Master's Thesis PHYTOPHTHORA MEGAKARYA and P. PALMIVORA

FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN

Master’s thesis

Kudjordjie, Enoch Narh

PHYTOPHTHORA MEGAKARYA AND P. PALMIVORA ON

THEOBROMA CACAO: ASPECTS OF VIRULENCE AND THE

EFFECTS OF TEMPERATURE ON GROWTH AND

RESISTANCE TO FUNGICIDES

Academic advisor: Michael Foged Lyngkjær (Associate professor) Submitted: 30th July, 2015

Name of department: Department of Plant and Environmental Sciences

Author: Enoch Narh Kudjordjie

Student ID: tvq528

Title / Subtitle: Phytophthora megakarya and P. palmivora on Theobroma cacao: Aspects of virulence and the effects of temperature on growth and resistance to fungicides

Subject description: This thesis investigates the dynamics of black pod disease of cocoa caused by Phytophthora megakarya and P. palmivora species in Ghana. It explores the virulence and aggressiveness of the oomycetes, their response to temperature as well as the sensitivity to the fungicide, Ridomil Gold.

Academic advisor: Michael Foged Lyngkjær Associate Professor Section for Plant Biochemistry Department of Plant and Environmental Sciences Faculty of Science University of Copenhagen

Date submitted: 30th July, 2015

Weight of Thesis: 45 ECTS

Front page image: Healthy cocoa pods (left) and heaped detached cocoa pods with severe black pod infections. Courtesy A. Y Akrofi.

Grade:

ABSTRACT

The effect of climate change is not only expected to cause a decline in areas of suitable crop production, but also impact the occurrence and severity of agricultural diseases. Theobroma cacao, the plant behind the global chocolate industry, and the source of livelihood of an estimated 40 – 50 million people is threatened by this phenomenon. In West Africa, Phytophthora palmivora and P. megakarya are the two causal agents of black pod disease, the most destructive of cocoa diseases, with P. megakarya described to be highly virulent. The aim of this study was to ascertain the current dynamics of cocoa black pod disease caused by the two Phytophthora species in Ghana. Sixteen Phytophthora isolates collected from the six cocoa growing regions were used in this study. Using inoculation methods and microscopic analysis, it was found that P. palmivora isolates were more aggressive, virulent and produced many zoospores and chlamydospores than P. megakarya. These results contradict earlier reports, a clear indication of an emerging trend in black pod disease levels caused by the two Phytophthora species. Temperature has considerable effects on the growth of Phytophthora isolates, with optimum temperatures of highest mycelia growth in P. megakarya being 25 ˚C while that of P. palmivora ranges from 25 ˚C to 28 ˚C. P. megakarya was found to be more sensitive to the higher temperatures and recorded no mycelial growth at 31 ˚C and 34 ˚C. On resistance to fungicides, Ridomil Gold (6 % metalaxyl M + 60% Cupper (I) oxide) applied at the recommended rate (3.3g/L), was very effective against Phytophthora isolates in vitro at varied temperatures with 100% inhibition of mycelia growth. The interactive effect of higher temperatures and fungicide (Ridomil Gold) on P. palmivora and P. megakarya in vitro is appreciable, and should be further investigated in order to establish minimum Ridomil Gold dosage for effective and safe disease control in the future. The findings of the present study provide new insights of P. palmivora and P. megakarya infections on cocoa that require further investigations for effective disease management in Ghana Keywords: Theobroma cacao, Black pod disease dynamics, P. megakarya, P. palmivora, virulence climate change, temperature.

PREFACE

This thesis was written as part of the requirements for the completion of my master education in Agricultural Development at the Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Denmark. Both field and laboratory works were carried out at the Cocoa Research institute of Ghana (CRIG) from the 5 January, 2015 to March 20, 2015.

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ACKNOWLEDGEMENT

I am so grateful to the Danish International Development Agency (DANIDA) for the scholarship, the Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen for making it possible for me to study here. I would like to express my deepest gratitude to my supervisor, Associate Professor Michael Foged Lyngkjær, you have been a tremendous mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. Your advice and help on both the current research as well as on my future research career have been priceless. My special appreciation and thank also goes to Professor Andreas de Neegaard, my study director for his support and encouragement. Any time I bounced into your office, you will turn from your seat, smile and say “hi! You again?” Thank you for being there for me always. I would also like to thank Mr. A. Y Akrofi, and Mr. Ishmael Amoako-Atta for their guidance, contributions and supervision of my field and laboratory work at the Cocoa Research Institute of Ghana (CRIG). I am very grateful for your support and may the Lord bless you. Dr. Ameyaw- Akumfi, I am very grateful for the accommodation. Also, a special thanks to Mr. Eric Kumi Asare, the laboratory technician at the mycology laboratory at CRIG. You have been a very good friend and always ready to help and give your best suggestions. To the mycology staff, Beatrice and Mary, the service personnel, Prince Kwakye and Habiba Osumanu. My research would not have been possible without your helps. And it would have been a lonely lab and stay without you all. I very much appreciate your contributions and daily support. Many thanks also goes to the staff at Danida Fellowship Centre (DFC) especially Lene Mosegaard, Eva Thaulow, and Henrik Bech for efficiently handling all the practical issues relating to my studies and accommodation. The junior staff organizes all the parties, the cakes and the tours just to make our stay in Denmark enjoyable. Sara, Camilla, Niels, Anne Marie, Troels, Sasha and Thomas, you were amazing! This fond memories will never fade. Last but not the least, to my family and friends, you are the sources of my motivation, and I will forever remain grateful to you all.

TABLE OF CONTENTS

ABSTRACT ...... II

PREFACE ...... III

ACKNOWLEDGEMENT ...... IV

LIST OF FIGURES ...... VIII

LIST OF TABLES ...... IX

LIST OF ABBREVIATIONS ...... X

1. INTRODUCTION...... 1

1.1 Background ...... 1

1.2 Objectives ...... 3

2.0 LITERATURE REVIEW ...... 4

2.1 Origin and spread of Theobroma cacao ...... 4

2.2 Botany and types of cocoa ...... 4

2.3 Economic importance of cocoa ...... 8

2.4 Nutritional attributes of Theobroma cacao ...... 8

2.5 Cocoa production in Ghana ...... 9

2.6 Areas of production ...... 10

2.7 Climate requirements ...... 11

2.8 Temperature scenarios in the cocoa producing zones of Ghana ...... 11

2.9 Effects of temperature on the development of plant pathogens ...... 13

2.10 Phytophthora species ...... 14

2.11 Biology and evolutionary placement ...... 15

2.12 Phytophthora species of cocoa ...... 15

2.13 P. palmivora and P. megakarya distinction ...... 15

2.14 The black pod disease ...... 16

2.15 Disease cycle ...... 17

2.16 Spore germination and host penetration, colonization and sporulation ...... 19

2.17 Resistance mechanisms in cocoa ...... 20

2.18 Black pod disease symptoms ...... 21

2.19 Strategies for disease management ...... 22 2.19.1 Quarantine control...... 22 2.19.2 Cultural control ...... 23 2.19.3 Chemical control ...... 23 2.19.4 Biological control ...... 26 2.19.5 Use of resistant cocoa varieties ...... 27 2.19.6 Integrated disease management ...... 28

3. MATERIALS AND METHODS ...... 29

3.1. Source of Phytophthora isolates ...... 29

3.2. Viability and pathogenicity of Phytophthora isolates ...... 30

3.3. Virulence test using leaf disc inoculation method ...... 30 3.3.1. Leaf sampling ...... 30 3.3.2. Leaf disc inoculation assay ...... 31

3.4. Determination of virulence of Phytophthora isolates on detached cocoa pods...... 32

3.5. Mycelial growth of Phytophthora isolates on agar at different temperatures ...... 33

3.6. Evaluation of ridomil gold against Phytophthora isolates...... 33

3.7. Data analysis...... 34

4. RESULTS ...... 35

4.1 Pathogenicity and aggressiveness of Phytophthora isolates ...... 35

4.2. Virulence of Phytophthora isolates on leaf disc...... 36

4.3. Virulence of Phytophthora isolates on detached pods ...... 39

4.4. Effect of temperature on the growth of Phytophthora isolates ...... 40

4.5. Growth patterns of Phytophthora isolates under different temperatures ...... 42

4.6. Formation of chlamydospore in relation to temperature ...... 44

4.7. Determination of resistance of Phytophthora isolates to ridomil gold with increase in temperature ...... 45

5. DISCUSSION ...... 47

5.1. Pathogenicity and aggressiveness of Phytophthora isolates ...... 47

5.2. Virulence screening of Phytophthora isolates ...... 48

5.3. Mycelia growth of Phytophthora isolates in relation to temperature ...... 50

5.4. Effects of temperature on chlamydospore formation ...... 52

5.5. Fungicide resistance of Phytophthora isolates in relation to temperature ...... 53

6. CONCLUSION ...... 54

7. PERSPECTIVES ...... 56

REFERENCES ...... 57

APPENDICES ...... 67

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LIST OF FIGURES

Figure 2.1 Cocoa (Theobroma cacao. L) in the wild and under cultivation...... 5 Figure 2.2 Cocoa at different stages ...... 6 Figure 2.3 Symptoms of cocoa diseases ...... 7 Figure 2.4 Zones suitable for cocoa production in Ghana ...... 10 Figure 2.5 Characteristic colony morphology of Phytophthora species on V8 juice agar media ... 16 Figure 2.6 Disease cycle of P. megakarya on cacao highlighting the main spore types and infective propagules ...... 18 Figure 2.7 Symptoms of Black pod disease on cocoa ...... 22 Figure 2.8 Depiction of fungicide resistance development in a pathogen population ...... 26 Figure 3.1 Map of Ghana (excluding the North) showing districts where Phytophthora isolates were collected ...... 29 Figure 3.2 Pod inoculation with mycelial plugs cut from 4 day actively growing Phytophthora cultures ...... 30 Figure 3.3 Leaf sampling for test of virulence ...... 31 Figure 3.4 An incubation tray containing leaf discs inoculated with zoospore suspension of Phytophthora isolates ...... 32 Figure 3.5 Pod inoculation using mycelia plugs of Phytophthora isolates ...... 33 Figure 4.1 Cocoa pods with black pod symptoms after inoculation with Phytophthora isolates ...... 35 Figure 4.2 Leaf discs showing black pod symptoms after incubation for 7 days ...... 37 Figure 4.3 Mean symptom rating of Phytophthora isolates on leaf discs ...... 38 Figure 4.4 Cocoa pods showing black pod symptoms after inoculation ...... 39 Figure 4.5 Mean mycelia growth of Phytophthora isolates at different temperatures ...... 41 Figure 4.6 Growth patterns of P. megakarya isolates on V8 juice agar at 25 ˚C and 28 ˚C ...... 43 Figure 4.7 Growth patterns P. palmivora isolates on V8 juice agar at 25, 28, 31 and 34 ˚C...... 43 Figure 4.8 Chlamydospores of P. palmivora viewed under electron microscope ...... 44 Figure 4.9 Screening for fungicide resistance ...... 46

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LIST OF TABLES

Table 2.1 Regional predictions for temperature in West Africa by the end of the 21st century ...... 13 Table 2.2 Characteristics that promote Phytophthora species as very successful pathogens in the tropics ...... 14 Table 4.1 Latent periods of infection of Phytophthora isolates ...... 36 Table 4.2 Amount of Zoospores and mean severity of Phytophthora isolates ...... 38 Table 4.3 Level of virulence of Phytophthora isolates on detached pods inoculated for 72 hours ... 40 Table 4.4 Effects of temperature on the growth of Phytophthora isolates ...... 42 Table 4.5 Chlamydospore rating in Phytophthora isolates ...... 45

LIST OF ABBREVIATIONS

% percent µl microliter ANOVA Analysis of Variance AR Ashanti Region BAR Brong Ahafo Region BCAs Biological Control Agents cm centimeters

CO2 Carbon dioxide CR Central Region CRI Crop Research Institute CRIG Cocoa Research Institute of Ghana DMRT Duncan’s Mean Range Test ER Eastern Region g grams GCM Global/ General Circulation Model ICCO International Cocoa Organization IPCC Inter- Governmental Panel on Climate Change m metre ml milliliters mm millimeters Pm Phytophthora megakarya Pp Phytophthora palmivora QTL Quantitative Traits Locus SCM Simple Climate Model VR Volta Region WR Western Region

1. INTRODUCTION

1.1 Background The Oomycete pathogens, Phytophthora palmivora (Butler) and P. megakarya (Brasier and Griffin) are the two main species of Phytophthora extensively reported as the causal agents of black pod disease of Theobroma cacao in West and Central Africa (Opoku et al., 2000), where more than 72% of the world’s cocoa is produced (ICCO, 2015). P. palmivora, a cosmopolitan species is described to be less virulent than P. megakarya. P. megakarya is restricted to only cocoa producing regions in Western and Central Africa. In Ghana, pod losses associated with P. palmivora have been estimated to range from 4.5% to 19%, while P. megakarya can cause losses ranging between 60- 80% and 100% in newly affected and old affected farms respectively (Opoku et al., 1999). The high incidence of the black pod disease is a major constraint to cocoa production especially in West Africa (Flood et al., 2004). Environmental conditions are known to influence the geographic distribution and seasonality of Phytophthora diseases, with temperature being reported to have the greatest influence on growth, reproduction and pathogenesis (Matheron and Matejka, 1992). A number of studies were carried out previously to study the correlation between climatic parameters and the incidence of the Phytophthora black pod (Thorold, 1967; Wood 1974,). According to Anim-Kwapong and Frimpong, (2005), the occurrence of black pod disease in Ghana is closely related to weather and climate. The disease is known to be more prevalent in damp conditions and is most destructive in years when the condition persist. In Cameroon, Ndoumbe- Nkeng (2002) also reported that rainfall, high moisture and low temperature create favourable conditions for the development of the disease. Climate change, the most discussed subject of the first decade of the 21 century (Cooper et al., 2008), is predicted to have a direct impact on the occurrence and severity of diseases of agricultural crops. This will have a serious impact on our food security (Gautam et al., 2013). Cocoa the source of cocoa beans for the global chocolate industry is known to be highly sensitive to environmental factors such as rainfall and water application, hours of sun, soil conditions and particularly to temperature due to effects on evapotranspiration (Anim-Kwapong and Frimpong, 2005). Thus, climate change could alter stages and rate of development of cocoa pests and pathogens, modify host resistance and lead to changes in the physiology of host pathogen/pests interactions (Anim- Kwapong and Frimpong, 2005). Under the climate change scenarios, temperature is expected to rise, thus modifying host-pathogen activity. The changes in plant physiology may alter the resistance mechanisms of both traditional and genetic engineered crops. In addition, temperature

