed under running tap and sieve was gently tapped to speed up the drainage. The nematode eggs were washed to a section of the sieve and later transferred into labelled 50 mL tubes and kept in a refrigerator at 4 °C prior to counting under compound microscope.

3.1.3. Isolation of Fungi Causing Tomato Wilt Disease

Tomato plants showing symptoms of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici was collected from the Sinna’s Garden, Department of Crop Science, University of Ghana and taken to the Plant Pathology laboratory, Legon. Sections from the stem of the infected tomato plant was cut and washed in distilled water to remove any soil on its surface.

The cut sections were then sterilized in 1% sodium hypochlorite for one minute. The cuttings were blotted on a clean tissue paper and plated on Water Agar in a Petri dish. The fungus that grew was sub-cultured onto Potato Dextrose Agar and incubated in the laboratory at room temperature. Slides of isolated fungus were prepared and its morphological features studied under compound microscope to confirm its identity.

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3.2 Determination of the Individual and Combined Effects of Fusarium oxysporum f. sp. lycopersici and Meloidogyne Spp. on the Growth and Yield of Tomato.

3.2.1 Experimental Site

The experiment was carried out on two locations; University of Ghana, Legon farm (latitude:

5.65704 longitude: -0.192661) and at the National Service Demonstration Farm at Papao

(latitude: 5.66433 longitude: -0.191885). Both fields had been used for the cultivation of vegetables and had high population of root- knot nematodes. The experimental area had a mean rainfall of 61.5 mm, a mean minimum and maximum temperature of 24.2 ⁰C and 31.2

⁰C respectively from September 2018 to December 2018 (Table 4). Soils at the two sites are classified under the Adentan series and are relatively light clayey soils with low fertility. The two fields were cleared, ploughed and harrowed to obtain a fine tilth of area.

Table 4. Climatic data of experimental area from August, 2018 to January, 2019 Month Rainfall (mm) Temperature (⁰C)

Minimum Maximum

August, 2018 33.3 22.9 28.6 September, 2018 34.8 23.6 29.7 October, 2018 133.3 23.7 30.9 November, 2018 71.8 24.5 31.7 December, 2018 6.0 24.6 32.5 January, 2019 0.0 24.9 33.0

Source: Ghana Meteorological Service Agency, Mempeasem, Legon, (2019)

3.2.2 Experimental Design

The experimental design on both fields was a 2 × 6 factorial experiment in a Randomized

Complete Block Design with three replications. Two tomato varieties, Mongal F1 and

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Petomech were used for this research with both varieties receiving a pathogenic treatment.

The treatments used for both fields were:

NF7 = Fusarium oxysporum inoculated 7 days after transplanting in field naturally infested with Meloidogyne incognita.

NF14 = Fusarium oxysporum inoculated 14 days after transplanting in field naturally infested with Meloidogyne incognita.

NF21 = Fusarium oxysporum inoculated 21 days after transplanting in field naturally infested with Meloidogyne incognita.

N = Plots naturally infested with Meloidogyne incognita but not inoculated with Fusarium oxysporum after transplanting (No Fusarium inoculation).

F = Fusarium oxysporum inoculated on field naturally infested with Meloidogyne incognita and treated with nematicide.

C = Fusarium oxysporum not inoculated on field naturally infested with Meloidogyne incognita and treated with nematicide (Control).

The planting distance of tomato plants for both fields was 80 cm x 50 cm with each plot having 25 plants and an area of 4 m x 2.5 m (10 m²). Each plot had 5 rows with 5 plants in each row. Distance between plots was 0.5 meters and 1meter between blocks. Individual plots were demarcated and soil samples collected from the well labelled plots for nematode estimations before tomato seedlings were transplanted

3.2.3 Nematicide Application

Three days after transplanting seedlings to the fields, a systemic nematicide (Velum Prime

400 SC) with active ingredient, Fluopyram was applied to treatments which required

Fusarium to be within the soil as control plots. The nematicide (100 mL) was applied per plant to all 25 plants within each plot. The rate of application was 8 mL of the nematicide in

15 litres of water.

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3.2.4 Nursing of Seeds

Well drained seedling trays were filled with soil and watered to moisten the soil. Holes with a depth of 1cm were created in the soils and seeds of two varieties sown (Mongal F1and

Petomech) were watered, thinned out when necessary and monitored till they were transplanted to the field after the fifth week.

3.2.5 Preparation of Micro Biological Media for Isolation of Fungi

Three grams of dehydrated agar (Oxoid Ltd., Basingstoke, Hampshire, England) was weighed into a 250 mL conical flask and topped up with 100 mL of distilled water. The flask was then plugged with cotton wool and covered with aluminum foil and autoclaved at 121°C for 15 minutes.

3.2.6 Potato Dextrose Agar (PDA) Preparation

Potato Dextrose Agar (Oxoid Ltd., Basingstoke, Hampshire, England) powder of 3.9 g was weighed into a 250 mL conical flask and 100 mL of distilled water added. The conical flask was plugged with cotton wool and covered with aluminum foil and then autoclaved at 121°C for 15 minutes.

3.2.7. Preparation of Fusarium oxysporum f.sp lycopersici Inoculum

Two weeks old culture of Fusarium oxysporum f.sp. lycopersici PDA for two weeks were scraped from three Petri dishes into a blender and topped up with 1000 cm3 of distilled water.

The mixture was blended and suspension of fungal spores was determined using a haemocytometer (Hawksley Cristalite, England) as 1.3 x 106cells per 5 mL.

3.2.8 Inoculation of Tomato Plant with Fusarium oxysporum f. sp. lycopersici Inoculum

Roots of the five weeks old tomato transplants on the field were exposed and approximately

5 mL of fugal spore suspension poured in the root zone. Inoculation was done after seven, 14 and 21 days to all 25 treatment plants.

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3.2.9 Re-Isolation of Fungi

Three plants from each plot were uprooted 60 days after the last treatment was applied from both fields and brought to the Pathology Laboratory of the Department of Crop Science,

University of Ghana for fungi re-isolation. Sections from the stem of the tomato plant were cut to pieces of 1 cm. This was washed in sterile distilled water to remove any soil on its surface and then sterilized in 1% sodium hypochlorite for one minute. Using the forceps, the cuttings were blotted on a clean tissue paper and plated first on Water Agar and then sub- cultured on PDA. Slides of Fusarium oxysporum were then prepared and fungi identified as

Fusarium oxysporum f. sp. lycopersici using morphological features.

3.2.10 Data Collection

3.2.10.1 Plant Height

The height of plants (cm) from all nine recordable plants were taken for each plot from the highest tip of the leaf to the soil level using a meter rule. The first plant height was taken four weeks after plants were transplanted to the field, with subsequent height taken after every two weeks till the tenth week.

3.2.10.2 Stem Girth

A venier caliper (Neiko, China) was used to take the diameter of the recordable plants in millimetres. Data was taken from the fourth week at two weeks interval till the tenth week after planting to the field.

3.2.10.3 Chlorophyll Content

The Chlorophyll meter (Apogee instruments, USA) was used to measure the Chlorophyll

Content Index (CCI) of the nine recordable plants for each treatment. Four weeks after the tomato plants were transplanted, the chlorophyll content was taken and then every two weeks for three more times.

