ABSTRACT
Cassava is one of the most important food crops globally and is a major staple food for more than 700 million people across the tropical and sub-tropical world. Cassava is adversely affected by diseases (caused by viruses, bacteria, nematodes and fungi), pests and adverse environmental constraints. Current research activities in cassava diseases are focused more on viral and bacterial diseases than on fungal diseases, yet phytopathogenic fungi play an important role in causing devastating disease epidemics leading to significant annual yield losses. One of the most important fungal diseases is the cassava brown leaf spot (BLS) disease. This study aimed at identification and characterization of the causative agents of BLS disease in Kenya and the reaction of cassava genotypes to infection by the pathogens. Experimental materials were sourced from the Kenya Agricultural and Livestock Research Organization (KALRO) Biotechnology Research Center, KALRO Kakamega and from a confined field trial of the Virus Resistant Cassava for Africa (VIRCA) project located at KALRO Kandara in Murang’a County. Fungi were isolated and purified on antibiotic-amended PDA media from the symptomatic leaf samples. Identification of the fungal pathogens was based on cultural and morphological characteristics of pure fungal cultures coupled with molecular characterization of individual pathogens. Results of the study delineated three pathogens from the genera Colletotrichum, Cladosporium and Alternaria, working in synergism to produce brown leaf spot disease as observed in the fields. The three fungal pathogens were used, in combination, to challenge five farmer-preferred cassava genotypes TME 204, TME 14, TME 7, Ebwanatereka 1 and Ebwanatereka 2. Symptoms were observed over a period of 56 days (eight weeks) at intervals of seven days, after which disease progress was determined. Analysis of Variance (ANOVA) was carried out using GenStat software, 15th Edition while molecular data was analyzed using Geneious Prime and MEGA 11 softwares. The cassava plants had varied responses depending on genotype. The highest area under disease progress curve (AUDPC) was 178.5 in TME 204 and lowest at 103.8 in Ebwanatereka 2. With regard to symptom severity, Ebwanatereka 2 exhibited a relatively slow response to infection compared to the other genotypes throughout the assessment period. On the other hand, TME 204 maintained a high infection response therefore indicating high level of susceptibility to cassava brown leaf spot disease. Findings of this study will add to the knowledge gap in the management approaches to cassava brown leaf spot disease. More research should be carried out to identify sources of resistance to the disease.
TABLE OF CONTENTS
DECLARATION ii
PLAGIARISM DECLARATION iii
DEDICATION iv
ACKNOWLEDGEMENTS v
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS AND ACRONYMS xiii
ABSTRACT xv
CHAPTER ONE: INTRODUCTION
1.1 Background to the study 1
1.2 Problem statement 2
1.3 Justification of the study 4
1.4 Objectives 4
1.4.1 Broad objective 4
1.4.2 Specific objectives 4
1.5 Hypotheses 5
CHAPTER TWO: LITERATURE REVIEW
2.1 Cassava production in the world 6
2.2 Economic importance of cassava 7
2.3 Constraints to cassava production 8
2.3.1 Arthropod pests infesting cassava 9
2.3.2 Major diseases affecting cassava 11
2.3.2.1 Viral diseases 11
2.3.2.2 Bacterial diseases 12
2.3.2.3 Fungal diseases 12
2.4 Cassava brown leaf spot disease (BLS) 13
2.4.1 Symptoms and life cycle of cassava brown leaf spot disease 14
2.4.2 Impact of cassava brown leaf spot disease 15
2.4.3 Identification of cassava brown leaf spot pathogens 16
2.4.4 Control of cassava brown leaf spot disease 17
CHAPTER THREE: MATERIALS AND METHODS
3.1 Description of the study site and experimental materials 18
3.2 Sampling of symptomatic cassava leaves 18
3.3 Preparation of potato dextrose agar modified with antibiotics 18
3.4 Isolation and purification of fungi 19
3.5 Identification of fungal pathogens 19
3.6 Conduct of pathogenicity test 20
3.7 Molecular identification of the fungi causing cassava brown leaf spot disease 22
3.7.1 DNA extraction 22
3.7.2 Polymerase chain reaction (PCR) and sequencing 23
3.7.3 Sequence analysis 24
3.8 Confirmation of the causal pathogen of the disease 24
3.9 Reaction of elite cassava genotypes to infection by fungi causing cassava brown leaf spot disease 25
3.9.1 Experimental design 26
3.9.2 Rating of cassava brown leaf spot disease severity on elite cassava genotypes 26
3.9.3 Data analysis 26
CHAPTER FOUR: RESULTS
4.1 Symptoms of cassava brown leaf spot disease under field conditions 28
4.2 Pathogen isolation, purification and identification 28
4.3 Growth and morphological characteristics of the pathogens on solid medium 30
4.4 Molecular identification and phylogenetic analysis of fungal pathogens 33
4.4.1. PCR amplification using universal primers 33