1 increases are predicted to lead to the geographic expansion of pathogen and vector distributions, bringing pathogens into contact with more potential hosts (Baker et al., 2000). The net effects of these changes are that, losses due to plant diseases will increase to an estimated 20% in the principal foods and cash crops worldwide (Rettinassababady and Jeyalakshmi, 2014). Ghana has experienced an increase in mean annual temperature of 1˚C since 1960, at an average rate of 0.21 ˚C per decade (McSweeney et al., 2010). A review of 15 different Global Circulation Models (GCM) indicate that the mean annual temperature is expected to increase by 1.0 ˚C - 3.0 ˚C by 2060 and by 1.5 ˚C – 5.2 ˚C by 2090 (De Pinto et al., 2012). In the cocoa producing regions, the predicted impact of climate change on cocoa production is expected to be great (Anim- Kwapong and Frimpong, 2005). The predictions under the General Circulation Model (GCM) for the cocoa producing forest belts are that, temperature will rise progressively from 2020 to 2080 (Anim- Kwapong and Frimpong, 2005). These climatic conditions are expected to aggravate soil moisture conditions during the dry season (November to March). The yield of cocoa is thus anticipated to decrease from 2020 to 2050, since cocoa is known to be highly susceptible to drought conditions, which are often associated with high temperatures (Anim- Kwapong and Frimpong, 2005). The close relationship between the environment and plant diseases suggest that, climate change will continue to cause modifications in the current phytosanitary measures as far as the change persist (Ghini et al., 2008). The need and the urgency to control black pod disease mainly in the advent of P. megakarya in the 1980’s, has added a new dimension to the management of the black pod disease leading to the heavily reliance on chemical control methods. Chemical control methods have been considered critical for sustaining the health of cocoa (Awudzi et al., 2012). Several fungicides have been recommended to farmers for the control of the black pod disease and several others are still being tested for their efficacy in the management of the disease. Currently, nine fungicides have been recommended for the control of black pod in Ghana (Andrews Y. Akrofi, personal communication, 11 February, 2015). But the heavy reliance on fungicides raises many concerns; issues of health and environment as well as fungicide resistance management in cocoa, especially in areas where the incidence of black pod is high. Insufficient application rate, inherently low effectiveness of the fungicides on the target pathogen, improper timing or application mode and excessive rainfall, may result in fungal pathogens resistance to fungicides (Damicone, 1996). The development of resistance to fungicides by fungal pathogens render chemical control methods ineffective, hence the need to adhere to recommended methods of fungicide application. In addition, knowledge of the environmental

2 parameters that govern Phytophthora infection is a critical aspect in making informed decisions on the timing of fungicide application. While the effects of temperature on disease epidemiology have been studied for many years in agricultural systems, it has also become imperative to ascertain the future of plant diseases in relation to predicted temperatures under the climate change scenarios. Several general circulation models (GCM) have been used to simulate global climate change scenarios and different levels of disease severity, with the aim of estimating yields, establish control tactics and strategies, aiding the decision making process. Understanding how plant pathogens retain their ability to cause disease may provide insight into reservoirs of disease and emerging infections. The analysis of the potential impacts of climate change on plant diseases is essential for the adoption of adaptation measures, as well as the development of resistant cultivars, new control methods or adapted techniques, in order to avoid more serious losses (Chakraborty and Pangga, 2004; Ghini, 2008). Thus, the aim of this study was to investigate the current dynamics of cocoa black pod disease caused by Phytophthora palmivora and P. megakarya in the different cocoa growing regions of Ghana.

1.2 Objectives The specific objectives were to;  Determine and compare virulence and aggressiveness of 16 Phytophthora isolates from the six cocoa growing regions in Ghana.  Determine the effects of varied temperature regimes, 25 ˚C, 28 ˚C, 31 ˚C and 34 ˚C on the growth of P. palmivora and P. megakarya isolates causing black pod disease on cocoa in Ghana.  Determine resistance of Phytophthora isolates to Ridomil Gold at 25 ˚C, 28 ˚C, 31 ˚C and 34 ˚C.

2.0 LITERATURE REVIEW

2.1 Origin and spread of Theobroma cacao Cocoa (Theobroma cacao L., Malvaceae, formerly Sterculiaceae) is a tree believed to originate from the neotropical rainforests, primarily in the Amazon basin and the Guyana Plateau (Lachenaud et al., 2007). Theobroma means “food of the gods” and cacao is derived from the Olmec and Mayan language (kawkaw) (ICCO, 2005). The Mayan and Aztec people of South America were known to consume chocolate as a beverage, which was prepared from ground cocoa beans mixed with maize, spices and water beaten into froth. In the 16th century, the Spanish introduced cocoa into Europe where it was made into a beverage with sugar called “chocolatl” (ICCO, 2005). The spread of cocoa continued through the 17th century to the British, French and Dutch territories, then to Brazil in the 18th century, São Tomé in 1822 and finally to the island of Fernando Po in West Africa around 1854 (Sundiata, 1974). Theobroma cacao, is the most commercially important among 22 species in the genus Theobroma. Mainly cultivated in the humid tropics, cocoa is a low altitude plant that grows from sea levels up to an altitude of 700 m (Oyekale, 2012). Cocoa production typically require annual rainfall levels of about 1250 – 3000 mm, with preferred temperature levels ranging from a minimum of 18-21 ˚C and a maximum of 30 – 32 ˚C (IITA, 2009). The principal producers of cocoa are Cote d’Ivoire, Ghana, Indonesia, Nigeria, Cameroon and Brazil. West Africa alone accounts for about 70% of the world’s cocoa with Cote d’Ivoire and Ghana being the main producers (ICCO, 2015).

2.2 Botany and types of cocoa The cocoa tree is a small, semi-deciduous plant that grows typically to be 5 to 10 m in height, with a canopy of around 4 to 5 m wide (FAO, 2007). In the wild, it may grow to about 25 m in height (Lachenaud et al., 2007) (Figure 2.1a), but when in cultivation it is usually kept under 7 m for easy management and harvest (Braudeau, 1975) (Figure 2.1b). Cocoa seedlings show dimorphic branching; a vertical (orthotropic) stem, called chupon, with spiral phyllotaxi, which terminates at the jorquette where three to five lateral (plagiotropic) fan branches develop (Carr and Lockwood, 2011). This branching formation where the buds grow out laterally from the terminal end of the main stem is called a “jorquette”. The crop also exhibits a flushing-type growth habit, with two to four growth flushes per year, and the leaves on new flushes are mostly pink to red and hang down vertically (Nyadanu, 2011).

Figure 2.1 Cocoa (Theobroma cacao. L) in the wild and under cultivation. (a) Theobroma spp. in the wild (b) Cocoa cultivated under shade (c) Flowers of cocoa on stems (d) cocoa in bearing

Theobroma cacao is a typical cross pollinated monoecious, cauliflorous plant with cluster of flowers formed on trunks and on main stems of the tree (FAO, 2007) (Figure 2.1 c). The flowers are borne in compressed dichasial cymes of a few to several individuals. Flowering which often tends to cycle with the wet and dry seasons (Winkel, 2013), normally starts when the plant is between three to five years old. Flowering is sensitive to temperature. Abundant flower formation occur when diurnal temperatures increase to the point where the night time temperature does not fall below 27 ˚C (Winkel, 2013). While constant temperatures for both day and night at 31 ˚C is known to severely impede florescence, as will temperatures below 23 ˚C. According to Braudeau, (1975), the above conditions are the optimal conditions for flowering, yet, it can still occur throughout the year, resulting in flowers and fruit occurring on the plant at the same time. The main agents of pollination are the midge Forcipomyia species, although other insects such as thrips, ants and aphids can cause accidental pollination (Chapman and Soria, 1983). Only about 1-5% of the flowers produced actually become pollinated, and finally develop into mature fruits (Young, 1994). Two

5 main reasons account for this; that the flowers only become receptive for approximately 48 hours, after which it is aborted from the tree (Braudeau, 1975). The second reason is attributed to self- incompatibility, i.e. the flowers cannot pollinate themselves (Rieger, 2012), but require out-crossing (Young, 1994). The fruit of cocoa at its initial stage of development is called “cherelle” which is later known as a ‘‘pod’’ when matured (FAO, 2007) (Figure 2.2 a). It is common to see both flowers and pods together on the same tree throughout the year. Cocoa pod is a type of indehiscent berry, weighs about 500 g on average depending on the clones, and contains about 30–60 seeds surrounded by sweet mucilage (Figure 2.2 b) (Lachenaud et al., 2007). Pods normally grow to an approximately 10-30 cm long and 8-10 cm wide. Fruits and seeds, depending on the cocoa variety may take 180 – 300 days to reach full maturation (FAO, 2007). The seeds are normally fermented and dried, (then known as ‘‘beans’’) (Figure 2.2 c), and used as the raw material for the chocolate industry (Figure 2.2 d).

Figure 2.2 Cocoa at different stages. (a) Matured cocoa pods (b) open cocoa pod with beans covered with sweet white mucilage (c) dried cocoa beans (d) cocoa products

Cocoa is usually grown under shade in newly thinned forest with little monetary capital investment especially in West Africa (Carr and Lockwood, 2011). It thrives well in loam, loamy silt, loamy clay, or loamy sand. Propagation is mainly by seed, but little use is made of cuttings or budded

6 clones, which are more difficult to grow in the early years of the plantation (Lachenaud et al., 2007). The seeds over the years have undergone improvements. In Ghana for example, the breeding and adoption of new seedling cultivars has led to large increases in productivity (Edwin and Masters, 2003). The planting densities vary from 400 to 2500 (plants/ha) with average yields between 200 and 800 kg of fermented dried cocoa per ha (Wood and Lass, 1985). When in cultivation, the cocoa tree is generally attacked by numerous insect pests and diseases: caterpillars, bugs of the mirid family, black pod, which is pantropical, witches’ broom and frosty pod rot in America, and Swollen-Shoot virus in Africa, etc (Lachenaud et al., 2007) (Figure 2.3).

Figure 2.3 Symptoms of cocoa diseases (a) black pod (Akrofi, 2015) (b) Witches broom (c) Frosty pod (d) cocoa swollen shoot (e) cocoa mirid and damage to pod (f) Rodents attack witches broom (ICCO image: www.dropdata.org), c and e (www.plantvillage.com).

Traditionally, cocoa has been distinguished between two geographical sub-species of the cocoa tree, T. cacao subsp. cacao and T. cacao subsp. sphaerocarpum, which correspond to the main two groups of historical cultivars: Criollo and Forastero (Cuatrecasas, 1964). Other classifications based on trade perceptions of its physical and sensory quality and associated botanical traits (Cheesman,

1944) put cocoa into three groups; Criollo, Forastero and Trinitaro (Carr and Lockwood, 2011). Criollo is known to have higher quality than the Forastero type but with lower yield and vigour and very susceptible to pests and diseases (Nyadanu, 2011). The bulk of the world’s cocoa is produced from the Forastero, because of its diverse populations and different geographic origins. The Amelonado cocoa is a Forastero type from the lower Amazon basin. Trinitario, commercially grown in the Carribean and Papua New Guinea is considered a natural hybrid between Criollo and the Amelonado population and is not found in the wild (Toxopeus, 1987; Carr and Lockwood, 2011). In a recent classification using microsatellite markers, Motamayor et al., (2008) identified 10 genetic groups which according to Carr and Lockwood (2011), reflects more accurately the genetic diversity of the cocoa germplasms. Lachenaud et al., (2007), stressed that good knowledge of the structuring of the species’ genetic diversity is therefore needed to produce high-yielding hybrid varieties.

2.3 Economic importance of cocoa The global economic importance of cocoa cannot be underestimated, as it plays key roles in producing and non- producing countries. The crop is cultivated in 57 countries on an estimated total area of 6.5 million hectares (Omont, 2001). Cocoa is predominantly a smallholder crop, as more than 90% of world cocoa production is produced from small farms (2- 5 hactares) mainly in Africa and Asia (ICCO, 2013). As a global traded commodity, cocoa contributes to the livelihoods of an estimated 40–50 million people (World Cocoa Foundation, 2012). The total production as at 2012 was 4.8 million metric tons (World Cocoa Foundation, 2014). Cocoa bean is used in the global food industry for the production of chocolate and powder (for drinking, baking and ice cream manufacture). The allure of chocolate and the love affair associated to it makes chocolate stand out among other confectionery products (ICCO, 2005). It is also used in the cosmetic and the pharmaceutical industries.

2.4 Nutritional attributes of Theobroma cacao The nutritional attributes of cocoa has been subjected to extensive scientific studies (ICCO, 2005). The cocoa component in chocolate is rich in essential minerals such as magnesium, copper, potassium and manganese, sodium, calcium, iron, phosphorus and zinc and vitamins including vitamin E and some of the vitamin B complex (thiamine, riboflavin and niacin). Cocoa butter is reported to contribute proportionally more stearic acid than any other naturally occurring fat (ICCO, 2005). Cocoa bean is rich in flavonoids, typically the flavanols, which are powerful antioxidants believed to help the body's cells resist damage by free radicals, which are formed by numerous

8 processes including when the body's cells utilize oxygen for energy. Oxidative damage to the body’s cells and tissues is believed to contribute to several diseases such as heart disease. Other research findings according to ICCO (2005), are beginning to emerge that the consumption of a flavonoid-rich cocoa beverage inhibit platelet activity and increase the time taken for blood to clot, contribute to reducing the risk of certain types of cancer. It is also known to contain substantial amounts of a class of biologically active compounds called methylxanthines, which include theobromine, caffeine and theophylline that enhance mental activity. These compounds cause physiological actions ranging from stimulation of the central nerve system (CNS), to stimulation of the cardiac muscle and relaxation of the muscles, in general. But despite the above benefits there are some perceived health risks of cocoa and chocolate such as allergies, diabetes, dental caries, acne and migraines (Details are beyond the scope of this review).