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3.2.10.4 Fresh Shoot and Root Weights

Three plants from each plot were uprooted and roots cut to separate the shoot from the root of the tomato plant. The shoot and roots were weighed separately and values recorded in grams using an electronic balance (Adam equipment, USA) to obtain the fresh shoot and fresh root weights, respectively.

3.2.10.5 Dry Shoot and Root Weights

Samples of tomato plants were uprooted and cut to separate the roots from the shoots. These were then dried in an oven at 70°C for five days to constant weight and weighed with an electronic balance (Adam equipments, USA) to obtain dry shoot and root weights.

3.2.10.6 Determination of Crop Yield

At plant maturity, tomato fruits were harvested and weighed to obtain, yield per plot. This was converted to yield per hectare (kg/ha) using the formula:

Yield (kg/ha) = Weight (kg) x 10000 m2 Harvested area (m2)

3.2.10.7. Wilt Incidence and Severity

To determine the wilt incidence, a wilt incidence scale (Table 5) was used (Nene et al.,

1981).

Percentage (%) wilt = Number of plants wilted× 100 Total number of plants

Wilt Severity (%) = Sum (rating number x number of plants in the rating) x 100% Total number of plants x highest rating

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Table 5. Rating scale for wilt incidence Scale Rating scale

No wilt 1

10% or less wilted 3

11 – 20% wilted 5

21 – 50% wilted 7

51% and more wilted 9

Source: Nene et al.,1981

3.3. Determination of Reproductive Ability of Meloidogyne incognita on Tomato, After

Fusarium oxysporum f. sp. lycopersici Infections.

3.3.1. Nematode Egg Population

Eggs extracted from the roots were counted per one gram of root and in 5 mL solution under a compound microscope using a tally counter.

3.3.2. Root Score

The roots from the tomato plants uprooted from each plot were scored using a diagrammatic root- knot scoring chart (Bridge & Page 1980).

3.3.3 Determination of Nematode Reproductive Index

The reproduction factor (Rf) was determined by dividing the final nematode count by the initial count for each plot. This was computed as;

Reproductive factor (Rf) = Final nematode population Initial nematode population

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3.4 Data Analysis

Data were analyzed using Analysis of variance (ANOVA) and where there was significant difference, the least significant difference (LSD 5%) was used to separate means using

GenStat (12th Edition).

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CHAPTER FOUR

4.0 RESULTS

4.1 Microorganisms Causing Root- Knot and Fusarium Wilt Diseases of Tomato

4.1.1 Meloidogyne incognita

Nematodes extracted from the soil were about 200 µm long, with a projecting neck and an anal body width 8 to 17 μm. The nematode had a relaxed stylet between 11 to 25 μm in length with a rounded well drawn out stylet knob. The tail length was between 15 to 60 μm, bluntly round and unstraited. Eggs laid by the females were enclosed in a gelatinous sac and were elongated, ellipsoid and stunted in shape. Based on the above morphological characteristics observed under the microscope, the nematode was identified as Meloidogyne incognita (Orton William, 1973) (×400) (Fig.1).

A B

Fig.1. Micrograph of Meloidogyne incognita, J2 (A) and nematode egg (B). 400 X magnification.

4.1.2 Fusarium oxysporum f. sp. lycopersici

Eight days after incubation on PDA, microconidia and macroconidia were observed. Fourteen days after, chlamydospore were also observed. The pathogen produced concentric, fluffy cotton pigment in media. As the culture grow, small single bi-celled conidia were also seen under microscope. The hyaline and multicelled macroconidia were sickle-shaped and

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septated. Fungi was identified as Fusarium oxysporum f.sp. lycopersici based on cultural and morphological characteristics (Barnett & Hunter, 1972) (Fig.2).

B A

Fig.2. Two weeks old culture of Fusarium oxysporum f.sp. lycopersici on potato dextrose agar(A) and Micrononidia of Fusarium oxysporum f.sp. lycopersici 400X (B).

4.2 Individual and Combined Effects of Fusarium oxysporum f. sp. lycopersici and Meloidogyne Spp. on the Growth and Yield of Tomato. 4.2.1 RESULTS FROM UNIVERSITY OF GHANA FARMS

4.2.1.1 Plant Height There were no significant differences (p ≤ 0.05) in plant height for Mongal F1 and Petomech plants that received various pathogen inoculation treatments but there was significant difference (p≤ 0.05) in the height between the two varieties (Fig.3). Mongal F1 had higher heights at from week 6 to week 10 after transplanting compared to Petomech.

The heights for Mongal F1 and Petomech tomato plants increased irrespective of treatments applied from week four till week ten with control having the least height. The least heights for Mongal F1 was between 27.3 cm and 36.9 cm at week four whiles the highest were between 46.7 cm and 57.8 cm at week ten (Fig.3). The least heights for Petomech were

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between 20.0 cm and 26.0 cm whiles the highest heights were between 27.5 cm and 39.3 cm

(Fig.3).

Plant Height 60

40 Mongal F1

Height (cm) Pectomech 35

25 0 4 6 8 10 Weeks after transplanting

Fig.3. Heights of Mongal F1 and Petomech plants inoculated with Fusarium oxysporum f. sp. lycopersci on University of Ghana farm from weeks 4 to 10. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.1.2 Plant Girth

There was no significant difference (p≤ 0.05) in plant girth measurements among the inoculated pathogen treatments for both Mongal F1 and Petomech tomato varieties.

The girth in both varieties of Mongal F1 and Petomech generally increased from the fourth week till the eighth week after transplanting.

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Mongal F1 had significantly thicker (p≤ 0.05) width of plants than Petomech. The highest and least girth values for Mongal F1 were 7.3 mm and 6.0 mm respectively. Pectomech also had 4.3 mm and 5.6 mm as its least and highest girths respectively (Fig.4).

Plant Girth

5 Mongal F1 4 Petomech Plant girth(mm) 3

2 0 4 6 8 10 Weeks after transplanting

Fig.4. Girths of Mongal F1 and Petomech plants inoculated with Fusarium oxysporum f. sp. lycopersci on University of Ghana farm weeks 4 to 10. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.1.3 Chlorophyll Content

There were no significant differences (p≤ 0.05) among the inoculated pathogen and interaction between the two tomato varieties and treatments in chlorophyll contents.

The chlorophyll content increased from the fourth week after transplanting to the sixth week.

After which it decreased till the tenth week (Fig.5).

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Fig.5. Chlorophyll of Mongal F1 and Petomech plants inoculated with Fusarium oxysporum f. sp. lycopersci on University of Ghana farm from weeks 4 to 10. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.1.4 Fresh and Dry Shoot Weight There were no significant differences (p≤ 0.05) in fresh shoot weights among the inoculated pathogen treatments of Mongal F1 and Petomech even though plants in the control treatment had the highest weights in both Mongal F1(169 g) and Petomech (60 g) varieties respectively

(Table 6). There was however significant difference (p≤ 0.05) between the varieties with

Mongal F1 having higher values in weight compared to Petomech.

There were also no significant differences (p≤ 0.05) in dry shoot weights among the inoculated pathogen treatments received by the two varieties, however there were significant differences (p≤ 0.05) between the varieties (Table 6).