4.4.2. Phylogenetic analysis 34
4.5 Pathogenicity tests 42
4.6 Phenotypic symptom assessment of elite cassava genotypes 47
4.6.1 Symptom development and disease incidence 47
4.6.2 Brown leaf spot disease severity and area under disease progress curves 48
CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 Discussion 51
5.2 Conclusion 54
5.3 Recommendations 54
REFERENCES 55
LIST OF TABLES
Table 1: Leading world cassava producing countries, quantity and acreage in 2019. 7
Table 2: Volumes of PCR components 23
Table 3: Cultural and morphological characteristics of fungal pathogens isolated from infected cassava leaves 30
Table 4: Comparison of BLS fungal pathogen sequences with sequences deposited in GeneBank sequences, showing sequences with best match, query coverage and similarity percentages for both ITS 1/4 and ITS 4/5 35
LIST OF FIGURES
Figure 1: Symptoms of brown leaf spot disease on cassava leaves 15
Figure 2: Cassava plants covered in humidity bags after inoculation for fungal infection 21
Figure 3: Symptoms of cassava brown leaf spot disease under field conditions 28
Figure 4: Incidence of fungal pathogens associated with cassava brown spot disease 29
Figure 5: Cultural and morphological characteristics of Colletotrichum sp. 31
Figure 6: Cultural and morphological characteristics of Cladosporium sp. 32
Figure 7: Cultural and morphological characteristics of Alternaria sp. 32
Figure 8: Purified PCR products for Colletotrichum sp., Cladosporium sp. and Alternaria sp. 34
Figure 9: Phylogenetic tree of Colletotrichum sp. (labelled Colletotrichum S7) isolated from cassava leaves and its near relatives 36
Figure 10: Phylogenetic tree of Colletotrichum sp. (labelled Colletotrichum S1) isolated from cassava leaves and its near relatives 37
Figure 11: Phylogenetic tree of Cladosporium sp. (labelled Cladosporium S3) isolated from cassava leaves and its near relatives 38
Figure 12: Phylogenetic tree of Cladosporium sp. (labelled Cladosporium S9) isolated from cassava leaves and its near relatives 39
Figure 13: Phylogenetic tree of Alternaria sp. (labelled Alternaria S5) isolated from cassava leaves and its near relatives 40
Figure 14: Phylogenetic tree of Alternaria sp. (labelled Alternaria S11) isolated from cassava leaves and its near relatives 41
Figure 15: Cassava leaves displaying chlorotic spots on surface at the beginning of symptom development at 7 days post-inoculation upon combined inoculation with Colletotrichum, Cladosporium and Alternaria species 43
Figure 16: Foliar disease symptoms on cassava plants inoculated with an isolate of Colletotrichum sp.; at 14 days post-inoculation 43
Figure 17: Foliar disease symptoms on cassava plants inoculated with an isolate of Cladosporium sp.; at 21 days post-inoculation 44
Figure 18: Foliar disease symptoms on cassava plants inoculated with an isolate of Alternaria sp.; at 14 days post-inoculation 44
Figure 19: Foliar disease symptoms on cassava plants inoculated with a combination of Colletotrichum and Cladosporium spp. isolates; at 14 days post-inoculation 45
Figure 20: Foliar disease symptoms on cassava plants inoculated with a combination of Colletotrichum and Alternaria spp. isolates; at 14 days post-inoculation 45
Figure 21: Foliar disease symptoms on cassava plants inoculated with a combination of Cladosporium and Alternaria spp. isolates; at 14 days post-inoculation 46
Figure 22: Foliar disease symptoms on cassava plants inoculated with a combination of Colletotrichum, Cladosporium and Alternaria spp. isolates; at 14 days post-inoculation 46
Figure 23: Experimental negative controls: (A and B) cassava plants sprayed with distilled water; (C) un-inoculated cassava plant 47
Figure 24: Percentage disease incidence over time for cassava genotypes TME 204, TME 14, TME 7, Ebwanatereka 1 and 2 48
Figure 25: Disease severity scores over time for five farmer preferred cassava genotypes. 49
Figure 26: Mean AUDPC scores of control (C) and fungal (E) treatments calculated from disease severity rating on TME 204, TME 14, TME 7, Ebwanatereka 1 and 2 cassava genotypes. 50
LIST OF ABBREVIATIONS AND ACRONYMS
ANOVA Analysis of Variance
BLAST Basic Local Alignment Search Tool
BLS Brown Leaf Spot
BRI Biotechnology Research Institute
CABI Centre for Agriculture and Bioscience International
CAD Cassava Anthracnose Disease
CBB Cassava Bacterial Blight
CBSD Cassava Brown Streak Disease
CMD Cassava Mosaic Disease
CRRD Cassava Root Rot Diseases
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
FAO Food and Agriculture Organization
FAOSTAT Food and Agriculture Organization Corporate Statistical Database
IFAD International Fund for Agricultural Development
IITA International Institute of Tropical Agriculture
ISAAA International Service for the Acquisition of Agri-biotech Applications
ITS Internal Transcribed Spacers
KALRO Kenya Agricultural and Livestock Research Organization