2.5 Cocoa production in Ghana Historically, cocoa was introduced into Ghana in 1879 and was first cultivated in the Eastern region (Akrofi, 2003), an area dominated by oil palm cultivation and rubber trading (Hill, 1963). By 1885, cocoa production saw a massive boost as a result of the fall in the world price of palm-oil, while a boom in rubber exports in 1890, provided the capital for the purchase of new land and the establishment of European produce-buying companies on the West African coast ready to trade the new crop, (Hill, op. cit.; Amanor, 2010; Gunnarsson, 1978). However, the outbreak of cocoa swollen shoot virus, capsid pests and loss of soil fertility led to a shift in production from the East to the South West. Although the crop has not been an unmitigated success, it has contributed significantly to the country’s economy. The country is the second largest producer of cocoa in the world with about 21% of the global total (FAOSTAT, 2012). Currently, cocoa is cultivated on an estimated total area of about 1.65 million hectares, generating about $2 billion in foreign exchange annually and is a major contributor to government revenue and GDP (COCOBOD, 2014). The sector employs over 800,000 smallholder farmers. In 2010, cocoa contributed 8.2% of the country’s GDP and 30% of total export earnings (Asante-Poku and Angelucci, 2013). Yet, Ghana’s cocoa yield, according to Mohammed et al., (2011) is estimated to be on average 25 percent less than the average yield level of the ten largest cocoa producing nations, and nearly 40 percent below the average yield level of neighbouring Cote d’Ivoire. Of the paramount reasons accounting for the low yield are pests and diseases including black pod and mistletoes, and poor cultural and management practices (Mohammed et al., 2011). Efforts by the Ghanaian government to boost cocoa production were held

9 back in the 2012/13 season, with the two main primary factors being weather conditions and the incidence of black pod diseases. An increased incidence of black pod disease has been recorded. The disease is estimated to cause pod losses of 200,000 tonnes (Oxford Business Group, 2014). However, in the longer term, the government of Ghana aims to boost production to 1 Million Mt on a sustained basis (Oxford Business Group, 2014).

2.6 Areas of production Profitable cocoa growing areas in Ghana are mainly the tropical rain forest belt which is divided into five major forest types namely: wet evergreen; upland evergreen; moist evergreen; moist semi- deciduous, and dry semi-deciduous (Hall and Swaine, 1981) (Figure 2.4). These zones have the highest annual rainfall in the country (1150 to over 2000 mm) covering the six cocoa producing regions of the country. The regions include the Eastern, Western, Ashanti, Brong Ahafo, Central and Volta. Thirty years interval regional production outputs is also shown in Figure 2.4. Currently, the Western region which is characterized with the wet and moist evergreen forest accounts for more than 50% of current production. Ashanti region accounts for about 16 percent of total production, while the Eastern and Brong Ahafo regions together account for about 19 percent of total production (COCOBOD, 2014).

Figure 2.4 Zones suitable for cocoa production in Ghana. Forest type abbreviations: WE= Wet Evergreen; ME= Moist Evergreen; UE= Upland Evergreen and DS= Dry Semi- Deciduous forest. (Modified) from Kemausuor et al., 2013).

2.7 Climate requirements

Climate change, the result of the acceleration of the increase in temperature and CO2 concentration over the last 100 years, has become the biggest threat, and the most discussed subject of the first decade of the 21 century (Gautam et al., 2013; Cooper et al., 2008). With strong evidence of accelerated global warming, agriculture specifically, in developing countries is known to be highly impacted by this phenomenon. The distribution of Theobroma cacao mainly throughout the warm and humid rain forests of the equatorial regions of the world (Ogata, 2008), revealed the significant correlations between cocoa production and climate. Cocoa production takes place under a limited set of climatic and biophysical conditions, situated in a narrow band of temperatures and relatively stable soil moisture content (Anim-Kwampong and Frimpong, 2005). Profitable growing temperatures vary from 30-32 ˚C mean maximum, 18-21 ˚C mean minimum and absolute minimum of 10 ˚C (Wood and Lass, 1985). The plant is very sensitive to drought, extreme temperature fluctuations and to patterns of rainfall distribution. Seasonal variations in temperature in Ghana are greatest in the north, with highest temperatures in the hot, dry season (April, May and June) at 27‐30˚C, and lowest in July, August and September at 25‐27˚C. Further south, temperatures reach 25‐27˚C in the warmest season January, February and March, and 22‐25˚C at their lowest in July, August and September (Mcsweeney et al., 2010). Average annual temperatures in the transitional zones are higher (27 ˚C) than the rainforest zones of the western region with average annual temperatures of about 25 ˚C. Rainfall distribution is bi-modal from April to July and September to November in all the six producing regions. The main cropping season starts in October-February/March while there is also a smaller/light mid-crop cycle, which occurs from around April/May to mid-September. The main dry season normally begins from November to February - March during which soils can be too dry, resulting in high seedling mortality especially when in establishment, and poor pod filling for bearing plants (Anim-Kwapong and Frimpong, 2005). The crop prefers relative humidity of 70% at night and up to 100% in the day (ICCO, 2011).

2.8 Temperature scenarios in the cocoa producing zones of Ghana Global mean temperature has been observed to have increased by 0.76 °C from the period of 1850 to 2005 (Frimpong et al., 2014). But a predicted further rise in temperature of 1–5 °C within and beyond this century is envisaged if global action is not taken to reduce greenhouse gas emissions, (Jones et al., 2007). The predicted annual changes in temperature that will occur by the end of the

21 century for the West African region according to IPCC using 21 GCM is summarized in the Table 2.1. Ghana has experienced an increase in mean annual temperature of 1˚C since 1960, at an average rate of 0.21 ˚C per decade (McSweeney et al., 2010). A review of 15 different Global Circulation Models (GCM) indicate that the mean annual temperature is expected to increase by 1.0 ˚C - 3.0 ˚C by 2060 and by 1.5 ˚C – 5.2 ˚C by 2090 (De Pinto et al., 2012). In the cocoa producing regions, the predicted impact of climate change on cocoa production is expected to be great. Anim- Kwapong and Frimpong, (2005) assessed the temperature scenarios for the semi-deciduous forest and evergreen rainforest zones of Ghana, using process-based methods that rely on the General Circulation Models (GCM) in conjunction with Simple Climate Models (SCM). The mean annual temperature change in the semi deciduous is expected to rise by 0.8, 2.5 and 5.4 in 2020, 2050 and 2080 respectively. While in the evergreen rainforest zones, the mean annual temperature is expected to change by 0.6, 2.0 and 3.9 C respectively for the same years. The consequence of predicted rise in temperature is very great as this is expected to cause a significant decline in the cocoa belts suitability for production. According to Läderach, et al. (2011), the implications of the projection are that, the distribution of suitability within the current cocoa-growing areas in Ghana will decrease significantly by 2050. A shift is expected to be seen in the suitable production areas of Brong Ahafo and Western regions by 2030, while conditions similar to the current climates will remain only in the areas between Central, Ashanti and Eastern regions (Läderach, et al., 2011). They further projected that by 2050, cocoa production will become concentrated in two areas in Ghana, between the Central and Ashanti regions, and in the mountain ranges of the Kwahu Plateau between the Eastern and Ashanti regions. An expected increase of up to 2°C in mean annual temperature will lead to a considerable decrease of suitability for cocoa growing-areas. The higher temperatures could intensify the dry season, to which cocoa is very susceptible and higher within-season water deficits caused by higher evaporative demand due to higher temperatures (Läderach, et al., 2011).

Table 2.1 Regional predictions for Temperature in West Africa by the end of the 21st century

*Maximum and minimum predictions of change are given together with the 25, 50 and 75 quartile values from the 21 GCM’s .Source: Cooper et al., (2008).

2.9 Effects of temperature on the development of plant pathogens Environmental conditions are known play important role in the development of plants and pathogens causing plant diseases. This makes weather a key production factor that is closely monitored in agriculture. Temperature and moisture are the most important environmental factors that affect the initiation and development of infectious plant diseases (Agrios, 2005). These factors in addition to soil nutrients and to some extent light and soil pH, affect disease development through their influence on the growth and susceptibility of the host. They also affect the multiplication and activity of the pathogen or on the interaction of host and pathogen as it relates to the severity of symptom development. Temperature, governs pathogen evolution rates such as the number of generations of pathogen reproduction per time interval (Garrett et al., 2006). Since the duration of an infection cycle determines the number of infection cycles as well as the number of new infections in one season, it is clear that the effect of temperature on the prevalence of a disease in a given season may be very great (Agrios, 2005). It has also been established that different temperature regimes affect the genetic machinery of the cell by favouring or inhibiting the expression of certain genes involved in disease resistance or susceptibility. Temperature requirement for infection however, differ among pathogens as some fungi for example require low or high temperatures to cause diseases (Garrett et al., 2006). The effects of temperature on the development of a particular disease after infection is also dependent on the host- pathogen combination (Agrios, 2005). In many instances, the optimum temperature promoting disease development may differ from those of both the pathogen and the host. The most rapid disease development, that is the shortest time required for the completion of an infection cycle, usually occurs when the temperature is optimum for the development of the pathogen but is above or below the optimum for the development of the host (Agrios, 2005).

2.10 Phytophthora species Phytophthora, the ‘plant destroyer’, is a cosmopolitan genus of Oomycete obligate plant pathogens with about 60 described species, attacking several plants in both the temperate and the tropical regions of the world (Drenth and Guest, 2004). The genera Phytophthora is considered one of the most destructive, and have been well studied in the temperate regions of the world, after the potato late blight epidemic in Europe in 1845–47, which led to the development of plant pathology as a scientific discipline. In the wet tropical regions also, Phytophthora diseases are prevalent and of high economic importance. They cause significant losses in many fruit crops in the form of stem cankers, leaf blights, root rots, collar rots, and fruit rots (Drenth and Sendall, 2001). Several host and pathogen factors together with features of their interactions make Phytophthora diseases so troublesome in the wet tropics. This is summarized in the table 2.2.

Table 2.2 Characteristics that promote Phytophthora species as very successful pathogens in the tropics

Source: Drenth and Guest (2004).

2.11 Biology and evolutionary placement Phytophthora belongs to the kingdom Stramenopiles, class Oomycetes and the order Peronosporales. Although they share many characteristics of ecology and life history with the true fungi, their genetics and reproductive mechanisms make them distinct ((Erwin and Ribeiro 1996). The recent placement of Phytophthora into the kingdom Stramenopiles was supported by distinct characteristics such as variation in the metabolic pathways, the presence of β-glucans rather than chitin in cell walls (Bartnicki-Garcia and Wang 1983), production of motile heterokont zoospores (Desjardins et al., 1969), and predominance of the diploid stage in the life cycle (Erwin and Ribeiro 1996).

2.12 Phytophthora species of cocoa Currently, eight species of Phytophthora have been isolated from cocoa (McMahon and Purwantara, 2004). They include P. palmivora (Butler), P. megakarya (Brasier and Griffin), P. capsici (Leonian emend.) (= tropicalis), P. katsurae (Ko and Chang), P. citrophthora (R.E. Smith and E.H. Smith), P. arecae (Coleman) Pethybridge, P. nicotianae (van Breda de Haan) and P. megasperma (Dreschler) (Erwin and Ribeiro 1996; Iwaro et al., 1997; Appiah et al., 2003). Phytophthora species thrive on all parts of the cocoa plant and can be found at all growth stages, from the seedling to mature stages causing a number of diseases (McMahon and Purwantara, 2004). P. palmivora, P. megakarya and P. citrophthora are known to cause the most damage, with P. Palmivora having a worldwide distribution while the latter two species are localized. In West Africa where most of the world’s cocoa is produced, P. megakarya has been identified as the most virulent Phytophthora species causing massive losses.

2.13 P. palmivora and P. megakarya distinction Although there have been difficulties separating P. megakarya and P. palmivora, recent advances involving the use of electrophoretic banding patterns of soluble proteins (Kaosiri & Zentmyer, 1980; Erselius & de Vallavieille, 1984; Bielenin et al., 1988), isoenzyme analyses (Oudemans & Coffey, 1991), genome-based techniques such as restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (Förster et al., 1990) and sequencing of the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) (Cooke & Duncan, 1997; Ristaino et al ., 1998), have been useful in distinguishing the two species. Some of the differences include the large nuclei of the gametangia containing 5-6 large chromosomes in P. megakarya, compared with P. palmivora with 9-12 much smaller chromosomes (Sansome et al., 1975, 1979); caducous sporangia, with medium-length stalks (10-30 µm) in P. megakarya, as against that of the short (1-5 µm) stalks and

15 long stalks (30->200 µm) produced by P. palmivora and P. capsici repectively (Zentmeyer, 1987); different RFLP, protein and isoenzyme patterns (Erselius and Shaw, 1982; Forster et al., 1990; Forster and Coffey, 1991). Morphological characteristics and sexual behavior have played very important roles in previous grouping of isolates to various species (Waterhouse, 1974), and still remain the most affordable option in countries affected with severe cocoa Phytophthora diseases. The colony characteristics such as the stellate striated (Figure 2.5 a) and cottony (Figure 2.5 b) pattern of P. palmivora and P. megakarya respectively, likewise their differential growth rate on V8 agar, are reasonably, characteristics that can be used to make preliminary identification to species level (Appiah et al, 2003). The sporangia of P. palmivora have short and stubby pedicels unlike the long and thin pedicels found in P. megakarya (Erwin and Ribeiro, 1996). The morphological distinction in growth of the two species could also be responsible for the differences in the way the disease develops.

Figure 2.5 Characteristic colony morphology of Phytophthora species on V8 juice agar medium: (a) P. Palmivora showing the stellate appearance in V8 juice agar medium. (b) P. megakarya showing cotton-like appearance in V8 juice agar medium. The short and long pedicels of the sporangium (insert) of the two Phytophthora isolates are shown by the arrow.

2.14 The black pod disease Black pod disease is considered the most destructive of the several cocoa diseases globally. The disease affects all plant parts including the pods, beans, flower cushions, leaves, stems and roots. In Ghana the disease is caused by P. palmivora and P. megakarya. P. palmivora is present in all cocoa producing regions while P. megakarya, is restricted to West Africa (Opoku et al., 1999; Nyadanu 2011). Pod losses due P. palmivora is estimated to range from 4.5% to 19%, while P. megakarya can cause losses ranging between 60-80% and 100% in newly affected and old affected farms respectively

(Opoku et al., 1999). Global annual losses due Phytophthora species is estimated to be about

450,000 tons, equivalent to $ 423 million (Bowers, et al., 2001).