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Table 6. Fresh and dry shoot weights for Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the University of Ghana farm. Treatments Fresh shoot weight (g) Treatment Dry shoot weight Treatment Mean (g) (g) Mean (g)

Mongal Petomech Mongal Petomech F1 F1

NF7 135.0 49.0 92.0 32.9 12.6 22.8

NF14 155.0 42.0 98.5 31.5 17.9 24.7

NF21 121.0 48.0 84.5 29.8 10.6 20.2

F 159.0 91.0 125.0 30.2 21.2 25.7

N 157.0 18.0 87.5 26.8 6.6 16.7

C 169.0 60.0 114.5 29.5 18.9 24.2

Variety 149.3 51.3 30.0 14.6 mean (g)

LSD (5%) V = 49.4 LSD (5%) V = 7.3 LSD (5%) T = NS LSD (5%) T = NS LSD (5%) V*T = NS LSD (5%) V*T = NS

NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.1.5 Fresh and Dry Root Weight There were no significant differences (p≤ 0.05) in fresh root weights among inoculated pathogen treatments applied to both Mongal F1 and Petomech. Treatment NF14 had the highest weights in of 34.6 g and 30.9 g in Mongal F1 and Petomech tomato varieties respectively (Table 7).

There was however significant difference between the fresh root weights of the two varieties with Mongal F1 ranging between 23.2 g and 34.6 g and Petomech ranging between 4.9 g and

30.9 g respectively.

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The dry root weight of Mongal F1 was significantly different (p≤ 0.05) from Petomech variety, with 11.6 g and 5.5 g as the highest and least weights of Mongal F1 respectively compared to Petomech which had the highest weight of 4.8 g and the least as 1.3 g (Table 7).

Table 7. Fresh and dry root weights for Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the University of Ghana farm

Treatments Fresh root weight(g) Treatment Dry root weight (g) Treatment mean (g) mean (g)

Mongal Petomech Mongal Petomech F1 F1

NF7 28.1 10.3 19.2 7.2 3.0 5.1

NF14 34.6 30.9 32.8 8.6 4.8 6.7

NF21 30.9 11.5 21.2 11.6 2.6 7.1

F 28.3 9.8 19.1 10.6 3.0 6.8

N 25.0 4.9 15.0 8.0 1.3 4.5

C 23.2 8.9 16.1 5.5 2.0 3.8

Variety mean 28.4 12.7 8.6 2.8 (g)

LSD (5%) V = 5.6 LSD (5%) V = 1.6 LSD (5%) T = NS LSD (5%) T = NS LSD (5%) V*T = NS LSD (5%) V*T = NS NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.1.6 Incidence and Severity of Tomato Wilt There were significant differences (p≤ 0.05) in wilt incidence between varieties and among the pathogen inoculation treatments of Mongal F1 and Petomech varieties.

In Mongal F1, treatment NF21 had the highest percentage (73%) wilt incidence whiles treatments N and C had the least percentage (20%) wilt incidence (Table 8) All pathogen

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inoculation treatments in Petomech, had plants that wilted with treatment NF7 having the highest wilt incidence of 93 % and least percentage wilt of 13.3 % in treatment C (Table 8).

There were significant differences (p≤ 0.05) in pathogen inoculation treatments applied to

Mongal F1 and Petomech varieties. Mongal F1 and Petomech had the highest disease severity of 66.7 % and 82.7 % respectively both in treatment NF21 (Table 8). The least percentage of disease severity was 14.0 % in Mongal F1 from treatments N and C while Petomech had 25.3

% in treatment N as the least disease severity (Table 8).

Table 8. Wilt incidence (%) and Wilt severity (%) in Mongal F1 and Petomech tomato varieties Fusarium after inoculations on the University of Ghana farm Treatments Wilt incidence (%) Treatment Wilt severity (%) Treatment mean (%) mean (%)

Mongal F1 Petomech Mongal F1 Petomech

NF7 26.7* 93.3 60.0 42.7 52.0 47.4

NF14 46.7 73.3 60.0 57.3 56.7 57.0

NF21 73.3 86.7 80.0 66.7 82.7 74.7

F 60.0 26.7 43.4 48.7 54.7 51.7

N 20.0 37.3 19.2 14.0 25.3 19.6

C 20.0 13.3 16.7 14.0 28.0 21.0

Variety 41.2 55.1 40.6 49.9 mean (%)

LSD (5%) V = 13.9 LSD (5%) V = NS LSD (5%) T = 24.1 LSD (5%) T = 21.7 LSD (5%) V*T = NS LSD (5%) V*T = NS NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide) *Wilt incidence was scored using wilt incidence scale (Nene et al., 1981)

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4.2.1.7 Yield There were significant differences (p≤ 0.05) between the two tomato varieties (Table 9).

Mongal F1 had relatively higher yields in pathogen inoculation treatments compared to

Petomech. None of the treatments in Petomech were above 1,000 kg/ ha (Table 9).

Mongal F1 and Petomech had the highest yield from treatment NF14 and the least from treatment C (Table 9).

Table 9. Yield for Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the University of Ghana farm. Treatments Yield (kg/ha) Treatment means (kg/ha) Mongal F1 Petomech

NF7 1,027.0 434.0 730.5

NF14 1,655.0 682.0 1168.5

NF21 1,273.0 477.0 874.5

F 914.0 373.0 643.5

N 1,172.0 332.0 752.5

C 557.0 220.0 388.5

Variety 1,099.5 419.7 means (kg/ha)

LSD (5%) V = 283.7 LSD (5%) T = NS LSD (5%) V*T = NS NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

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4.2.1.8 Re-Isolation of Fungi from Plant Stem

After re-isolating from the stem of plants, Fusarium oxysporum f.sp. lycopersici was observed in all treatments of Mongal F1 except treatments NF14 and C.

In Petomech, all treatments showed the presence of the fungus after re-isolating from the stem.

4. 2 .2 RESULTS FROM NATIONAL SERVICE FARM

4.2.2.1 Plant Height

There was significant difference (p≤ 0.05) between the two tomato varieties. Mongal F1 had higher heights compared to Petomech from week 4 till week 10 (Fig.6).

There was a general increase in the height of plants as the weeks progressed. Mongal F1 had the least heights ranging from 44.6 cm to 52.4 cm and the highest heights between 50.9 cm and 55.8 cm for the fourth and tenth week respectively (Fig.6).

Petomech variety had the least heights between 37.1 cm and 45.8 cm and the highest, between 47.4 cm and 52.7 cm in week four and week ten respectively (Fig.6).

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Plant height 60 55 50 45 40 35 Mongal F1 30 Height (cm) 25 Petomech 20 15 10 0 4 6 8 10 Weeks after transplanting

Fig.6 Heights of Mongal F1 and Petomech plants inoculated with Fusarium oxysporum f. sp. lycopersci on the National service farm from weeks 4 to 10. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.2.2 Plant Girth

There were significant differences (p≤ 0.05) in plant girth between the two varieties. Mongal

F1 had thicker girths of plants compared to Petomech (Fig.7).

Mongal F1 had the least girth of plants at week four between 5.2 mm and 6.8 mm and the highest plant girths ranging from 8.7 to and 11.2 mm (Fig.7).

Petomech had the least plant girths between 3.3 mm and 5.6 mm in week four and the highest girths ranging from 6.7 mm to 8.8 mm (Fig.7).