KEPHIS Kenya Plant Health Inspectorate Service
NaCl Sodium Chloride
NASE Namulonge Selection
NCBI National Center for Biotechnology Information
PCR Polymerase Chain Reaction
PDA Potato Dextrose Agar
PPD Post-harvest Physiological Deterioration
RNA Ribonucleic acid
rRNA Ribosomal Ribonucleic acid
SDS Sodium Dodecyl Sulphate
TME Tropical Manihot esculenta
TE TRIS EDTA
TRIS HCL Trisaminomethane hydrochloride
UPGMA Unweighted Pair Group Method with Arithmetic Mean
USDA United States Department of Agriculture
VIRCA Virus Resistant Cassava for Africa
CHAPTER ONE
INTRODUCTION
1.1 Background to the study
Cassava (Manihot esculenta) is a woody shrub that belongs to the family Euphorbiaceaea. It is widely cultivated in the tropics and subtropics as an annual crop and used as a major source of carbohydrates. Cassava produces edible starchy storage roots that are long and tapered and covered with a strong detachable skin that is rough and brown on the outside (Wassie, 2019). In developing countries, particularly in sub-Saharan Africa, cassava plays an essential role as a food security crop owing to its ability to grow well in low rainfall and on poor and marginal soils (Mtunguja et al., 2019). Being perennial, the crop can be harvested as need arises and has a wide harvesting window that allows it to act as a reserve for famine and is important in management of labour schedules. It serves either as a subsistence and/or a cash crop thus offering flexibility to resource-poor farmers. Having been described as one of the most drought-tolerant crops, cassava has gained popularity and is therefore cultivated widely in the cassava growing countries and in new production zones (Mtunguja et al., 2019). This is because the crop is adaptable to vast environments therefore can mitigate the effect of climate change.
In Kenya, cassava is used to manufacture commodities such as gluten-free flour, feeds for animals, sucrose substitute in beverages and confectionary products. The gluten-free carbohydrates in cassava is important in preventing gluten intolerance as well as food allergies. Due to the high fibre content of the crop, it is helpful in reducing cholesterol level. Cassava is also rich in minerals such as manganese, calcium and iron, which is helpful to expectant women (Ministry of Agriculture, 2019).
Cassava is one of the most important food crops globally and ranks fourth as a food crop in developing countries after rice, wheat and maize (Agricultural Research Council, 2014). It is a major staple food for more than 700 million people across the tropical and sub-tropical world and has gained popularity as a source of carbohydrates as well as income for millions of smallholder farmers in sub-Saharan Africa (Legg et al., 2014). For optimum growth and yield, cassava requires humid warm climates with temperatures between 25oC and 29oC and evenly distributed annual rainfall of between 1000-1500mm. Regardless of these ideal requirements, cassava is widely adaptable, growing in a range of soils and rainfall regimes (Tan, 2015).
Despite the vast production and utilization of cassava, there are biotic and abiotic stresses that adversely affect its yield thereby limiting the full realization of its immense potential (Bull et al., 2011). Much as cassava can survive harsh environmental conditions, its productivity is severely affected by; terminal drought, extremes of heat, salinity, pH and flooding (Tadele, 2018). Biotic stresses affecting the crop include insect pests the common ones being whiteflies, mealybugs, green- and red-spider mites, scales, shoot flies, fruit flies and cassava horn worm. Cassava diseases are mainly caused by viruses, nematodes, bacteria and fungi. The main viral diseases affecting cassava are cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) while bacterial diseases include cassava bacterial blight (CBB) and bacterial stem rot. Among the major fungal diseases are brown leaf spot (BLS), cassava anthracnose disease (CAD), phyllosticta leaf spot, white thread and super-elongation disease (Mwang’ombe et al., 2013; Titus and Lawrence, 2015). These challenges pose a threat to food security especially in the tropics and sub-tropics (Bull et al., 2011; Campo et al., 2011). As a way of reducing yield losses to insects and pests, Howeler et al. (2013) suggests the use of resistant cassava varieties, use of control agents and managing levels of crop nutrients in order to reduce insect reproduction. On the other hand, cassava diseases can be cotrolled through the use of clean planting materials, elimination of infected plants and crop rotations in order to suppress pathogens. One of the most important factors in effective disease control is accurate identification of the disease causative agent(s). This study was therefore carried out to identify the causative agents of one of the most important phytopathogenic fungal diseases namely cassava brown leaf spot.