2.15 Disease cycle The Phytophthora infection cycle is well described by Hardham (2001). Wang et al., (2011) observed similar infection stages in P. parasitica and the model plant Arabidopsis thaliana. Akrofi (2015) also described black pod disease cycle caused by P. megakarya (Figure 2.6). In general, the infection cycle typically involves four main stages: initial contact with a potential host, zoospore encystment and spore adhesion to the host plant, spore germination and host penetration and plant colonization and nutrient acquisition (for growth and sporulation) (Hardham, 2001). The various stages of the infection cycle are discussed later in this and next section. The life cycle of Phytophthora species involves both sexual and asexual stages. The sexual cycle result in the production of oospores requires two mating types (A1 and A2) in the heterothallic species P. megakarya and P. palmivora. But oospores are rarely observed in nature, possibly because opposite mating types are rarely found together (Appiah et al., 2003). The sexual phase is a potential problem as it produces genetically different offspring, any of which might be able to overcome the host’s resistance. A diploid vegetative mycelium and three types of spores: sporangia, zoopores and chlamydospores are formed in the asexual reproduction phase ((Erwin and Ribeiro, 1996; Goodwin, 1997; Drenth and Guest, 2004). The mycelium serves as the primary inoculum. It exist mainly in soils and bark cankers and develops into the sporangia which in the presence of moisture may germinate directly, or differentiate to produce 8–32 zoospores, each of which passes through a cycle of dispersal and encystment before germinating (Drenth and Guest, 2004). Successful infections are usually initiated by motile, biflagellate zoospores that are chemotactically attracted to nearby host tissues (Hardham, 2001). Zoospores that reach the leaf or root surface undergo encystment, a rapid process in which the flagella are detached, adhesive material is secreted and the cells round up and form a cell wall (Hardham, 2001). The cyst wall become strong within 5–10 minutes, to allow the cell to generate turgor pressure and the water expulsion vacuole disappears. Firm adhesion stops the spores from being dislodged and allows physical force to be exerted by the pathogen cell as it tries to invade the plant surface. Hardham (2001) suggested that if host plants will be able to secrete compounds that inhibit spore adhesion, then it is likely that the rate of successful infection would be reduced. P. megakarya is reported to emit zoospores earlier and also two times more than P. palmivora (Brasier et al. 1981). A single pod under humid conditions may produce about 4 million sporangia

17 containing motile spores that are disseminated by rain, movement of planting materials, insects and rodents, contaminated harvesting tools and pruning implements (Brasier et al., 1981). Both sexual and asexual spore types are potentially infective, with chlamydospores and oospores serving as overwintering or resting structures (Drenth and Sendall, 2001). Chlamydospores are the main long- term survival structures of P. megakarya and P. palmivora in soils (Brasier et al., 1981). Chlamydospores develop into mycelia under favourable conditions and infect cocoa tissue (Akrofi, 2015). The duration of survival in P. megakarya species were determined to be longer than in P. palmivora (Brasier et al., 1981. Black pod infested pods shrivel and become mummified and serves as a reservoir of inoculum for at least three years (Akrofi, 2015). Pod removal during routine pruning is recommended for effective control of black pod disease.

Figure 2.6 Disease cycle of P. megakarya on cacao highlighting the main spore types and infective propagules. In the cycle, sporangiophore bearing sporangia, sporangia containing zoospores, zoospores being discharged from sporangium, infection on cacao pod, infection on tree trunk, infection on leaf, different levels of infection on cacao pods, mycelia and encysted zoospores are shown. Source: Akrofi, (2015)

2.16 Spore germination and host penetration, colonization and sporulation Microscopically, Phytophthora penetration and hyphal development are similar in both root and leaf tissues (Wang et al., 2011). The penetration, colonization, nutrient acquisition and sporulation processes of the Phytophthora infection cycle as illustrated by Hardham, (2001) is summarized in this section. Phytophthora cysts can germinate within 20–30 min after zoospore encystment. When the zoospores settle in the grooves on the root surface, the germ tube often grows straight into the root, penetrating intercellularly along the anticlinal walls between the epidermal cells immediately below the spore. In other cases, the germ tube grows directly through the periclinal wall of the root epidermal cells. But it has been observed that intercellular growth may be a relatively easy process for the advancing germ tube, simply requiring degradation of the pectin component of the middle lamella. Penetration of the periclinal wall of root epidermal cells (and perhaps even more so of the cuticle-covered leaf or stem epidermal cells) will require the degradation of other wall components. This may take time, either because the necessary enzymes are not immediately available or because it takes more time for the enzymes to degrade the wall sufficiently to allow penetration. After initial invasion, Phytophthora hyphae as mentioned earlier may grow intercellularly within the root cortex or, less frequently, they may grow ‘intracellularly’. In intracellular growth, haustoria (small globular or finger-like cells) develop in cortical cells. The haustoria in leaf or root cells lack nuclei, a feature that has been used to distinguish haustoria from intracellular hyphae. The development of haustoria is preceded by the accumulation of electron-dense material in the plant cell wall at the site of future invasion. The nature and origin of the electron-dense material is unknown, although its pattern of radiation from the pathogen side of the wall suggests that it is a result of pathogen secretory activity. As the haustorium develops, the electron-dense material persists and apparently becomes the extrahaustorial matrix. The degree of compatibility of the interaction influences the number of haustoria that are formed, fewer developing if the plant is resistant to infection. Wall appositions are often deposited by the plant (in compatible and incompatible interactions) at sites in contact with pathogen cells. In resistant plants, rapid encasement of developing haustoria may inhibit their growth and function.

In susceptible hosts, nutrients acquired from the plant allow the pathogen to ramify quickly through the plant tissues. Within 2–3 days of initial invasion, soilborne Phytophthora species are able to sporulate, forming chlamydospores in cortical cells and sporangia on the root surface. Sporangia develop at the apices of hyphae as cytoplasm flows into the enlarging apex from the subtending hypha. In vitro, sporulation can be triggered by a transfer of mycelium from nutrient broth to a

19 nutrient-free mineral salts solution and so it seems likely that reduction in available nutrients might also stimulate sporulation in planta. The sporangia are multinucleate, sealed off with a basal septum, and apparently contain all components needed for zoospore formation and function. Zoosporogenesis is achieved through rapid cytokinesis of the multinucleate sporangium into uninucleate domains.

2.17 Resistance mechanisms in cocoa Plants possess a range of defences that can be actively expressed in response to abiotic and biotic stresses mainly pathogens and parasites. The mechanisms of defence or resistance to diseases is governed by a combination of morphological characteristics and biochemical reactions of the host plant (Nyadanu et al., 2012). The structural characteristics act as physical barriers and inhibit the pathogen from gaining entrance and spreading through the plant while the biochemical reactions take place in the cells and tissues of the plant and produce substances that are either toxic to the pathogen or create conditions that inhibit growth of the pathogen in the plant (Agrios, 2005). The kind of resistance a host plant employs against a pathogen or an abiotic agent is controlled directly or indirectly by the genetic material of the host and of the pathogen (Agrios, 2005). In cocoa, resistance to Phytophthora species infection is described to operate at two distinct stages of infection: penetration stage, which restricts the entry and establishment of the pathogen, thus reducing the frequency of lesions and the post-penetration stage, which reduces the rate of spread of the pathogen and hence the rate of lesion expansion (Iwaro et al., (1997). According to Agrios (2005), the thickness and toughness of the outer wall of epidermal cells are evidently important factors in the resistance of some plants to pathogens. A strong correlation between pod husk thickness, moisture content and resistance to P. palmivora have been reported by Nyadanu et al., (2011). Further, Nyadanu et al., (2012) investigating the histological mechanisms of resistance to black pod disease in cocoa observed the associations between differential genotypic responses to black pod disease being dependent on the variation in pod husk anatomical characters. They concluded that the number, width and length of vascular bundles, epicarp thickness, distance between vascular bundles and epicarp, number of cells in epicarp and mesocarp are associated with resistance to black pod disease. Lower epicarp thickness and higher number of vascular bundles were observed in susceptible genotypes suggesting their porosity to Phytophthora species. Cells in epicarp and mesocarp were more compactly arranged in resistant genotypes than in susceptible ones. The presence of extra thickness of phloem fibre and its gritty nature in resistance genotypes were proposed to act as strong mechanical barrier for penetration and absortion of sap from phloem.

The role of epicuticular waxes as defence weapons against black pod disease have also been studied in cocoa pods and leaf surfaces (Nyadanu et al., 2012). From their studies, Nyadanu et al., (2012) found that cocoa genotypes with higher amount of wax layer were more resistant to black pod than cocoa genotypes with lesser amount of wax. They suggested that epicuticular wax layer provide an extra defence against Phytophthora species. Surface waxes in previous studies have also been reported to protect plants from pathogens (Jenks et al., 1994; Carver et al., 1996). Presence of lignin in the cell walls of resistant cocoa genotypes have demonstrated to be an important mechanisms of resistance to fungal penetration (Vance et al., 1980; Nyadanu et al., 2012).

2.18 Black pod disease symptoms Symptoms of black pod begins on the surface of the pod starting as a discoloured spot, then develops into a small brown or black lesions with a well- marked boundary normally appearing two to three days after infection (Nyadanu, 2011). Lesions grow rapidly covering the whole pod surface and internal tissues, including the beans of susceptible genotypes within few days (Guest 2007). A whitish mycelium with sporangia starts to develop on the surface of the diseased pod after about 5 to 10 days of infection, with the fruit turning black and mummified (Nyadanu, 2011)(Figure 2.7). Infection on older pods normally starts at either the tip or the stem end of the pods. Equatorial infections are usually associated with damage to the pod surface or wounds. The rot involves the whole of the fleshy tissue of the husk as well as the pulp and seeds. The infected pod is later colonized by secondary fungi. It is possible to distinguish between black pod caused by P. megakarya and P. palmivora. Black pod caused by P. megakarya produces lesions with irregular edges on the fruit whereas lesions caused by P. palmivora have regular borders and are generally smaller (Erwin and Ribeiro, 1996). Moreover, multiple infections are common phenomena associated with P. megakarya, resulting from rain splashing sporangia from sporulating pods onto healthy ones (Figure 2.7b). ten Hoppen et al. (2011) also observed more infection foci at the bottom of the plantation and in areas with heavy shade for P. megakarya.

Figure 2.7 Symptoms of Black pod disease on cocoa. (a) Black pod caused by P. palmivora* (b) Multiple lesions on cocoa pods- symptoms of P. megakarya infections (c) P. megakarya infection symptoms at different stages (d) stem canker lesions before scraping (e) Stem canker lesions after scraping showing scarlet colouration* (f) infested cocoa beans. Pictures: courtesy of Akrofi, A.Y (2015). Asare, E.K (unpublished). * Guest, 2007.

2.19 Strategies for disease management The impacts of Phytophthora diseases are of global concern. Losses attributed to black pod on cocoa can reach up to 100% in smallholders’ plantations when no control measures are taken (Despreaux et al., 1998; Berry and Cilas, 1994). The losses and cost of controlling Phytophthora diseases have thus placed a huge financial burden, serious socioeconomic and environmental consequences on governments and agricultural enterprises wherever these pathogens are found (Akrofi, 2015). Current strategies adopted for the effective and sustainable management of black pod disease are discussed below. 2.19.1 Quarantine control Minimizing or preventing the movement of Phytophthora spp. into new cocoa regions or regions of less black pod incidence is crucial for effective control of black pod diseases. Although P. palmivora is ubiquitous, pathogenic diversity within the species exists and the introduction of exotic

22 isolates poses a significant threat to cocoa production (Appiah et al., 2003). P. megakarya is restricted to West and central Africa and effective quarantine measures are required to prevent its spread to other cocoa regions globally. In Ghana for example, the spread of P. megakarya from one region to the other has been linked with the movement of planting materials (Akrofi et al., 2003). Transporting of soils between cocoa-growing areas must be avoided and cocoa germplasm intented for exchange must be subjected to effective testing procedures via quarantine facilities (Opoku et al., 2000). In Ghana, the Plant Protection and Regulatory Services Directorate of the Ministry of Food and Agriculture, inspect and give phytosanitary certificates for both imported and exported plant materials. 2.19.2 Cultural control Cultural practices are regarded as the first and the least expensive disease control option for managing Phytophthora diseases on cocoa farms (Akrofi, 2015). In West Africa where production is done on small scale with low inputs, poor farmers mostly rely on cultural practices. Traditionally, cultural control methods, such as removal of chupons, good farm sanitation and appropriate shade management have been proven effective, as a first line of defense against pests and diseases (Awudzi et al., 2012). Practices such as phytosanitary pod removal, which consists of removing diseased pods, have proven relatively efficient in reducing the secondary inoculum (Ndoumbe`- Nkeng et al., 2004). Akrofi (2015) mentioned that frequent harvesting as a control measure partly saves infected mature pods, encourages removal of infected pods and reduces sources of sporangial inoculum. In addition, it also helps in reducing cushion cankers. Other practices include pruning and appropriate tree spacing, increases aeration and reduce humidity, thus reducing sporulation and infection on farms (Akrofi, 2015). Cultural control methods when used in combination with chemical control have been reported to give successful results in West Africa, (Djiekpor et al., 1982; Tondje et al., 1993). 2.19.3 Chemical control Chemical control using systemic and copper-based contact fungicides is the most widely-used control method (Deberdt et al., 2008). Protectant fungicides that are mainly “fixed” copper compounds e.g. copper hydroxides and copper oxides, or systemic fungicides containing copper and metalaxyl as mixtures are routinely sprayed onto pods with lever-operated knapsack sprayers for Phytophthora pod rot disease control (Akrofi, 2015). The control of black pod caused by P. megakarya requires frequent applications of copper or copper-metalaxyl mixtures, but this practice is too expensive for both local Ghanaian farmers and others in Africa (Opoku et al., 2000, 2007b; Sonwa et al., 2008).