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Plant girth

11 10 9 8 7 6 Mongal F1

Girth (mm) 5 Petomech 4 3 2 0 4 5 8 10 Weeks after transplanting

Fig.7 Girths of Mongal F1 and Petomech plants inoculated with Fusarium oxysporum f. sp. lycopersci on the National service farm from weeks 4 to 10. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.2.3 Chlorophyll Content

There were no significant differences (p≤ 0.05) in chlorophyll content among the pathogen inoculation treatments and varieties of both tomato varieties. There was a general decrease in the chlorophyll content of both Mongal F1 and Petomech variety as the weeks progressed

(Fig.8).

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Mongal F1 Petomech

34 34 29 29 NF7 NF7 24 24 NF14 NF14 19 19 NF21 NF21 14 14 F F 9 9 4 N 4 N Chlorophyll content(CCI) 0 4 6 8 10 C Chlorophyll content (CCI) 0 4 6 8 10 C Weeks after transplanting Weeks after transplanting

Fig.8. Chlorophyll content of Mongal F1 and Petomech plants inoculated with Fusarium oxysporum f. sp. lycopersci on the National service farm from weeks 4 to 10. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.2.4 Fresh and Dry Shoot Weights

There was significant difference (p≤ 0.05) in the fresh shoot weight of Mongal F1 and

Petomech varieties. Mongal F1 had higher weights compared to Petomech. Mongal F1 had

285.0 g as the highest fresh shoot weight for treatment NF14 and the least as 163.0 g in treatment N (Table.10). Petomech also, had the highest fresh shoot weight as 220.0 g and least fresh shoot weights as 137.0 g in treatments NF14 and N respectively (Table.10).

There were no significant differences (p≤ 0.05) in dry shoot weights of pathogen inoculation treatments and variety. Mongal F1 had 41.7 g as the highest dry shoot weight and 33.8 g as the least weight in treatments F and C respectively (Table.10). Petomech had 40.6 g for treatment C as the highest dry shoot weight and 29.7 g in treatment F as the least dry shoot weight (Table.10).

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Table 10. Fresh and dry shoot weights for Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the National service farm Treatments Fresh shoot weight(g) Mean fresh Dry shoot weight Mean dry shoot (g) (g) shoot (g)

Mongal Petomech Mongal Petomech F1 F1

NF7 181.0 149.0 165.0 37.1 35.8 36.45

NF14 285.0 156.0 220.5 37.2 30.9 34.1

NF21 227.0 108.0 167.5 54.2 26.4 40.3

F 233.0 72.0 152.5 41.7 17.8 29.6

N 163.0 112.0 137.5 38.4 28.3 33.4

C 203.0 198.0 200.5 33.8 47.3 40.6

Variety mean 215.3 397.5 40.4 31.1

LSD (5%) V = 52.7 LSD (5%) V = NS LSD (5%) T = NS LSD (5%) T = NS LSD (5%) V*T = NS LSD (5%) V*T = NS

4.2.2.5 Fresh and Dry Root Weights

There were no significant differences (p≤ 0.05) in the fresh root weight among pathogen inoculation treatments however, there was significant differences (p≤ 0.05) between the two tomato varieties (Table 11). Mongal F1 had higher weights in grams than Petomech. The highest weight in Mongal F1 was 128.1 g and in Petomech, 102.2 g which were both observed in treatments NF14 (Table 11). The lowest fresh root weights however were 66.7 g

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in Mongal F1, observed in treatment NF7 and 20.4 g for treatment NF21 in Petomech (Table

11).

There was significant difference (p≤ 0.05) in the dry root weights in both tomato varieties and interaction between the varieties and pathogen inoculation treatments. Generally, Mongal F1 had higher dry root weights than Petomech except for treatment C where Petomech had 10.2 g and Mongal F1 had 8.0 g (Table.11).

Table 11. Fresh and dry root weights for Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the National service farm Treatments Fresh root weight Treatment Dry root weight (g) Treatment (g) mean (g) mean (g)

Mongal Petomech Mongal Petomech F1 F1

NF7 66.7 56.3 61.5 8.3 5.8 7.1

NF14 128.1 102.2 230.3 13.6 7.4 10.3

NF21 123.4 20.4 71.9 13.6 5.6 9.6

F 87.9 39.4 63.7 12.4 4.9 8.7

N 86.0 70.8 31.4 9.0 5.6 7.3

C 91.6 43.0 67.3 8.0 10.2 9.1

Variety 97.3 55.4 10.8 6.5 mean (g)

LSD (5%) V = 28.9 LSD (5%) V = 2.0 LSD (5%) T = NS LSD (5%) T = NS LSD (5%) V*T = NS LSD (5%) V*T = 4.8

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In Mongal F1 variety, the weights of the dry roots were lesser in treatments that had both organisms present (NF7, NF14 and NF21) and only Nematode present (N) than in treatments that had only Fungi present (Fig.9).

18 16 14 12 10 8 MONGAL F1 6 PETOMECH Dry root (g) weight 4 2 0 NF7 NF14 NF21 F N C Treatments

Fig.9 Interaction in dry root weight of Mongal F1 and Petomech tomato varieties and pathogen inoculation treatments. NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)

4.2.2.6 Incidence and Severity of Tomato Wilt

There were significant differences (p≤ 0.05) in wilt incidence between Mongal F1 and

Petomech tomato variety (Table 12). All pathogen inoculation treatment for both tomato varieties had more than 50% of the total plant population wilted (Table 12).

Petomech however had higher percentage wilt incidence than Mongal F1 for all treatments with the exception of treatment N (Table 12).

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There was significant difference (p≤ 0.05) in Fusarium wilt severity between two varieties.

Petomech had higher percentage wilt severity for all pathogen inoculation treatments compared to Mongal F1. None of the treatments in Mongal F1 had wilt severity above 50%

(Table 12).

The least wilt severity was observed in treatment C for both Mongal F1 and Petomech as 6.7

% and 12.7 % respectively (Table 12).

The highest disease severity was 29.3 % observed in treatment NF14 and the highest was

54.0 % observed in treatment F for Mongal F1 and Petomech respectively (Table 12).

Table 12. Wilt incidence (%) and Wilt severity (%) in Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the National service farm Treatments Wilt incidence (%) Treatment Wilt severity (%) Treatment mean (%) mean (%)

Mongal Pectomech Mongal F1 Petomech F1

NF7 86.7* 100.0 93.4 16.0 38.7 27.4

NF14 100.0 100.0 100.0 29.3 41.3 35.3

NF21 93.3 100.0 96.7 20.7 5.27 13.0

F 93.3 100.0 96.7 14.7 54.0 34.4

N 66.7 60.7 63.2 22.0 23.3 22.7

C 60.0 73.3 66.7 6.7 12.7 9.7

83.3 89.0 18.2 37.1 Variety mean (%)

LSD (5%) V = NS LSD (5%) V = 8.9 LSD (5%) T = 21.8 LSD (5%) T = 2.1 LSD (5%) V*T = NS LSD (5%) V*T = N NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide)*Wilt incidence was scored using wilt incidence scale (Nene et al., 1981)

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4.2.2.7 Yield

There were significant differences (p≤0.05) in yield between Mongal F1 and Petomech tomato varieties.