1.2 Problem statement
Current research activities in cassava diseases are focused more on viral and bacterial diseases than on fungal diseases, yet phytopathogenic fungi play an important role in causing devastating disease epidemics leading to significant yield losses. This has made plant pathogenic fungi a serious economic factor and a threat to food security thus attracting the attention of all agricultural stakeholders including farmers, breeders and scientists across the globe (Li et al., 2020). Cassava fungal diseases include root rots, foliar diseases and stem necrosis disease. These diseases cause a significant loss of planting materials thus making them unsuitable for planting (Boas et al., 2017). Cassava root rot diseases (CRRD) are caused by a complex of soil-borne fungi including Fusarium spp. Phytophthora spp., Pythium spp., Neoscytalidium spp. and Lasiodiplodia spp. (Boas et al., 2017). These fungi at times occur in different compositions of species and in some cases restricted to a geographical region. These root rot diseases are a major constraint responsible for up to 80% of yield losses (Boas et al., 2017). Foliar diseases include cassava super-elongation disease, white leaf spot and cassava brown spot diseases while cassava stem diseases include cassava bud necrosis and cassava anthracnose disease (McCallum et al., 2017; Legg and Álvarez, 2017). Super-elongation disease, caused by the fungus Sphaceloma manihoticola causes more than 80% losses in susceptible cassava genotypes while cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides (Kunkeaw et al., 2010) causes a viability loss of 50-75% in infected planting materials (Legg and Álvarez, 2017).
One of the most important fungal diseases is the cassava brown leaf spot (BLS). It is characterized by large, brown, necrotic spots appearing on older leaves and the infected leaves have a tendency to drop early (Msikita et al., 2000). The disease is spread to new leaves and plants by wind or rain splash. BLS disease epidemics in cassava are reported worldwide majorly on lower canopy of crops that are more than five months old. The disease is favored by high humidity and temperature resulting in about 78% disease incidence in the wet savannahs and 98% in the case of transition forest of West African countries. The importance of cassava brown leaf spot disease may be underestimated due to its being confined to the lower canopy leaves. However, the disease causes defoliation which may have a significant effect on yield especially in areas where cassava is extensively grown for commercial production. The effect of defoliation becomes aggravated when the infection is followed by a period of drought (Hillocks and Wydra, 2002).
Since the disease is usually favored by high temperature and humidity, high rainfall areas are more prone to the disease than areas of relatively low rainfall (Hillocks and Wydra, 2002). In this regard therefore, it is advisable to use cultivars that are less susceptible to the disease. The highly susceptible germplasms are often attacked quite early. In these cultivars, the brown spots can cover a great surface area of the leaves and this could significantly reduce photosynthetic activities, thus reducing yields (Moses et al., 2015). The causative pathogen of cassava brown leaf spot disease has been identified and characterized as reported in studies conducted in various countries but in Kenya little has been done to identify and/or characterize the pathogen.
1.3 Justification of the study
Studies on cassava brown leaf spot (BLS) disease have been carried out in various regions including China (Pei et al., 2014), Thailand (To-Anun et al., 2011), Asia, North- and Latin America and some parts of Africa (Lozano and Booth, 1974). However, little has been done in Kenya to understand the pathogen(s), yet there is increase in cassava production in the country including areas which were not main production zones. In all the production areas, the disease is rampant and has also been observed in recent outbreaks in cassava field trials in the country (H. Obiero, personal communication, September, 2016). Besides, the farmer-preferred cassava genotypes which are currently in use for consumption and research work are susceptible to BLS disease. All these factors necessitate the need to pay more attention to BLS disease in order to enhance its management. The need to identify the causative agent(s) of cassava brown leaf spot disease cannot be overemphasized. Identification of the causative agent(s) is an important pre- requisite in better management of cassava BLS disease thereby avoiding devastating crop yield losses. In the various regions where the disease has been studied, the causative agent has been reported to be a fungus in the genus Cercospora. In Kenya, the causative agent(s) has not been studied comprehensively and may or may not be the same fungus. The aim of this study was therefore to identify the causative agent(s) of cassava brown leaf spot in Kenya and to determine the reaction of popular cassava genotypes to infection by the pathogen(s).
1.4 Objectives
1.4.1 Broad objective
To contribute to better management of cassava brown leaf spot disease through identification of the causative agent and assessment of phenotypic reaction of elite cassava genotypes to the pathogen.
1.4.2 Specific objectives
i. To identify and characterize the causal agent of cassava brown leaf spot in Kenya
ii. To determine the phenotypic response of elite cassava genotypes to infection by cassava leaf spot pathogen(s)
1.5 Hypotheses
i. The causative agent for the cassava leaf spot is a fungal pathogen
ii. The selected cassava germplasm have no significant difference in their response to infection by cassava leaf spot pathogen(s)
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