Different recommendations required for black pod control as fungicide applications are based on local factors such as specie of pathogen, climatic conditions, cocoa variety, planting density, and social and economic considerations (Wood and Lass, 1985). In Ghana, nine agro chemicals mainly containing copper and copper-metalxyl mixtures have been recommended for the control of cocoa pests and diseases mainly black pod (Akrofi, A. Y; personal communication) (see appendix 6). Ridomil Gold 66 WP is the most widely used fungicide for the control of black pod in Ghana. It is a systemic fungicide with mefenoxam or metalaxyl-M (6%) and Cupper (I) oxide (60%) as active ingredients. Metalaxyl inhibits ribosomal RNA synthesis and inhibits the pathogen at various stages in its life cycle; mycelia growth and spore formation stages. The mode-of-action of copper ions (cu2+) is the nonspecific denaturation (disruption) of cellular proteins, specifically disrupting enzymes in the Oomycetes spores in such a way as to prevent germination. The recommended application dosage is 50g/15L. Recommended maximum intervals between sprays is 3-4 weeks. The frequent usage of copper based fungicides (3-4 weeks intervals) for the control of black pod however, raises a growing concern about the harmful effects of fungicides especially on the health of farmers and consumers. The practices are also considered as not environmentally friendly and unsustainable (Akrofi, 2015). Addo-Fordjour et al., (2013) for example, reported copper accumulation and contamination of soils and also detected copper residues in cocoa leaves and beans, resulting from copper-based fungicide sprayed on cocoa plantations in Ghana. Other practices such as annual trunk injections of the inexpensive inorganic salt potassium phosphonate have been reported to be very effective against P. palmivora, particularly in reducing cankers, in very wet areas of Papua New Guinea (Guest et al, 1994), and in Ghana against both P. palmivora and P. megakarya (Opoku et al., 2007b). For effective usage of fungicides, Akrofi (2015) mentioned that targeting disease foci and using information on disease dynamics to plan for spraying regimes can limit the amount of fungicides sprayed on farms and, thereby, reduce copper accumulation and contamination in the production chain.

2.19.3.1 Development of fungicide resistance – Experimental theory Fungicides have been an integral part in the management of fungal diseases in crop production for several years. However, the introduction of site-specific fungicides in the late 1960s, has led to resistance within the pathogen population, which has become a major problem in crop protection (Brent, 1995). Fungicide resistance is a genetic adjustment by a fungus, resulting in reduced sensitivity of the fungus to the fungicide. Resistance may arise from single or multiple gene mutations. Resistant isolates typically arise from a very low natural rate of genetic mutation, and

24 these isolates are less sensitive or not inhibited by a labeled application rate of a fungicide (Ma and Michailides, 2005). Since the fungicide can effectively control sensitive isolates, resistant isolates may become dominant in pathogen populations under selection pressure of fungicide use over time. This leads to disease control failures eventually. Fungicides with a single-site mode of action, that is fungicides active against only one point in one metabolic pathway in a fungus, have relatively higher risk for resistance development as compared to those with multi-site mode of action (McGrath, 2004). The shift toward resistance in a pathogen population occurs at different rates depending on the number of genes conferring resistance. When single gene mutations confer resistance, a rapid shift toward resistance may occur, leading to a population that is predominantly resistant and where control is abruptly lost (Figure 2.8a). Where multiple genes are involved, the shift toward resistance progresses slowly, leading to a reduced sensitivity of the entire population (Figure 2.8b). The gradual shift with the multiple gene effect may result in reduced fungicide activity between sprays, but the risk of sudden and complete loss of control is low. But it is difficult to clearly distinguish between sensitive and resistant sub-populations with field sampling during the early shifts. Integrating cultural practices and optimum fungicide use patterns have been recommended as effective strategies for managing fungicide resistance (Damicone, 1996). The aim is to minimize selection pressure through a reduction in time of exposure or the size of the population exposed to the at-risk fungicide. On cocoa farms, fungicide resistance management can be achieved by maintaining appropriate tree spacing, height and canopy structure to enhance uniform fungicide applications. Other strategies include adhering to the spray regimes and using recommended fungicide dosages with the correct applications methods. Further it is also important to consider the prevailing weather conditions before spraying as wind and rainfall can interfere with fungicide application and action respectively.

Figure 2.8 Depiction of fungicide resistance development in a pathogen population. (a) Qualitative resistance (b) quantitative resistance

2.19.4 Biological control The use of antagonistic microorganisms is frequently considered as one of the safest and most affordable control strategies of controlling plant diseases. Some successes have been made which led to the development and registration of several biological control products (Fravel, 2005; Kaewchai et al., 2009). Many species of Trichoderma, such as Trichoderma asperellum, T. harzianum, T. polysporum, T. viride and T. virens have been used successfully as biological control agents against a variety of phytopathogenic fungi (Almeida et al., 2007; Benitez et al., 2004; Harman et al., 2004; Hermosa et al., 2013; Kaewchai et al., 2009). The mechanisms employed by biological control agents (BCAs) are complex, and what has been defined as biocontrol is the final result of different mechanisms acting synergistically to achieve disease control (Howell, 2003). Even within the genus Trichoderma, previous research indicates that the mechanisms are many and varied (Howell, 2003). Biocontrol results either from competition for nutrients and space or as a result of the ability of BCAs to produce and/or resist metabolites that either impede spore germination (fungistasis), kill the cells (antibiosis) or modify the rhizosphere, e.g. by acidifying the soil, so that pathogens cannot grow (Benitez et al., 2004). Biocontrol may also result from a direct interaction between the pathogen itself and the BCA, as in mycoparasitism, which involves physical contact and synthesis of hydrolytic enzymes, toxic compounds and/or antibiotics that act synergistically with the enzymes. Trichoderma BCAs can

26 even exert positive effects on plants with an increase in plant growth (biofertilization) and the stimulation of plant-defense mechanisms (Benitez et al., 2004). Although some Trichoderma spp. such as Trichoderma virens, T. harzianum, Pseudomonas putida biotype A, P. aeruginosa, P. spinosa, Burkholderia gladioli, Burkholderia sp., Bacillus sphaericus, B. polymyxa, and Serratia marcescens were antagonistic to P. palmivora in in-vitro experiments (Hanada et al., 2009; Mpika et al., 2009), none of these microorganisms has been further developed for commercial application in T. cacao fields (Akrofi 2015). According to Guest (2007), the short life cycle, phenomenal reproductive capacity, complex disease cycle, and zoospore motility of Phytophthora generates explosive epiphytotics in cocoa which have so far rendered inundative biological control agents ineffective. The use of Trichoderma asperellum isolate PR 11, for the control of P. megakarya in Cameroon was found promising, but not as effective as chemical control (Tondje et al., 2007a). Deberdt et al., (2008), using Trichoderma asperellum biocontrol agents against P. megakarya concluded that research on the biological control against P. megakarya is promising but still requires further confirmation. They suggested that setting up multiple trials under different agro-ecological conditions will facilitate the process of developing an effective and durable method. In addition to the above, a parallel economic survey needs to be carried out in order to show the benefit generated by the use of such microbial agents compared to the current chemical control.

2.19.5 Use of resistant cocoa varieties Obtaining cocoa resistant to black pod disease has become the goal of many breeding programs as this has long been regarded as the most economical; environmentally friendly and effective control method (Iwaro et al., 2004). But the process of incorporating durable resistance into cultivars with desirable agronomic and quality attributes has been slow (Guest 2007). Cocoa is genetically variable and as a perennial tree, its improvement presents significant challenges to breeders. Moreover, most breeding programs have focused on yield and quality under intense management regimes and correspondingly low rates of disease, thus neglecting the impact of disease on yields under smallholder farm conditions (Guest 2007). The genetic variability of pathogen population and the influence of environmental conditions (Van der Vossen, 1997) have also slowed the breeding process. Efforts have been made to isolate naturally occurring resistant clones against black pod via field surveys in cocoa farms and methodologies to assess such resistance have been developed (Pokou et al., 2008; Efombagn et al., 2011). Resistance to black pod disease has been described to be additive

27 and polygenically inherited and could be improved by recurrent selection (Iwaro et al., 1999; Adomako 2006). Field observations of the cocoa cultivars have shown consistent differences in levels of infection; although no selection has shown complete immunity, but sufficient variability for incorporation into breeding programmes. Adomako (2006) also reported significant differences between cocoa genotypes in the levels of black pod disease in two field trials. Recent advances such as genome mapping, has been used to identify and localise quantitative trait loci (QTL) involved in disease resistance (Lanaud et al., 2004). In addition, multiple QTLs have been identified to be involved in resistance to P. palmivora, P. megakarya and P. capsici (Clement et al. 2003; Risterucci et al., 2000; 2003). These tools offer the possibility of improving durability of resistance in T. cacao to P. megakarya by a possible accumulation of many different resistance genes located in different chromosome regions (Akrofi, 2015).

2.19.6 Integrated disease management The management of black pod disease using the above methods individually has been unsatisfactory as pod losses are still high. Hence a combination of adequate cultural and quarantine methods, chemical, biological and use of resistant varieties in an integrated program has been recommended as the best alternative for a sustainable management of cacao black pod disease (Deberdt et al., 2008). Developing an integrated approach also means addressing issues such as understanding the environmental parameters, epidemiology of the disease and the fate of the biological control agent, as well as the targeted pathogen. In particular, factors such as genetic background of the pathogen and ability of the biocontrol agent to be established and to spread could have an impact on the efficiency of this integrated approach (Deberdt et al., 2008).

3. MATERIALS AND METHODS

3.1. Source of Phytophthora isolates Twenty Phytophthora isolates (10 each of P. palmivora and P. megakarya) were initially screened by culturing on Campbell vegetable juice agar. Phytophthora isolates which grew in cultures after three days were selected for the study. Eight isolates each of P. palmivora (Pp-BAR 143, Pp-ER 370, Pp-ER372, Pp-CR 281, Pp-CR 276, Pp-CR 293, Pp-WR 431 and Pp-WR 432) and P. megakarya (Pm-BAR 152, Pm-BAR 158, Pm-ER 371, Pm-WR 425, Pm-VR 116, Pm-AR 320, Pm- AR 334 and Pm-AR 351) were used in the study. All isolates were obtained from the laboratory of Plant Pathology Division of Cocoa Research Institute of Ghana (CRIG), New Tafo-Akim, Eastern region, Ghana. These isolates are among other Phytophthora isolates collected from Brong Ahafo (BAR), Ashanti (AR), Eastern (ER), Volta (VR), Western (WR) and Central (CR) the cocoa growing regions of Ghana (Figure 3.1).

Figure 3.1 Map of Ghana (excluding the North) showing districts where Phytophthora isolates were collected. Regional boundaries shown by wavy lines. District abbreviations: A – Asutifi, TS - Tano South, TN - Tano North, THLD- Twifo Hemang Lower Denkyira, AYE – Ajumako Enya Essiam, SS - Sekyere South, EJ – Ejisu Juabeng, AAN- Ashanti Akim North, H - Hohoe, EA - East Akim, BC – Birim Central, WW - Wassa West and WAE – Wassa Amenfi East. Pm-431place of collection is Camp, district (?) not certain known.

3.2. Viability and pathogenicity of Phytophthora isolates Mycelia plugs of Phytophthora isolates maintained in sterile distilled water at 25±2oC for about two years were blot-dried between two sterile filter papers and plated on Campbell Vegetable juice agar o (200 ml V8 juice, 800 ml sterile distilled water, 15 g agar, 2.5 g CaCO3 and autoclaved at 121 C for 15 minutes). The plates were incubated in the dark at 28±2oC for two days. Hyphal tips of emerging Phytophthora colonies were aseptically transferred onto fresh V8 plates and incubated at 28±2oC to obtain pure cultures. Mycelia plugs (10 mm in diameter) were taken from the periphery of the 4- days old actively growing Phytophthora isolates using sterile cork borer and inserted into 1 cm depth wounds created with sterile cork borer at proximal and distal ends of approximately equal sizes and age (about 2 months old) of carefully washed hybrid cocoa pods (Figure 3.2). Inoculated pods were watered with sterile distilled water and arranged on wet foams (moistened with 500 ml sterile distilled water) in plastic trays. Beakers of water were put in the trays and sealed to provide humidity. The trays were incubated at 25±2oC and observed daily for black pod lesion development. Internal tissues were excised from the infected pods onto sterile V8 plates and incubated in the dark at 28±2oC.

Figure 3.2 Pod inoculation with mycelial plugs cut from 4 day actively growing Phytophthora cultures

3.3. Virulence test using leaf disc inoculation method

3.3.1. Leaf sampling New flushing leaves of mixed cocoa hybrid (Amazon x Amazon) were tagged (Figure 3.3a) at CRIG’s experimental plot ‘R 2’ to obtain eight weeks old leaves for this experiment. Young lignified leaves, upper part of pedicel brown and lower part green (arrowed in Figure 3.3b) without insect attack, were later randomly harvested before noon into transparent polyethylene bags. The

leaves were washed thoroughly but carefully under running water, rinsed in three changes of sterile distilled water and allowed to air-dry at room temperature.

Figure 3.3 Leaf sampling for test of virulence. (a) Leaf flushes tagged to determine leaf age. (b) Young lignified leaf about two months old (upper part of pedicel brown and lower part green) in good physiological condition a b 3.3.2. Leaf disc inoculation assay Leaf discs (1.5 cm in diameter) were cut out from the neatly washed leaves with a leaf disc cutter and arranged on wet foam (moistened with 500 ml sterile distilled water) with their abaxial surface upwards in plastic incubation trays. Each tray contained 16×15 leaf discs for each Phytophthora isolate and replicated seven times. The trays were partially covered to allow the leaf discs to air-dry over night at room temperature. Leaf disc inoculation was carried out according to the methodology developed by Nyassé et al. (1995). The center of each row of 15 leaf discs were inoculated with 10 μl zoospore suspension of each Phytophthora isolate and labeled (Figure 3.4). The experiment was repeated twice. The zoospore suspensions were prepared by flooding 10 days-old culture plates of the Phytophthora isolates with 20 ml of chilled sterile distilled water. The flooded culture plates were placed in a refrigerator at 10 ˚C for 45 minutes and then transferred into an incubator of 25±2 C for 45 minutes. Concentrations of harvested zoospores were determined with a haemocytometer by filling the chambers with 200 μl of immobile (addition of three drops of 70% ethanol to 1 ml zoospore suspensions) and counted under the light microscope (Leica) at x 10 magnification. The concentration of Zoospores in the mixture was calculated as: (Number of zoospores / 5) x 25 x 104

Inoculated leaf discs were covered and sealed to provide humidity and incubated at 25±2oC. After seven days of incubation, disease severity symptoms were scored on a 0-5 scale of the infection level developed by Nyassé et al., (1995) as follows; 0 = no symptoms 1 = isolated small brown or dark brown spots 2 = small brown spots with few connections between them 3 = brown spots forming coalescing lesions of intermediate size 4 = large coalescing lesions with light or dark brown points 5 = large brown uniform lesions covering the whole leaf disc

Figure 3.4 An incubation tray containing leaf discs inoculated with zoospore suspension of Phytophthora isolates

3.4. Determination of virulence of Phytophthora isolates on detached cocoa pods. Healthy green hybrid (Amelonado x Amazon) cocoa pods, approximately three months old were randomly sampled from CRIG’s experimental plot ‘V 5’ for this experiment. A set of sixteen (16) carefully washed and rinsed cocoa pods in three changes of sterile distilled water were arranged on wet foam (moistened with 1000 ml sterile distilled water) in a metal tray. The pods were allowed to air-dry at room temperature and laterally wounded with a cork borer to a depth of 0.5 cm. The wounds were inoculated with 10 mm diameter mycelia disc plugs taken from the periphery of 8 days old cultures of the sixteen (16) Phytophthora isolates (Figure 3.5a). In another setup, a set of sixteen unwounded (16) cocoa pods were laterally inoculated with 10 mm diameter mycelia disc plugs taken from the periphery of the same Phytophthora cultures used in the first setup (Figure 3.5b). The pods were tagged and sprayed with sterile distilled water. Beakers

32 containing 100 ml of water were placed in the trays, covered and sealed to provide humidity and incubated at room temperature. The experiment was repeated twice. Lesion developments and sporulation on the pods were measured and assessed after 3 days of incubation.