Treatment NF21 had 1,810.0 kg/ha as the highest yield and 1,411.0 kg/ha as the least in treatment N for Mongal F1 (Table 13). Yields for Mongal F1 were above 1,000.0kg/ha.

Petomech had lower yields compared to Mongal F1. The least yield of Petomech was observed in treatment NF21 as 330.0 kg/ha and the highest yield was 1,402.0 kg/ ha in treatment N (Table 13).

Table 13. Yield for Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the National service farm Treatments Yield (kg/ha) Treatment mean (kg/ha)

Mongal F1 Petomech

NF7 1,532.0 464.0 998.0

NF14 1,784.0 1,020.0 1,402.0

NF21 1,810.0 330.0 1,070.0

F 1,751.0 490.0 1,110.5

N 1,411.0 1,402.0 1,406.5

C 1,585.0 746.0 1,165.5

Variety 1,645.5 738.7 means(kg/ha)

LSD (5%) V = 457.8 LSD (5%) T = NS LSD (5%) V*T = NS NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide.

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4.2.2.8 Re-Isolation of Fungi from Plant Stem After re-isolating from the stem of selected plants on each plot, Fusarium oxysporum f.sp. lycopersici was not present in treatments N and C of both Mongal F1 and Petomech.

Treatment NF7 in Mongal F1 also did not have the fungi present. All other treatments in

Petomech however had the fungi present.

4.3 Reproductive Ability of Meloidogyne on Tomato After Fusarium oxysporum f. sp. lycopersici Infections.

4.3.1. RESULTS FROM UNIVERSITY OF GHANA FARM

4.3.1.1 Root Galling Score and Egg Count

There was significant difference (p≤ 0.05) in root galls scored between Mongal F1 and

Petomech varieties (Table. 14).

No galls were observed on the roots of plants in all pathogen inoculation treatments for

Mongal F1 after roots were scored.

In Petomech, galls were observed on root samples for all treatments except in treatments

NF21 and C. Treatment N had the highest galling (2.0) on root samples (Table. 14).

There were no significant differences (p≤ 0.05) in nematode eggs counted between Mongal

F1 and Petomech varieties and in pathogen inoculation treatments (Table. 14).

The highest number of eggs counted in Mongal F1was 15.0 from treatment NF21 and the least was 0.0 from both treatments NF7 and C (Table. 14).

Nematode eggs were counted in all pathogen inoculation treatments of Petomech tomato variety with 1.0 as the least number of eggs in both treatments NF7 and F and the highest number of eggs was 13.0 from treatment N (Table. 14).

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Table 14. Root gall score and Egg count in Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the University of Ghana farm Treatments Root gall Score Treatment Egg Count Treatment mean mean

Mongal Petomech Mongal Petomech F1 F1

NF7 0.0 1.0 0.5 0.0 6.0 3.0

NF14 0.0 1.0 0.5 3.0 1.0 2.0

NF21 0.0 0.0 0.0 15.0 9.0 12.0

F 0.0 1.0 0.5 1.0 1.0 1.0

N 0.0 2.0 1.0 7.0 13.0 10.0

C 0.0 0.0 0.0 0.0 2.0 1.0

Variety 0.0 0.8 4.3 5.3 means

LSD (5%) V = 0.3 LSD (5%) V = NS LSD (5%) T = NS LSD (5%) T = NS LSD (5%) V*T = NS LSD (5%) V*T = NS NF7 = fungus inoculated 7 days after transplanting to naturally infested nematode infested, NF14 = fungus inoculated in 14 days after transplanting to naturally infested nematode field, NF21= fungus inoculated 21days after transplanting to naturally infested nematode field, F= fungus inoculated on naturally infested nematode field treated with nematicide, N =Fusarium oxysporum not inoculated on tomato plants after transplanting in Meloidogyne incognita infested field, C = Control (Fusarium oxysporum not inoculated onto tomato plants and soil treated with nematicide

4.3.1.2 Nematode Count and Reproductive Factor

Nematode numbers in general reduced for all treatments in the final nematode count at the

end of the experiment.

The least reproductive factor of 0.0 was observed in the control and 0.2 was observed in all

other treatments for Mongal F1 (Table 15).

All treatments had a reproductive factor of 0.1 except treatment NF21, which had a higher

reproductive factor of 0.3 for Petomech (Table 15).

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Table 15. Initial nematode count, final nematode count and reproductive factor of Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the University of Ghana Farm Treatment Mongal F1 Petomech

Initial Final Reproductive Initial Final Reproductive Count Count Factor Count Count Factor

NF7 501.0 106.0 0.2 1,021.0 139.0 0.1

NF14 435.0 70.0 0.2 695.0 98.0 0.1

NF21 610.0 109.0 0.2 649.0 218.0 0.3

F 349.0 78.0 0.2 923.0 55.0 0.1

N 487.0 93.0 0.2 793.0 102.0 0.1

C 896.0 36.0 0.0 912.0 107.0 0.1

4.3.2 RESULTS FROM NATIONAL SERVICE FARM

4.3.2.1 Root Gall Score and Egg Count

There was significant difference (p≤ 0.05) in root galls scored between the two tomato varieties.

The roots of Mongal F1 did not show galling in any of the treatments after inoculations

(Table 16).

In Petomech, all pathogen inoculation treatments had galls on the roots except treatments

NF7 and F. Treatments NF21 and N had the highest galling of 3.0 on plant root (Table 16).

In the Egg count, there were no significant differences (p≤ 0.05) among pathogen inoculation treatments and varieties, in both Mongal F1 and Petomech (Table 16).

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After extraction of eggs from the roots, treatments NF7 and the C had no eggs in Mongal F1 variety. The highest number of eggs in Mongal F1 was 29.0 which was from treatment NF14

(Table 16). Petomech on the other hand, had eggs in all treatments except in treatment NF7.

Treatment C had 58.0 as the highest number of eggs (Table 16).

Table 16. Root score and Egg count in Mongal F1 and Petomech tomato varieties after Fusarium inoculations on the National Service farm Treatments Root Scoring Mean root Egg Count Mean Egg score count Mongal Petomech Mongal Petomech F1 F1

NF7 0.0 0.0 0.0 0.0 0.0 0.0

NF14 0.0 2.0 1.0 29.0 8.0 18.5

NF21 0.0 3.0 1.5 2.0 3.0 2.5

F 0.0 0.0 0.0 1.0 1.0 1.0

N 0.0 3.0 1.5 6.0 47.0 26.5

C 0.0 2.0 1.0 0.0 58.0 29.0

Variety mean 0.0 1.7 6.3 19.5

LSD (5%) V = 1.0 LSD (5%) V = NS LSD (5%) T = NS LSD (5%) T = NS LSD (5%) V*T = NS LSD (5%) V*T = NS

4.3.1.2 Nematode Count and Reproductive Factor

Nematode numbers in general reduced for all treatments in the final nematode count at the end of the experiment.

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In Mongal F1, the least reproductive factor was 0.0 observed in treatment N whiles all other treatments had 0.1 as the reproductive factor (Table 17).

In Petomech, the least reproductive factor was 0.0 in treatment F whiles the highest was 0.2 observed in treatment N (Table 17).