Figure 3.5 Pod inoculation using mycelia plugs of Phytophthora isolates: (a) wounded pods (b) unwounded pods

3.5. Mycelial growth of Phytophthora isolates on agar at different temperatures The centre of three replicated 9 cm V8 agar plates were inoculated with 5 mm diameter mycelia disc plugs taken at 1.5 cm from the centre of 4-days old actively growing cultures of P. palmivora and P. megakarya isolates sealed with parafilm and incubated in the dark at 25±2oC for 4 days. Replicated plates were setup and incubated in the dark at 28±2oC, 31±2oC and 34±2oC. Radial growth of Phytophthora colonies on the media were measured after every 24 hours along two perpendicular lines drawn at the bottom of the plates. The experiment was repeated twice. Sporulation of the isolates at the various incubation temperatures were observed microscopically after ten days of incubation. Mycelia disc plugs, 5 mm in diameter, were taken at 9.6 mm and 8.2 mm from the centre of P. palmivora and P. megakarya cultures respectively using sterile cork borer. Mycelia mat from the plugs were mounted in drops of 2% lactophenol blue on slides, covered with slips and mounted under light microscope at x4 to estimate the amount of spores.

3.6. Evaluation of ridomil gold against Phytophthora isolates An equivalent dosage of 50g/15l of Ridomil Gold (6% metalaxyl M + 60% Cupper (I) oxide) (3.33g) was dissolved in 1000 ml molten Campbell Vegetable juice agar (V8) at 50oC, swirled to mix and poured into Petri dishes. The plates were allowed to set and left to stand overnight to test its sterility. Mycelia plugs (5 mm in diameter) from 4 days old V8 agar cultures of the Phytophthora isolates were each placed upside down at the centre of 90 mm plates. Three replicated plates per each isolate were setup and incubated at 25 ºC, 28 ºC, 31 ºC and 34 ºC. After 24 hours of

33 incubation, diameter growth of each isolate was observed and measured along two perpendicular lines drawn at the bottom of the plates every 24 hours for five days.

3.7. Data analysis Data were subjected to analysis of variance (ANOVA) at a significance level of 5% using Genstat® 11 Edition statistical software (VSN International Limited). The treatment means were separated whenever the F test values were significant using the Duncan’s Multiple Range Test (DMRT).

4. RESULTS

4.1 Pathogenicity and aggressiveness of Phytophthora isolates Symptoms of black pod lesions were detected on detached cocoa pods inoculated with mycelia plugs of Phytophthora isolates (Figure 4.1). All Phytophthora isolates used in this study were viable, causing lesions on detached pods when inoculated. Rate of symptoms development on inoculated pods were observed to differ. This may be due to differences in pathogenicity between Phytophthora species and among isolates of the same species. Phytophthora isolates with least latent period were observed to be more aggressive as they produced symptoms of black pod early. P. palmivora isolates recorded the shortest latent periods of infection (2 days) after inoculation. All cocoa pods inoculated with P. megakarya showed black pod disease symptoms after 5 days, except Eastern region isolate Pm-ER 371 which gave disease symptoms at day 3. Pm-ER 371 may be more aggressive than the other P. megakarya isolates. The latent periods of infections for all the isolates is shown in Table 4.1.

Figure 4.1 Cocoa pods with black pod symptoms after inoculation with Phytophthora isolates

Table 4.1 Latent periods of infection of Phytophthora isolates

(Pm) - Phytophthora megakarya; (Pp) - Phytophthora palmivora; > more than

4.2. Virulence of Phytophthora isolates on leaf disc Phytophthora isolates caused varying degrees of infection when inoculated on leaf discs (Figure 4.3). The difference in pathogenicity may be due to the varying amounts of zoospores (the infective propagules) (Table 4.2) produced by the Phytophthora isolates. Overall, P. palmivora isolate Pp-CR 276 and Pp-CR 293 from the Central region produced the highest and least number of zoospores respectively. In P. megakarya, isolates Pm-ER 371 and Pm-AR320 recorded the highest (1.00) and least (0.47) number of zoospores respectively. Isolates with the same latent period but different amounts of zoospores gave different varying degrees of lesions on leaf discs. Interestingly, isolates Pm-BAR158 and Pm-AR 351 have the same number of zoospores and latent period, but produced different levels of disease symptoms, 0.77 and 0.76 respectively. The number of zoospores was observed to correlate with lesion size in most cases, but there are some exceptions. In P. palmivora inoculated leaves, lesion size for Pp-BAR 143 was higher, although it has fewer number of zoospores when compared with Pp-WR 431 and Pp-WR 432. This trend was also detected in pods inoculated with P. megakarya isolates (Pm-BAR 158 and Pm-AR- 320). Isolate Pp-WR 431 gave

36 higher disease symptoms (2.76) but has fewer zoospores when compared with Pm-ER 371 (1.00) (Table 2.2). Pp-WR 431 has a shorter latent period (2 days) but in Pm-ER 371, this is longer (3 days). The shorter the latent period, the faster lesions develop. The number of zoospores and latent period of infection are very important components of aggressiveness in Phytophthora species.

Figure 4.2 Leaf discs showing black pod symptoms after incubation for 7 days

Figure 4.3 Mean symptom rating of Phytophthora isolates on leaf discs

Table 4.2 Amount of Zoospores and mean severity of Phytophthora isolates Isolate name Amount of zoospores Mean symptom rating in 1 ml suspension

culture (x 104) Pm-BAR 152 338 0.64 c

Pm-BAR 158 225 0.77 c Pm-ER 371 1788 1.00 c Pm-WR 425 213 0.48 c Pm-VR 116 188 0.55 c Pm-AR 334 113 0.47 c

Pm-AR 320 50 0.59 c Pm-AR 351 225 0.76 c Pp-BAR 143 1513 3.07 ab

Pp-ER 370 888 2.44 b

Pp-ER 372 438 1.84 bc Pp-CR 281 150 1.74 bc Pp-CR 276 9813 3.95 a Pp-CR 293 100 0.95 c Pp-WR 431 1550 2.76 ab

Pp-WR 432 2875 2.96 ab Data followed by the same letter in each column do not differ significantly (p <0.05) according to DMRT.

4.3. Virulence of Phytophthora isolates on detached pods Symptoms of black pod on detached wounded and unwounded cocoa pods is shown in Figure 4.4. Least infections without sporangia were observed in unwounded pods (Figure 4.4a) while infection in wounded pods (Figure 4.4b) was very high with sporangia present. Defense mechanisms against black pod disease have been described to occur at both penetration (on pod surface) and post penetration stages. Pod husk characteristics such as cuticle thickness and waxes are key defense weapons against plant pathogens. Wounded cocoa pods have lost this first line of defense, making Phytophthora entry and establishment easier, resulting in increased disease levels. Wounded pods inoculated with P. palmivora isolates had bigger lesions with a lot of sporangia than those inoculated with P. megakarya. Once the pod is injured Pm-AR334 is capable of causing the highest infections among the P. megakarya. In the unwounded pods, only two P. megakarya isolates, Pm- BAR 158 and Pm-ER 371 gave disease symptoms with the latter recording the highest disease levels for all the isolates. This means that black pod disease caused by P. megakarya may be more prevalent in the Brong Ahafo and the Eastern regions. P. megakarya isolates from the other regions are not aggressive enough to infect healthy pods. But this assertion may be on the contrary, as factors that promote disease development may differ among the various cocoa production regions.

Figure 4.4 Cocoa pods showing black pod symptoms after inoculation: (a) Unwounded pods with less disease symptoms (b) wounded pods showing high disease symptoms with sporangia.

Table 4.3 Level of virulence of Phytophthora isolates on detached pods inoculated for 72 hours Unwounded Wounded Isolate name Diameter sporulation Diameter growth sporulation growth (cm) (cm)

Pm-BAR 152 0.0 n.a (2) x 3.10 +/-1.0 x Pm-BAR 158 2.25 +/-0.8 (2) x 4.55+/-0.9 + Pm-ER 371 3.45 +/-0.5 (2) x 4.75 +/-0.4 + Pm-WR 425 0.0 n.a (2) x 5.40+/-0.5 + Pm-VR 116 0.0 n.a (2) x 4.85+/-0.2 + Pm-AR 334 0.0 n.a (1) x 6.60 n.a + Pm-AR 320 0.0 n.a (2) x 3.30+/-0.9 x Pm-AR 351 0.0 n.a (2) x 3.40+/-0.2 + Pp-BAR 143 3.20+/-0.6 (2) x 9.15+/-0.9 + Pp-ER 370 2.25+/-0.8 (2) x 7.05+/-2.2 + Pp-ER 372 3.10 n.a (1) x 6.70 n.a + Pp-CR 281 2.15+/-0.4 (2) x 8.30+/-0.4 + Pp-CR 276 3.00+/-0.02 (2) x 9.40+/-0.9 + Pp-CR 293 1.75+/-0.06 (2) x 4.65+/-1.6 + Pp-WR 431 2.45 n.a (1) x 6.35 n.a + Pp-WR 432 3.30+/-1.1(2) x 8.05+/-1.3 +

(-) No mycelial growth, (x) No sporulation (+) Presence of sporangia, n.a- not applicable, ( )- number of replicates

4.4. Effect of temperature on the growth of Phytophthora isolates All isolates of P. megakarya and P. palmivora showed varied degrees of mycelial growth at 25 ˚C, 28 ˚C, 31 ˚C and 34 ˚C after 96 hours of incubation (Figure 4.5). Statistical data is also shown in Table 4.4. Highest mycelial growth was recorded in P. palmivora isolates at all temperatures. For P. palmivora, optimum mycelial growth occurred at 25 ˚C and 28 ˚C with isolates Pp-BAR 143 and Pp-CR 281 showing greatest mycelial growth of 44 mm and 45 mm respectively at 25 ˚C and 28 ˚C. For P. megakarya, Pm-AR 334 recorded the highest growth of 35 mm at 25 ˚C, while Pm-BAR 152 had the least growth of 24 mm. At 28 ˚C however, mycelia growth of isolate Pm-AR 334 saw appreciable decline recording 27 mm with the least being 13 mm for isolate Pm-BAR 152.

At higher temperatures, 31 ˚C and 34 ˚C, no mycelial growth was observed in P. megakarya isolates. Appreciable mycelial growth was detected at 31 ˚C for P. palmivora isolates, with the exception of isolate Pp-CR 293 which recorded a sharp decline in growth (19 mm). Mycelial growth of P. palmivora at 34 ˚C however, saw a significant decline for all P. palmivora isolates, with the maximum growth being 15 mm in Pp-ER 370. This growth is less than the minimum growth observed for isolate Pp-CR 293 (19 mm) at 31 ˚C. From the present results, P. megakarya is very sensitive to higher temperatures than P. palmivora.

Figure 4.5 Mean mycelia growth of Phytophthora isolates at different temperatures.

Table 4.4 Effects of temperature on the growth of Phytophthora isolates Isolate Name Temperatures

25 ˚C 28 ˚C 31 ˚C 34 ˚C Pm - BAR 152 24 h 13 k 0 f 0 d Pm -BAR 158 34 ef 25 fg 0 f 0 d Pm - ER 371 33 efg 22 gh 0 f 0 d

Pm -WR 425 33 efg 17 ij 0 f 0 d Pm - VR 116 32 efg 21 hi 0 f 0 d Pm - AR 334 35 de 27 f 0 f 0 d

Pm - AR 320 31 fg 15 jk 0 f 0 d Pm - AR 351 33 ef 20 hi 0 f 0 d Pp - BAR 143 44 a 40 bc 35 bcd 10 bc Pp - ER 370 42 ab 43 ab 38 ab 15 a

Pp - ER 372 35 de 37 cd 33 d 4 d Pp - CR 281 41 bc 45 a 40 a 8 c Pp - CR 276 38 cd 42 ab 37 bc 12 abc

Pp - CR 293 30 g 31 e 19 e 0 d Pp - WR 431 37 d 36 d 34 bcd 13 ab Pp - WR 432 42 ab 42 ab 34 cd 9 bc Means in the same column for each isolate followed by the same letter are not significant different.

4.5. Growth patterns of Phytophthora isolates under different temperatures Although growth of Phytophthora isolates was observed to increase with time in vitro, their growth patterns differ under the different temperature regimes. P. megakarya isolates Pm-AR 320, Pm-AR 334 and Pm-AR 351 had two days exponential growth with the stationary growth beginning at the third day. This occurred at both 25 ˚C and 28 ˚C (Figure 4.6). Growth of isolates Pm-WR 425 and Pm-VR 116 at 28 ˚C, also became constant after two days of exponential growth. P. megakarya isolates Pm-BAR 152, Pm-BAR 158 and Pm-ER 371 had steady mycelial growth without decline at day four. The steady growth rate, short latent period and high number of zoospores in isolate Pm- ER 371, could account for the higher virulence of the isolate during leaf disc inoculations. This features also made the isolate more aggressive causing the greatest lesions in unwounded cocoa pods. Brong Ahafo isolates Pm-BAR 152 and Pm-BAR 158 exhibit similar growth patterns to that of Pm-ER 371, but with lesser number of zoospores produced relatively higher disease levels on leaves. P. palmivora isolates however, had relatively similar growth patterns at all temperatures except isolate Pp-CR 293, which did not grow at 34 ˚C (Figure 4.7).