The reproductive factor for all treatments were below one (Table 17).

Table 17. Initial nematode count, final nematode count and reproductive factor of Mongal F1 Petomech tomato varieties after Fusarium inoculations on the National service farm Treatment Mongal F1 Petomech

Initial Final Reproductive Initial Final Reptoductive count count factor count count factor

NF7 288.0 24.0 0.1 252.0 13.0 0.1

NF14 243.0 18.0 0.1 252.0 23.0 0.1

NF21 271.0 28.0 0.1 343.0 37.0 0.1

F 267.0 17.0 0.1 712.0 3.0 0.0

N 361.0 5.0 0.0 208.0 31.0 0.2

C 116.0 9.0 0.1 209.0 25.0 0.1

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CHAPTER FIVE

5.0 DISCUSSION Meloidogyne incognita and Fusarium oxysporum f.sp. lycopersici are both soil borne plant pathogens so they initially infect root epidermis after which the fungi invades the vascular tissue of the plant. The simultaneous presence of both pathogens in roots resulted in lower dry root weights compared to the individual presence of either nematode or fungi for Mongal

F1 tomato variety. In Petomech tomato variety, however, the occurrence of nematode only in roots resulted in lower dry root weight than when both pathogens were simultaneously present. Bhagwati and Goswami (2000) observed reduced vigour in tomato growth when both Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici were present simultaneously in roots. The presence of both Meloidogyne incognita and Fusarium oxysporum f. sp. lentis in pea also reduced plant growth compared to their individual presence in roots (Haseeb et al., 2006). The two findings above, corroborates that of the two pathogens in Mongal F1 tomato in this study. There was therefore detrimental effect on the growth of Mongal F1 tomato variety when the two pathogens were present in soils. Tomato farmers should therefore control both pathogens if present in their soils. They may rotate tomato plants with other crops like groundnut and cassava on their farms or allow fields to fallow for four years when the Fusarium populations would have dwindled.

Both inoculation of tomato plants with nematode 10 days prior to fungi inoculation and simultaneous inoculation of both pathogens, resulted in the highest tomato wilt severity

(Bhagawati & Goswami, 2000). In the present study, however, the highest wilt severity occurred when fields naturally infected with nematode were inoculated with fungi, 21 days after transplanting in both tomato varieties. This may indicate that when nematode is initially present before fungi, the wilt severity is high. Synergy between Meloidogyne spp. and other

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pathogens have been known to cause more damage worldwide (Rivera & Aballay, 2008).

Increased damage by both organisms might have been caused by prior wounds created by

Meloidogyne incognita favouring the fungi infection by creating rich metabolic substrates

(Bhabesh et al., 2007). The synergism by both pathogens could have caused malfunction in the roots of plants that weaken the tomato making it more susceptible to the fungi (Ganaie &

Khan 2011). The plants exhibit wilt as a result of the root damage, stunting occurs and eventually leads to death (Nehal et al., 2007). Surprisingly, wilt was observed in fields free of

Meloidogyne incognita and Fusarium oxysporum f.sp. lycopersici in the study. Rekah &

Katan (1999), studied the migration of Fusarium oxysporum on tomato fields and after geostatistical analysis found that, Fusarium oxysporum f. sp. radicis- lycopersici can move a distance of about 1.1 m to 4.4 m during a growing season. This was a deviation from the mono cyclic nature of many non-zoosporic pathogens that are soil borne. This may have resulted in the wilts observed on plants in fields free of both pathogens.

Naresh et al. (2017) studied the interaction between Meloidogyne incognita and Fusarium oxysporum f.sp. lycopersici using the susceptible tomato cultivar, Pusa Ruby. They observed the highest number of eggs (45.67) in plots with nematode inoculum only. In this study also, the highest number of eggs in Petomech variety were 13.0 and 47.0 on plots naturally infested with nematode only at the University of Ghana farm and National service farm respectively. This could have been because the plant roots were damaged by the fungus resulting in reduced root system available to the nematodes for feeding. The interaction between Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita reduces galling and nematode reproduction on tomato plants (Nagesh et al., 2006; Ganaie & Khan, 2011).

The low number of nematode eggs in roots might be because some plants died whiles others had early senescence hence the nematodes were not able to complete their life cycles

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reducing the number of second stage juveniles as well as their reproductive factor. Patel et al.

(2000) and Senthamarai et al. (2006) attributed low reproductive factors in chickpea and coleus respectively, to low numbers of second stage juveniles of nematode.

In this study, no galls were seen on the roots of Mongal F1 tomato plant. Mongal F1 could indeed be resistant or have some level of tolerance to Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita. Susceptibility of tomato to Fusarium wilt is as a result of physiological changes and root exudates that prevent the fungi from penetrating resistant cultivars (Juliatti et al., 1994). Resistant varieties could be varieties that are able to compensate for loss of carbohydrates associated with Fusarium infections. Also, the ability of plants to resist root- knot nematodes, depends on their ability to allow second stage juvenile nematodes to penetrate roots (De ley & Mando, 2004; Gharabadiyan et al., 2012) and even after penetrating, cause juveniles to die before females are able to reproduce or even form galls (Gharabadiyan et al., 2012).

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CHAPTER SIX

6.0 CONCLUSION AND RECOMMENDATION 6.1 CONCLUSION

The following conclusions can be drawn from this study:

1. Meloidogyne incognita and Fusarium oxysporum f.sp. lycopersici were the causal agents of root- knot and Fusarium wilt diseases respectively.

2. The presence of Meloidogyne incognita and Fusarium oxysporum f sp. lycopersici in the soil either as individuals or combined had similar effect on plant height, girth, fresh root weight and yield of tomato.

• When both Meloidogyne incognita and Fusarium oxysporum f.sp. lycopersici were together, their interaction resulted in reduced root weight and severe wilt in both tomato varieties.

3. Fusarium oxysporum f. sp. lycopersici reduced the reproductive ability of Meloidogyne incognita in tomato plants when both pathogens existed simultaneously in the soil.

6.2 RECOMMENDATIONS

The following recommendations can be drawn from this study;

1. Tomato varieties other than Mongal F1 and Petomech should be used to study the interaction between Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita since both varieties responded differently. 2. This study should be repeated in a pot experiment with different levels of Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita inoculum to better understand the interaction between Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita. 3. Mongal F1 is tolerant to both Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita therefore, further screening of Mongal F1 to various Fusarium inocula levels should be undertaken to ascertain the level of tolerance.