Figure 4.6 Growth patterns of Phytophthora megakarya isolates on V8 juice agar at 25 ˚C and 28 ˚C

Figure 4.7 Growth of Phytophthora palmivora isolates on V8 juice agar at 25, 28, 31 and 34 ˚C.

4.6. Formation of chlamydospore in relation to temperature Microscopic analysis revealed varying number of chlamydospores produced by only P. palmivora isolates in a thick mycelial matrix at the various 25 ˚C (Figure 4.8). The number of chlamydospores found in a matrix, were graded as ++++, +++, ++, + and – representing extremely abundant spores, too many spores, many spores, few spores and no spores respectively (Table 4.5). The amount of chlamydospores was observed to decrease with increase in temperature for most of the isolates. Isolates Pp-CR 276 and Pp-WR 432 gave the highest number of chlamydospores while the least was observed in isolates Pp-CR 293. No chlamydospore was detected in the mycelia mats of P. megakarya isolates at the various temperatures. Chlamydospore count was not done at 34 ˚C because of limited growth of P. palmivora isolates on the 10 day cultures.

Figure 4.8 Chlamydospores of Phytophthora palmivora viewed under light microscope. (a) low power (x 4) (b) high power (x10)

Table 4.5 Chlamydospore rating in Phytophthora isolates

(-) No chlamydospore; (+) few spores; (++) many spores ;(+++) too many spores; (++++) extremely abundant spores.

4.7. Determination of resistance of Phytophthora isolates to ridomil gold with increase in temperature Phytophthora isolates were very sensitive to the media Ridomil-V8 juice agar media and thus, did not grow, 96 hours after incubation at the different temperatures (25 ˚C, 28 ˚C, 31 ˚C and 34 ˚C). Ridomil Gold 66 WP is known to have a direct toxic action on Phytophthora species mainly through persistence of the metalaxyl, which, after spraying, penetrates inside the pods and so prevents the infection. The recommended dosage (3.3g/L) used for the media preparation is very high and hence very effective against the Phytophthora isolates. This result is unlikely to be achieved on the field. Mycelial plugs used for the inoculation were observed to change colour from yellow to green (Figure 4.9) with a sheath of water around them. When the mycelial plugs were transferred from the Ridomil-V8 juice agar onto freshly prepared V8 juice agar media and left for 10 days, growth did not occur. The colour of the plugs were rather observed to change to the initial colour (yellow), 24 hours after inoculation.

Figure 4.9 Screening for fungicide resistance. (a) Mycelial plug on a V8 medium (b) plugs on ridomil-V8 juice agar medium after 24 hours (note the colour difference). a

5. DISCUSSION

5.1. Pathogenicity and aggressiveness of Phytophthora isolates Pathogenicity is a product of pathogens virulence and its host susceptibility (Scheffer, 1997). In this study, establishing pathogenicity tests was aimed at selecting the most viable and virulent Phytophthora isolates for the experiment. Variability in pathogenicity was found in Phytophthora isolates. This confirms earlier reports of strains of P. palmivora differing in pathogenicity to various host plants (Ashby, 1929; Orellavar and Some, 1957; Turner, 1960). The differences in pathogenicity in P. palmivora and P. megakarya could be due to differences in aggressiveness. Aggressiveness is the quantitative variation of pathogenicity on susceptible hosts, without any restriction related to specificity. Components often measured to determine aggressiveness include the elementary quantitative traits of the pathogen life cycle, such as infection efficiency, latent period, sporulation rate, infectious period or lesion size (Pariaud, et al., 2009). Aggressive Phytophthora isolates were observed to have short latent period. The latent period is an important parameter, since it is related to the generation time of the pathogen, and thus greatly influences the rate of epidemic development and pathogen fitness (Lannou and Soubeyrand, 2012). Results from the current work revealed differences in latent periods of infection among strains of P. palmivora and P. megakarya, resulting in different levels of black pod disease symptoms on detached cocoa pods. Isolates with short latent periods were found to be more virulent and aggressive as they require short time to establish on pods to cause infection. P. palmivora isolates Pp-BAR 143, Pp-ER 370, Pp-ER 372, Pp-WR 431, and Pp-WR 432 had the least latent periods of infection (2 days). This makes the above isolates more virulent when compared to the others. Subsequently, the above mentioned P. palmivora isolates produced the highest infections when inoculated on leaf discs. Similarly, P. megakarya isolate Pm-ER 371, which recorded the least latent periods of infection of three days, had the highest mean symptom rating (1.00) when inoculated on leaf discs. Shaw (1990) mentioned that variability in latent period among pathogen genotypes could reveal heterogeneity in both the time at which the first sporulation occurs and the dynamics of lesion development. The findings of the present work confirms that of Shaw’s, as Phytophthora isolates of the same species with different latent periods gave different levels of disease symptoms on cocoa leaves and pods. Isolates that establish and infect early (short latent period) develop bigger lesions and sporulate faster. Early sporulation leads to higher disease spread that can lead to epidemics if not controlled. Thus, results from the present study imply that, latent periods of infection is an important determinant of aggressiveness in Phytophthora isolates.

5.2. Virulence screening of Phytophthora isolates The environment may affect the rate of growth or multiplication and degree of virulence of a pathogen, as well as its dispersal by wind, water, vector, etc. (Agrios, 2005). Under favourable conditions, in the presence of moisture, sporangia of Phytophthora species release zoospores, the infective propagules (Guest, 2007). In the field, sporangia and zoospores are the main infective propagules of both P. palmivora and P. megakarya (Akrofi, 2003). Microscopic analysis from the present study revealed varied amount of zoospores per ml suspension culture produced by Phytophthora isolates, with some isolates (Pp-CR 276) having extremely high number of zoospores. Zoospores produced in the natural environment as primary inoculum may vary in number, and as such inoculations were done with equal volumes and not equal concentrations of zoospore suspensions. The number of zoospores per ml zoospore suspensions prior to inoculation was observed to correlates with the level of infection in some isolates. Virulence screening of the Phytophthora isolates using leaf disc test revealed different levels of infection. There were significant differences (P <.001) in mean severity among some P. palmivora isolates, Pp-BAR 143, Pp-ER 370, Pp-CR 276, Pp-WR 431, Pp-WR 432 and that of P. megakarya isolates. Isolates Pp-BAR 143, Pp-ER 370, Pp-CR 276, Pp-WR 431 and Pp-WR 432 gave higher levels of infections which appeared on leaf discs as lesions. Isolate Pp-CR 276 produced the highest amount of zoospore and at the same time recorded highest disease severity. This was also observed in isolates Pp-WR 431 and Pp-WR 432. P. megakarya isolate Pm- ER 371, produced a contrasting result in terms of the amount of zoospore and infection levels when compared to P. palmivora isolates except Pp-CR 293. On the contrary, some Phytophthora isolates (Pp-BAR 143, Pp-BAR 158 and AR 320) with fewer zoospores gave higher disease symptoms, a clear indication of other contributing factors to the pathogens aggressiveness. Results from the present study, suggest that the amount of zoospores produced does not always correlate with the disease severity caused by a Phytophthora species. According to Lannou and Soubeyrand (2012), all compatible pathotypes do not cause the same amount of disease on a given variety; some are more aggressive than others and express a higher rate of reproduction. Moreover, Kontradowitz and Verreet, (2010) stressed that the difference in disease symptoms between pathogen populations is due to differences in aggressiveness. The variability in aggressiveness measurements may result from the physiological state of the pathogen (Pariaud et al., 2009). Storage or multiplication conditions may alter pathogen aggressiveness. This was clearly shown for P. infestans By Day & Shattock (1997) in which isolates collected in different years and stored in liquid nitrogen were compared with ‘standard’ reference isolates.

Jeffrey et al., (1962) and Jinks & Grindle (1963) also observed a decrease in aggressiveness of P. infestans strains after repeated transfers on chickpea medium, and reported that different strains underwent these changes to varying extents and at differing rates. Phytophthora isolates used in the present study were collected from regions with different agro climatic conditions that may influence isolates aggressiveness. But it can also be argued that stored isolates may acclimatize to the new storage conditions and for that matter it will be difficult to have data that reflect regional aggressiveness of Phytophthora isolates. That notwithstanding, Phytophthora isolates may also require different preferred conditions such as relative humidity to efficiently cause infections. Moreover in vitro conditions are likely to favour some isolates due to alterations in Phytophthora isolates’ physiological status, resulting in variability in aggressiveness. Yet some of the Phytophthora isolates revealed differences peculiar to their regions. Obtaining regional pathogen aggressiveness data is very important. It can be used as a basis to develop regional disease profiles which is important in disease modelling and predictions. Inoculation tests performed on both wounded and unwounded detached cocoa pods to ascertain the infection levels caused by Phytophthora isolates on leaf discs yielded similar results. Inoculated cocoa pods (wounded and unwounded) gave varied disease symptoms. This was measured in terms of lesion size. The size of lesions is considered a quantitative trait that is measured as an aggressiveness component (Mundt et al., 2002b). Lesion size is defined as the surface area that produces spores (Pariaud, et al., 2009). Pariaud, et al., (2009) further noted that, for some pathogens, lesion size is reported to accounts for a large part of the quantitative development of epidemics and lesion growth rate is a key factor in pathogen competition for available host tissue. Lesion size in wounded pods were bigger than that of unwounded pods. Agrios (2005) mentioned that the thickness and toughness of the outer wall of epidermal cells are evidently important factors in the resistance of some plants to certain pathogens. In this study, the epidermal cells providing the first line of defense and inhibition to disease establishment were removed, hence infection levels were higher in the wounded pods than unwounded pods. In cocoa, resistance to Phytophthora is reported to operate at two distinct stages of infection: penetration stage (occurring on pod surfaces), which restricts the entry and establishment of the pathogen, thus reducing the frequency of lesions and the post-penetration stage, which reduces the rate of spread of the pathogen and hence the rate of lesion expansion (Iwaro et al., 1997). The first barrier encountered by plant pathogens during infections are mainly cuticle and cell wall (Agrios, 2005), which in cocoa are found in the pod husk. Thus from the current study it is clear that the thickness and the composition of pod husk play important role in disease infection and severity.

Nyadanu et al., (2011), investigating thickness of cocoa pod husk and its moisture content as resistance factors to black pod also observed that genotypes of cocoa with thicker pod husks tended to be more resistant than genotypes with thinner pod husk. Pod surface waxes have also been reported to play imported defense roles against black pod disease development. Genotypes with higher amount of wax layer were found to be more resistant to black pod than cocoa genotypes with lesser amount of wax (Nyadanu et al., 2012). Hardham (2001) suggested that if host plants will be able to secrete compounds that inhibit spore adhesion (occurring roots, leaves, pods etc), then it is likely that the rate of successful infection would be reduced. Moreover, successful Phytophthora infection cycle begin with the firm adhesion of motile zoospores to the host tissue (leaves, pods, roots) surface. Hardham (2001) suggested that if host plants are able to secrete compounds that inhibit spore adhesion, then it is likely that the rate of successful infection would be reduced. Hence breeding for resistant cultivars targeting cocoa pod husks may significantly minimize black pod. Characteristic pod husks that can delay or resist Phytophthora penetration will significantly minimize disease initiation in the unwounded pods. Moreover, cocoa hybrids with thick pod husks, will have fewer tendencies to be injured by animals, pests, man etc. Injuries results in a breakdown of resistance at the penetration stage (pod husk surfaces), rendering the pod highly susceptible to black pod. A delay in lesion development subsequently delays sporulation, reducing disease epidemics.

5.3. Mycelia growth of Phytophthora isolates in relation to temperature Results from the current study revealed significant differences (p<.001) in growth response between P. palmivora and P. megakarya isolates at the various range of temperatures where growth occurred. Optimum mycelial growth (35 mm) for P. megakarya occurred 25 ºC while that of P. palmivora, 44 mm and 45 mm occurred at 25 ºC and 28 ºC respectively. This varied response of growth to temperature has been reported to occur among Phytophthora species and isolates of the same species (Milus et al., 2006). Other studies on temperature relation to isolates of Phytophthora of cocoa, areca nut, coconut and black pepper showed temperature optima of 25-30 ºC for P. palmivora, P. meadii, and P. nicotianae var. nicotianae (Sastry and Hegde, 1987). Ribeiro (1978) also observed an optimum range of 28-30 ºC for these species. Hence the current reported range of temperature optima for the two Phytophthora species agrees with earlier studies. Results of the present study also demonstrate that 31 ˚C and 34 ˚C (higher temperatures) negatively affect the growth of P. palmivora and P. megakarya. P. megakarya is more sensitive to higher temperatures than P. palmivora.