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APPENDICE

Analysis of Variance on Growth Parameters for University of Ghana Farm

Appendix 1 Plant Height University of Ghana Farm Variate: Plant Height 4 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 863.18 863.18 20.83 <.001 TREATMENT 5 350.25 70.05 1.69 0.179 VARIETY.TREATMENT 5 26.20 5.24 0.13 0.985 Residual 22 911.75 41.44 Total 35 2356.43

Variate: Plant Height 6 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 1495.24 1495.24 24.11 <.001 TREATMENT 5 959.50 191.90 3.09 0.029 VARIETY.TREATMENT 5 122.08 24.42 0.39 0.848 Residual 22 1364.59 62.03 Total 35 4223.14

Variate: Plant Height 8 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 2148.63 2148.63 29.37 <.001 TREATMENT 5 527.89 105.58 1.44 0.248 VARIETY.TREATMENT 5 154.53 30.91 0.42 0.828 Residual 22 1609.42 73.16 Total 35 4833.67

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Variate: Plant Height 10 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 3337.76 3337.76 68.68 <.001 TREATMENT 5 365.31 73.06 1.50 0.229 VARIETY.TREATMENT 5 264.59 52.92 1.09 0.394 Residual 22 1069.20 48.60 Total 35 5358.00

Appendix 2 Plant Girth University of Ghana Farm

Variate: Plant Girth 4 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 27.878 27.878 22.37 <.001 TREATMENT 5 3.178 0.636 0.51 0.766 VARIETY.TREATMENT 5 6.580 1.316 1.06 0.411 Residual 22 27.416 1.246 Total 35 68.227

Variate: Plant Girth 6 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 28.320 28.320 20.14 <.001 TREATMENT 5 6.549 1.310 0.93 0.480 VARIETY.TREATMENT 5 2.631 0.526 0.37 0.861 Residual 22 30.937 1.406 Total 35 71.912

Variate: Plant Girth 8 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 24.651 24.651 19.44 <.001 TREATMENT 5 2.656 0.531 0.42 0.831 VARIETY.TREATMENT 5 3.809 0.762 0.60 0.700 Residual 22 27.904 1.268 Total 35 66.105

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Variate: Plant Girth 10 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 43.1211 43.1211 56.73 <.001 TREATMENT 5 8.7731 1.7546 2.31 0.079 VARIETY.TREATMENT 5 3.3992 0.6798 0.89 0.502 Residual 22 16.7236 0.7602 Total 35 75.8298

Appendix 3 Chlorophyll Content University of Ghana Farm

Variate: Chlorophyll content 4 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 0.13 0.13 0.01 0.935 TREATMENT 5 90.72 18.14 0.95 0.471 VARIETY.TREATMENT 5 49.80 9.96 0.52 0.759 Residual 22 421.60 19.16 Total 35 692.64

Variate: Chlorophyll content 6 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 27.25 27.25 1.37 0.255 TREATMENT 5 48.32 9.66 0.48 0.784 VARIETY.TREATMENT 5 53.01 10.60 0.53 0.750 Residual 22 438.66 19.94

Total 35 762.75

Variate: Chlorophyll content 8 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 49.59 49.59 0.71 0.410 TREATMENT 5 282.66 56.53 0.81 0.558 VARIETY.TREATMENT 5 196.98 39.40 0.56 0.729 Residual 22 1544.46 70.20 Total 35 2154.64

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Variate: Chlorophyll content 10 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 116.89 116.89 3.51 0.074 TREATMENT 5 191.22 38.24 1.15 0.365 VARIETY.TREATMENT 5 89.49 17.90 0.54 0.746 Residual 22 732.84 33.31 Total 35 1245.94

Appendix 4 Wilt Incidence on University of Ghana Farm

Variate: Wilt Incidence

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 2700.8 2700.8 6.73 0.017 TREATMENT 5 20415.4 4083.1 10.18 <.001 VARIETY.TREATMENT 5 7680.0 1536.0 3.83 0.013 Residual 21 8425.8 401.2 Total 34 43817.1

Appendix 5 Wilt Severity on University of Ghana Farm

Variate: Wilt severity

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 784.0 784.0 2.40 0.136 TREATMENT 5 13748.9 2749.8 8.40 <.001 VARIETY.TREATMENT 5 272.0 54.4 0.17 0.972 Residual 22 7197.9 327.2 Total 35 22140.2

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Appendix 6 Fresh Shoot Weight on University of Ghana Farm

Variate: Fresh shoot

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 86926. 86926. 17.04 <.001 TREATMENT 5 7830. 1566. 0.31 0.903 VARIETY.TREATMENT 5 5553. 1111. 0.22 0.951 Residual 22 112228. 5101. Total 35 249618.

Appendix 7 Fresh Root Weight on University of Ghana Farm

Variate: FRESH_ROOT

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 3108.43 3108.43 44.99 <.001 TREATMENT 5 324.28 64.86 0.94 0.476 VARIETY.TREATMENT 5 44.77 8.95 0.13 0.984 Residual 22 1520.13 69.10 Total 35 5554.33

Appendix 8 Dry Shoot Weight on University of Ghana Farm

Variate: Dry shoot

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 2168.5 2168.5 19.22 <.001 TREATMENT 5 340.0 68.0 0.60 0.699 VARIETY.TREATMENT 5 191.8 38.4 0.34 0.883 Residual 22 2482.4 112.8 Total 35 7585.3

Appendix 9 Dry Root Weight on University of Ghana Farm

Variate: Dry root

Source of variation d.f. s.s. m.s. v.r. F pr.

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VARIETY 1 304.502 304.502 57.17 <.001 TREATMENT 5 57.725 11.545 2.17 0.095 VARIETY.TREATMENT 5 39.189 7.838 1.47 0.239 Residual 22 117.169 5.326 Total 35 566.143

Appendix 10 Yeild on University of Ghana Farm

Variate: Yield

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 4153718. 4153718. 24.95 <.001 TREATMENT 5 1994418. 398884. 2.40 0.074 VARIETY.TREATMENT 5 403616. 80723. 0.48 0.783 Residual 20 3329011. 166451. Total 33 10177763.

ANALYSIS OF VARIANCE ON REPRODUCTIVE PARAMETERS ON UNIVERSITY OF GHANA FARM

Appendix 11 Initial Nematode Count on University of Ghana Farm Variate: Initial nematode count

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 81701. 81701. 4.96 0.036 TREATMENT 5 50357. 10071. 0.61 0.692 VARIETY.TREATMENT 5 45447. 9089. 0.55 0.735 Residual 22 362130. 16460. Total 35 706219.

Appendix 12 Final Nematode Count on University of Ghana Farm

Variate: Final nematode count

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 1356.7 1356.7 1.83 0.190 TREATMENT 5 4609.5 921.9 1.24 0.323 VARIETY.TREATMENT 5 1741.5 348.3 0.47 0.795 Residual 22 16308.4 741.3

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Total 35 24683.0

Appendix 13 Egg Count on University of Ghana Farm

Variate: Egg count

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 9.00 9.00 0.10 0.757 TREATMENT 5 698.33 139.67 1.52 0.224 VARIETY.TREATMENT 5 157.67 31.53 0.34 0.881 Residual 22 2019.83 91.81 Total 35 3047.00

Appendix 14 Root Gall Score on University of Ghana Farm Variate: Root score

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 1.7778 1.7778 8.80 0.007 TREATMENT 5 0.8889 0.1778 0.88 0.511 VARIETY.TREATMENT 5 0.8889 0.1778 0.88 0.511 Residual 22 4.4444 0.2020 Total 35 8.2222

Analysis of Variance of Growth Parameters on National Service Farm

Appendix 15 Analysis of variance of plant height on national service farm

Variate: Plant height 4 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 96.86 96.86 2.52 0.127 TREATMENT 5 382.60 76.52 1.99 0.120 VARIETY.TREATMENT 5 67.04 13.41 0.35 0.878 Residual 22 846.40 38.47 Total 35 1576.76