The effects of climatic parameters mainly temperature and relative humidity on the expression of disease have been extensively described in the literature (Pariaud et al., 2009). Temperature is considered an important parameter for mycelial growth, sporulation and spore germination in Phytophthora (Gisi, 1983). Tucker (1931) emphasized the value of sporangial and gametangial characteristics as taxonomic criteria together with temperature relations. Knowledge of temperature requirements of Phytophthora species on cocoa is essential in the management and control of black pod diseases. According to Guest (2007), unlike P. palmivora, light inhibits the growth of P. megakarya. Temperature is related to light use efficiency and hence infection due P. megakarya is likely to reduce especially on farms where shade and canopy management practices are effective. It has also been reported that rapid black pod disease development occurs when relative humidity during the day remained higher above 80% under the tree canopy, with well distributed rainfall and low temperatures (Akrofi 2015). In the Western region of Ghana, the wettest region in the country where about 50 % of the country’s cocoa is produced, the crop is cultivated with very little or no shade. This cultivation practice does not favour black pod disease development and may in part explain the higher level of production from that region. In the other production regions, shade management is however very poor as farmers find it difficult to prune cocoa tree branches that can bear fruits. Phytophthora isolates growth graphs also revealed distinctively, isolates daily growth patterns at the different temperatures. Similar to plant fungi growth pattern, some isolates of P. megakarya experience stationary growth early (at day 3) at both 25 ºC and 28 ºC, unlike in P. palmivora where all the isolates are still at the exponential phase even at day 4. Isolates that experience stationary phase may have depleted organic nutrient in their culture media and subsequently experience a phase of decline. But this is likely not to be so as growth of P. megakarya isolates on culture media was small and the media was fresh. Alterations in the physiological status of the isolate may also affect the rate of growth of the isolate in the culture medium and might explain the current trend. P. megakarya isolates grew faster at their optimal temperature (25 ºC) than at 28 ºC. P. palmivora tolerates a wider range of temperature as isolates growth was similar at 25 ºC, 28 ºC and 31 ºC. An exception to this observation is Pp-CR 293. The higher tolerance of P. palmivora makes it ubiquitous. The difference in growth rate at different temperatures may, in part, also explain why disease severity differs among the isolates. In regions such as the Western and Central regions where temperatures (25-27 ºC) tend to highly favour both P. megakarya and P. palmivora, black pod disease incidence is likely to be higher. Although results obtained in this study contradicts earlier reports that P. megakarya emit zoospores earlier and also two times more than P. palmivora (Brasier et al., 1981; Opoku et al., 2000), the

51 early stationary phase attained by some P. megakarya isolates in vitro may confirm the above observation. P. megakarya isolates after reaching stationary phase, progress to the phase of decline, the phase where spores are produced in order to start a new cycle. The shorter the life cycle, the higher potential of severe disease epidemics.

5.4. Effects of temperature on chlamydospore formation Studies on pathogen aggressiveness and climate are limited to the effect of temperature (Pariaud et al., 2009), thus making temperature an important parameter in disease epidemiology. The effect of temperature on aggressiveness components has been established for many pathogen species and presents an optimum for spore germination, lesion development and sporulation. Spores are formed as a fungus depletes its energy sources and as such the environment becomes an important determinant on whether sexual or asexual spores are formed. Chlamydospores, the thick-walled, resistant structures produced by many Phytophthora species can be a key component in the ecology and epidemiology of Phytophthora species, by ensuring pathogen survival during adverse conditions. Abundant production of chlamydospores by P. palmivora isolates indicates the persistence of the pathogen at different temperature regimes. This means that P. palmivora will survive longer and continue to cause infections in all cocoa regions of Ghana. Moreover, the abundant production of chlamydospores by P. palmivora may also contribute to it being ubiquitous, since it can remain in soils for longer periods and become infective when conditions are favourable. The high number of zoospores and chlamydospores, as well as the high virulence detected from this work suggest that P. palmivora isolates are becoming more troublesome in terms of disease severity than P. megakarya in the different cocoa regions of Ghana. This finding contradicts earlier report suggesting that P. megakarya is more destructive. Akrofi (2003), indicated that P. megakarya produces sporangia faster and in much greater quantities (4-6 times more) than P. palmivora and also discharges its zoospores from the sporangia earlier (Opoku et al., 2000). The fact that findings from this present study contradict previous reports is a clear indication of a new dynamics of black pod disease infections caused by P. palmivora and P. megakarya. This must be carefully monitored and investigated as it may present a new dimension in disease control measures. In addition, isolates from the Central region for example, Pp-CR 276 and Pp-CR 293 collected from the same district, Ajumako-Enya- Essiam, were observed to show some unusual traits. While Pp- CR 276 was the most virulent among all the Phytophthora isolates, with the highest number of zoospores and chlamydospores, in the mycelial mat, Pp-CR 293 was producing the least zoospore/ml and causing the least infections among P. palmivora isolates. Pp-CR 281 was behaving

52 as an intermediary between these two although it gave the highest mycelial (45 mm) at 28 ºC. From this observation, it can be suggested that P. palmivora population in the Ajumako-Enya- Essiam district may be experiencing changes in its pathogen population. Alteration in pathogen population is an integral part of pathogen evolution and is mainly triggered by any of the forces of evolution, which has the potential of a wide distribution over a large geographical area (McDonald and Linde, 2002). Molecular study to elucidate the Phytophthora diversity and continuous monitoring for any changes in the Phytophthora population are suggested. Molecular techniques are much faster, more specific, more sensitive, and more accurate, than other methods (Capote et al., 2012). Such tools also make it possible to gain an insight at the molecular level into the processes which underlie pathogenesis (Griffith, 2000).

5.5. Fungicide resistance of Phytophthora isolates in relation to temperature Economic losses attributable to Phytophthora diseases have been reduced through the use of chemical fungicides. Poor disease control using fungicides may however, result in resistance (lack of sensitivity) of the fungal pathogens (Damicone, 1996). In order to maximize disease control with these fungicides, applications should coincide with periods favourable for pathogenic activity and disease development (Matheron and Matejka, 1992). In previous studies, extreme temperatures was observed to limit disease development by direct effect on Phytophthora species in soil (Matheron and Matejka, 1992), by reducing infection or subsequent disease development on several plant genera. Hence temperature may be useful indicators of periods of pathogen inactivity or arrested disease development when fungicide application is not needed. Results from this study showed 100% inhibition after 96 hours of incubation on Ridomil Gold-V8 juice agar at all temperatures. This implies that Ridomil Gold is very effective for the control of P. megakarya and P. palmivora in vitro. On the field however, factors such as the amount and frequency of rainfall, insufficient application rate, inherently low effectiveness of the fungicide on the target pathogen, improper timing or application method, as root/soil environment etc. may affect the efficacy of fungicide treatments (Akrofi, 2015., Damicone, 1996). This study showed that higher temperatures have inhibit growth of P. palmivora and P. megakarya. The interactive effect of temperature and fungicide on P. palmivora and P. megakarya was very appreciable. This means that with predicted rise in temperature, current recommended fungicide dosage for spraying should be minimized in the future for effective and safe disease control.

6. CONCLUSION

This study provides empirical data with implications for the different aspects of Phytophthora black pod epidemics in cocoa production at the different cocoa production zones of Ghana. Knowledge of how P. palmivora and P. megakarya retain their ability to cause disease provides useful insights into reservoirs of black pod infections. From the findings of the presented study, it can therefore be concluded that, latent period of infection is an important aggressive component of Phytophthora species causing black pod disease of cocoa. All isolates of P. palmivora and P. megakarya with least latent period of infection produced the highest infections on both cocoa pods and leaf discs. Thus, the shorter the latent periods of infection, the higher the virulence among Phytophthora isolates. The pathogenic diversity found to exist among Phytophthora isolates may also be attributed to the type and number of zoospores produced by the various isolates. These also determine the aggressiveness of the various Phytophthora isolates on cocoa pods and leaf discs. In general, P. palmivora isolates with the exception of Pp-CR 293, were more virulent as they produced many infective zoospores and chlamydospores and recorded the highest mean disease symptoms than P. megakarya. These results contradict earlier reports, a clear indication of an emerging trend in black pod disease caused by the two Phytophthora species. This findings is however, not conclusive since only few (16) isolates from few locations were used in this study. Furthermore, mean disease symptoms was found to be greater in wounded cocoa pods than in unwounded pods. The fact that disease was lesser in unwounded pods when compared to wounded pods, gives a strong evidence of the role of outer wall of epidermal cells found on cocoa husk in disease development. Thus, breeding for resistant cocoa genotypes with pod husks that can inhibit Phytophthora development will significantly minimize black pod disease. The study also demonstrated that temperature is an important environmental factor with considerable effect on P. palmivora and P. megakarya growth. Generally, mycelial growth decreases with increasing temperatures for both Phytophthora species. But P. megakarya was found to be more sensitive to temperature than P. palmivora. Optimum temperature for greatest mycelial growths (24 - 35 mm) for P. megakarya occurred at 25 ˚C, while for P. palmivora, greatest mycelial growths (29- 45 mm) occurred between 25 ˚C to 28 ˚C. The fact that only P. palmivora isolates were less sensitive and produced chlamydospores, at the different temperatures 25 ˚C, 28 ˚C and 31 ˚C is an indication of how pod losses due P. palmivora may become prevalent in the future. Again,

P. palmivora is ubiquitous, and with the emerging high level of virulence found in this study, as well as its tolerance to higher temperatures, global pod losses due it may increase in the future. Lastly, all Phytophthora isolates were very sensitive to Ridomil Gold 66 WP (6 % metalaxyl M + 60% Cupper (I) oxide) when applied at the recommended rate (3.3g/L) in vitro at different temperatures. There was 100% inhibition to isolates mycelial growth on Ridomil-V8 agar media. With the observed considerable effects of temperature on mycelia growth from this study, recommended Ridomil Gold dosages in the future should be minimized in anticipation of predicted rise in temperature. This will enhance effective and safe black pod disease control and management.

7. PERSPECTIVES

The current study revealed pathogenic diversity between P. megakarya and P. palmivora and among isolates of the same species. The variability in aggressiveness may result from the alteration in physiological state of the Phytophthora isolates during storage. Also since isolates were collected from different regions, the pathogenic diversity may be regionally based. This was not clearly observed in this study due to the fewer number of isolates used, three per region. Hence for future research, an extensive regional survey and isolates collection should be carried out. Employing molecular techniques, the virulence and aggressiveness factors in P. megakarya and P. palmivora should be studied. Data and knowledge generated will be useful in developing regional-based disease profiles, which at the present are not available in Ghana. With regional disease profiles, it will be relatively easy to detect the dynamics within P. megakarya and P. palmivora populations and the underlying factors responsible. Regional based disease profiles will be a key component of disease supporting system (DSS) for effective disease management. At the same time, it will also be very useful in the development of models for black pod disease forecasting. Moreover, breeding works should be intensified into developing cocoa pods with characteristic cocoa pod husk surfaces as well as exploring other defense mechanisms that will delay or prevent Phytophthora disease establishment. A prerequisite to this is the reliance on modern molecular and genetics tools to elucidate the mechanisms involved in black pod disease development on cocoa. In addition, Ridomil Gold has been found to be very effective against P. palmivora and P. megakarya at different range of temperatures 25 ˚C, 28 ˚C, 31 ˚C and 34 ˚C. Phytophthora isolates were also found to be sensitive to higher temperatures. Hence it is important to investigate further the interactive effects of different temperature regimes, and other climatic factors (precipitation, relative humidity etc.) against different fungicides concentrations in all recommended fungicides. This will help ascertain the minimum fungicide dosages required for effective and safe disease control.

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APPENDICES

Appendix 1: Analysis of variance table for radial growth at 25 ˚C Analysis of variance

Variate: Rad_growth_at_25_˚C

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

ISOLATE 15 1197.083 79.806 23.66 <.001

Residual 32 107.917 3.372

Total 47 1305.000

Appendix 2: Analysis of variance table for radial growth at 28 ˚C Analysis of variance

Variate: Rad_growth at 28˚C

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

ISOLATE 15 5474.728 364.982 82.35 <.001

Residual 32 141.833 4.432

Total 47 5616.561

Appendix 3: Analysis of variance table for radial growth at 31 ˚C

Analysis of variance

Variate: Rad_growth at 31˚C

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

ISOLATE 15 14617.703 974.514 324.70 <.001

Residual 32 96.042 3.001

Total 47 14713.745

Appendix 4: Analysis of variance table for radial growth at 34 ˚C

Analysis of variance

Variate: Rad_growth at 34˚C

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

ISOLATE 15 1437.863 95.858 14.33 <.001

Residual 32 214.042 6.689

Total 47 1651.905

Appendix 5: Phytophthora isolates data REGIO CODE Type Date of Place of District District co- N storage collection ordinates Western GH-13-WR - Pp 18/11/2013 Wantram Wassa 5° 46′ 22.8″ N 425 station Amenfi East 2° 5′ 31.2″ W Western GH-13-WR- Pm 18/11/2013 Camp ? 431 Western GH-13-WR- Pm 18/11/2013 Afranse Wassa West 5° 18′ 0″ N 432 1° 59′ 38.4″ W Eastern GH-13- ER - Pm 18/11/2013 Abease Birim Central 5° 55′ 29.89″ N 370 0° 58′ 55.78″ W Eastern GH-14- ER- Pm 19/01/2014 Tafo East Akim 6° 10′ 4.8″ N 371 0° 33′ 7.2″ W Eastern GH-14- ER- Pp 19/01/2014 Tafo East Akim 6° 10′ 4.8″ N 372 0° 33′ 7.2″ W Brong GH-13- Pp 16/10/2013 Manfo Ahafo Ano 7° 0′ 0″ N Ahafo BAR- 143 Nkwanta North 2° 10′ 0″ W Brong GH-13- Pm 16/10/2013 Bechem/SP Tano South 7° 11′ 0″ N Ahafo BAR- 152 U 2° 0′ 0″ W Brong GH-13- Pm 16/10/2013 Acherensua Asunafo 6° 48′ 0″ N Ahafo BAR-158 North 2° 31′ 0″ W Central GH-13-CR- Pp 22.11.2013 Anyinase Ajumako- 5° 27′ 48.96″ N 276 Enya- Essiam 0° 56′ 12.48″ W Central GH-13-CR- Pp 22.11.2013 Nkwantado Twifo- 5° 37′ 0″ N 281 Hemang 1° 33′ 0″ W Lower Denkyira Central GH-13-CR- Pp 22.11.2013 Ajumako Ajumako- 5° 27′ 48.96″ N 293 Enya- Essiam 0° 56′ 12.48″ W Ashanti GH-13-AR- Pm 10.10.2013 Agogo Ashanti Akim 6° 37′ 5″ N 320 North 1° 12′ 36″ W Ashanti GH-13-AR- Pm 10.10.2013 Wiamoase Sekyere 7° 4′ 0″ N 334 South District 1° 24′ 0″ W Ashanti GH-13-AR- Pm 10.10.2013 Ejisu Ejisu Juaben 6° 43′ 0″ N 351 1° 28′ 0″ W Volta GH-13-VR- Pm 14.09.2013 Akpafo Hohoe 7° 9′ 9″ N 116 Odomi 0° 28′ 36″ E Pp- P. palmivora; Pm- P. megakarya

Appendix 6: Details of COCOBOD approved fungicides, insecticides, herbicides and fertilizers for use on cocoa in Ghana as at October 2013.

*Formulation type: DF = Dry Flowable; WP = Wettable Powder and SC = Suspension Concentrate #: NA=Not available