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Variate: Plant height 6 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 234.40 234.40 4.89 0.038 TREATMENT 5 285.12 57.02 1.19 0.346 VARIETY.TREATMENT 5 100.38 20.08 0.42 0.830 Residual 22 1054.02 47.91 Total 35 1922.98

Variate: Plant height 8 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 208.95 208.95 8.87 0.007 TREATMENT 5 126.81 25.36 1.08 0.400 VARIETY.TREATMENT 5 103.91 20.78 0.88 0.509 Residual 22 518.35 23.56 Total 35 984.85

Variate: Plant height 10 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 227.41 227.41 10.82 0.003 TREATMENT 5 131.82 26.36 1.25 0.319 VARIETY.TREATMENT 5 96.98 19.40 0.92 0.485 Residual 22 462.59 21.03 Total 35 967.45

Appendix 16 Analysis of variance of plant girth on national service farm Variate: Plant girth 4 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 18.821 18.821 9.24 0.006 TREATMENT 5 12.062 2.412 1.18 0.349 VARIETY.TREATMENT 5 4.582 0.916 0.45 0.809 Residual 22 44.831 2.038 Total 35 113.828

Variate: Plant girth 6 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 17.5421 17.5421 26.22 <.001 TREATMENT 5 5.6120 1.1224 1.68 0.182

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VARIETY.TREATMENT 5 2.1318 0.4264 0.64 0.674 Residual 22 14.7213 0.6692 Total 35 43.8097

Variate: Plant girth 8 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 25.2171 25.2171 28.06 <.001 TREATMENT 5 7.8869 1.5774 1.76 0.164 VARIETY.TREATMENT 5 4.6184 0.9237 1.03 0.426 Residual 22 19.7701 0.8986 Total 35 58.7215

Variate: Plant girth 10 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 35.641 35.641 16.65 <.001 TREATMENT 5 16.421 3.284 1.53 0.220 VARIETY.TREATMENT 5 4.744 0.949 0.44 0.814 Residual 22 47.098 2.141 Total 35 110.828

Appendix 17 Analysis of variance of chlorophyll content on national service farm Variate: Chlorophyll content 4 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr. VARIETY 1 147.62 147.62 6.21 0.021 TREATMENT 5 58.03 11.61 0.49 0.781 VARIETY.TREATMENT 5 58.84 11.77 0.50 0.776 Residual 22 522.78 23.76 Total 35 817.74

Variate: Chlorophyll content 6 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 41.24 41.24 1.56 0.225 TREATMENT 5 77.97 15.59 0.59 0.708 VARIETY.TREATMENT 5 190.40 38.08 1.44 0.250 Residual 22 582.40 26.47 Total 35 1120.96

Variate: Chlorophyll content 8 weeks after transplanting

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Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 48.42 48.42 2.00 0.172 TREATMENT 5 89.29 17.86 0.74 0.604 VARIETY.TREATMENT 5 165.90 33.18 1.37 0.274 Residual 22 533.34 24.24 Total 35 901.00

Variate: Chlorophyll content 10 weeks after transplanting

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 155.3 155.3 0.90 0.354 TREATMENT 5 646.5 129.3 0.75 0.597 VARIETY.TREATMENT 5 737.8 147.6 0.85 0.528 Residual 22 3808.2 173.1 Total 35 5764.5

Appendix 18 Analysis of variance of wilt incidence on national service farm

Variate: Wilt incidence

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 277.8 277.8 0.84 0.370 TREATMENT 5 8188.9 1637.8 4.93 0.004 VARIETY.TREATMENT 5 455.6 91.1 0.27 0.922 Residual 22 7311.1 332.3 Total 35 19855.6

Appendix 19 Analysis of variance of wilt severity on National service farm

Variate: Wilt severity

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 3211.1 3211.1 19.38 <.001 TREATMENT 5 3200.0 640.0 3.86 0.012 VARIETY.TREATMENT 5 1688.9 337.8 2.04 0.113 Residual 22 3646.0 165.7 Total 35 13812.0

Appendix 20 Analysis of variance of fresh shoot weight on national service farm

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Variate: Fresh shoot

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 61805. 61805. 10.71 0.004 TREATMENT 5 28681. 5736. 0.99 0.445 VARIETY.TREATMENT 5 29074. 5815. 1.01 0.438 Residual 21 121185. 5771. Total 34 236709.

Appendix 21 Analysis of variance of fresh root weight on national service farm

Variate: Fresh root

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 15848. 15848. 9.09 0.007 TREATMENT 5 11982. 2396. 1.37 0.274 VARIETY.TREATMENT 5 8677. 1735. 1.00 0.444 Residual 21 36612. 1743. Total 34 187001.

Appendix 22 Analysis of variance of dry shoot weight on national service farm

Variate: Dry shoot

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 762.2 762.2 3.97 0.060 TREATMENT 5 528.9 105.8 0.55 0.736 VARIETY.TREATMENT 5 1728.2 345.6 1.80 0.157 Residual 21 4033.9 192.1 Total 34 7162.3

Appendix 23 Analysis of variance of dry root weight on national service farm

Variate: Dry root

Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr.

VARIETY 1 161.706 161.706 19.98 <.001 TREATMENT 5 52.596 10.519 1.30 0.302 VARIETY.TREATMENT 5 110.397 22.079 2.73 0.047 Residual 21 169.996 8.095

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Total 34 495.087

Appendix 24 Analysis of variance of yield on national service farm

Variate: Yield

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 7234899. 7234899. 16.49 <.001 TREATMENT 5 886602. 177320. 0.40 0.841 VARIETY.TREATMENT 5 1976137. 395227. 0.90 0.498 Residual 22 9650012. 438637. Total 35 20368779.

Analysis of Variance on Reproductive Factors

Appendix 25 Analysis of variance of initial nematode count on national service farm

Variate: Initial nematode count

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 5136. 5136. 0.51 0.481 TREATMENT 5 39006. 7801. 0.78 0.575 VARIETY.TREATMENT 5 34305. 6861. 0.69 0.639 Residual 22 220073. 10003. Total 35 308549.

Appendix 26 Analysis of variance of final nematode count on national service farm

Variate: Final nematode count

Source of variation d.f. s.s. m.s. v.r. F pr.

VARIETY 1 26.69 26.69 0.98 0.333 TREATMENT 5 179.81 35.96 1.32 0.291 VARIETY.TREATMENT 5 199.14 39.83 1.46 0.241 Residual 22 598.28 27.19 Total 35 1008.97

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Appendix 27 Analysis of variance of egg count on national service farm Variate: Egg count

Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr.

VARIETY 1 1530. 1530. 1.24 0.278 TREATMENT 5 5347. 1069. 0.87 0.519 VARIETY.TREATMENT 5 6725. 1345. 1.09 0.394 Residual 21 (1) 25859. 1231. Total 34 (1) 43183.

Appendix 28 Analysis of variance of root gall score on national service farm Variate: Root score

Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr.

VARIETY 1 21.286 21.286 9.34 0.006 TREATMENT 5 9.549 1.910 0.84 0.538 VARIETY.TREATMENT 5 9.549 1.910 0.84 0.538 Residual 21 (1) 47.883 2.280 Total 34 (1) 85.143

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Appendix 29 Root–Knot Nematode Rating Chart – Bridge and Page

Source: Bridge and Page, 1980