ABSTRACT
Tomato, a major vegetable widely used in Kenya faces a number of production challenges some of them being diseases like late blight, early blight and bacterial wilt. Chemical compounds which have environmental and health concerns are mostly used to control early blight in tomato production. In this study, two Trichoderma isolates, two Bacillus isolates and commercial Pseudomonas fluorescens were used in the management of early blight, a major disease of tomato. These were tested for their effectiveness in managing Alternaria solani in vitro. The dual culture technique was used and consisted in growing the antagonists together with the pathogen. Diameter of A. solani colony was measured and used to calculate the percent growth inhibition. Trichoderma isolates were the most effective against the radial growth of A. solani with percent growth inhibition of 80.9 and 82.2%. These were followed by Bacillus isolates with percent growth inhibition of 56.6 and 54.1%. Pseudomonas fluorescens also suppressed A. solani radial growth but with a lower percent growth inhibition of 47.6%. Trichoderma isolates, Bacillus isolates and commercial Pseudomonas fluorescens were also evaluated for their effectiveness in managing tomato early blight under greenhouse and field conditions. Water and Tower 72 WP® (Metalaxyl 8% and Mancozeb 64%) were used as control and standard check respectively. Data were collected on disease parameters and yield of marketable fruits. In the greenhouse, the percent disease index by the 90th day after transplanting was significantly lower in all treatments than in the control. Isolate CB12 recorded the lowest percent disease index of 28.3% which was comparable to the standard chemical at 30.5% and both were significantly different from the control at 61.6%. The highest mean quantity of marketable fruits of 0.21 kg/plant was recorded from Tricho 7, followed by the standard chemical with a comparable yield of 0.20 kg/plant. Control treatment recorded significantly lower marketable fruit weight of 0.06 Kg/plant. At both experimental sites, on the 90th day after transplanting, the percent disease index was significantly lower in all the treatments compared to the control. The lowest percent disease index recorded for the antagonists was from Tricho 10 at 35.0% and was comparable to the standard chemical at 30.3%. The two were significantly lower than the control at 68.8%. As for yield of marketable fruits, Tricho 10 recorded significantly higher mean weight at 10.5 tons/hectare compared to the control which recorded 3.8 tons/hectare. However, the standard chemical recorded significantly higher yield at 11.7 tons/hectare compared to Tricho 10. Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens are effective in managing early blight under in vitro, greenhouse and field conditions and they are able to reduce the effects of early blight on tomato production. They should be used for a sustainable production of tomatoes.
Key words: Trichoderma spp., Bacillus spp., Pseudomonas fluorescens, management and early blight of tomatoes.
TABLE OF CONTENTS
DECLARATION i
DECLARATION OF ORIGINALITY ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF PLATES xii
LIST OF APPENDICES xiii
LIST OF ABBREVIATIONS xiv
GENERAL ABSTRACT xvi
CHAPTER ONE: GENERAL INTRODUCTION
1.1. Background information 1
1.2. Problem statement 2
1.3. Justification of the study 3
1.4. Objectives 5
1.4.1. Main objective 5
1.4.2. Specific objectives 5
1.5. Hypotheses 5
CHAPTER TWO: LITERATURE REVIEW
2.1. Economic importance of tomatoes 6
2.2. Constraints to tomato production in Kenya 9
2.2.1. Physiological disorders 9
2.2.2. Pests and weeds 10
2.2.3. Diseases 10
2.3. Limitations in the management of tomato diseases in Kenya 13
2.4. Description of early blight of tomatoes 14
2.4.1. Economic importance of early blight of tomatoes 14
2.4.2. Description of tomato early blight causative agent 14
2.4.3. Infection process of early blight in tomatoes 15
2.4.4. Symptoms caused by early blight on tomatoes 16
2.4.5. Source of inoculum 17
2.4.6. Factors favoring early blight of tomatoes 18
2.4.7. Management of early blight of tomatoes 19
2.4.8. Management of early blight using microbial antagonists 20
2.4.8.1. Modes of action for Bacillus subtilis 21
2.4.8.2. Description and modes of action for Pseudomonas fluorescens 22
2.4.8.3. Modes of action of Trichoderma species 24
2.4.8.4. Description and modes of action of Streptomyces species 25
2.5. Use of microbial based pesticides in plant disease management 26
CHAPTER THREE: EFFECTS OF TRICHODERMA SPP., BACILLUS SPP. AND PSEUDOMONAS FLUORESCENS ON RADIAL GROWTH OF ALTERNARIA SOLANI
3.1. Abstract 29
3.2. Introduction 30
3.3. Materials and methods 31
3.3.1. Isolation and identification of Alternaria solani 31
3.3.2. Isolation and identification of Trichoderma spp. 31
3.3.3. Isolation and identification of Bacillus spp. 32
3.3.4. Preparation of Pseudomonas fluorescens 32
3.3.5. Preparation of Alternaria solani inoculum 32
3.3.6. Pathogenicity test of Alternaria solani 32
3.3.7. In vitro activity of the antagonists against Alternaria solani 33
3.3.8. Screening of Trichoderma isolates and Bacillus isolates 34
3.3.9. Data analysis 35
3.4. Results 35
3.4.1. Morphological features of isolated Alternaria solani and early blight symptoms on tomato plants inoculated with Alternaria solani conidia 35
3.4.2. Morphological features and isolation frequency of Trichoderma isolates 36
3.4.3. Effects of Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens on the radial growth of Alternaria solani 38
3.4.3.1. Screening of Bacillus isolates against Alternaria solani 38
3.4.3.2. In vitro activity of Bacillus subtilis isolates and Pseudomonas fluorescens
against Alternaria solani 38
3.4.3.3. Screening of Trichoderma isolates against Alternaria solani 40
3.4.3.4. In vitro activity of Trichoderma isolates against Alternaria solani 41
3.5. Discussion 42
CHAPTER FOUR: EFFECTIVENESS OF TRICHODERMA SPP., BACILLUS SUBTILIS AND PSEUDOMONAS FLUORESCENS IN THE MANAGEMENT OF EARLY BLIGHT OF TOMATOES
4.1. Abstract 47
4.2. Introduction 48
4.3. Material and methods 49
4.3.1. Description of the study sites 49
4.3.2. Preparation of culture filtrates from the antagonists 50
4.3.2.1. Preparation of cultures filtrates from Bacillus subtilis isolates 50
4.3.2.2. Preparation of culture filtrates from Trichoderma isolates 50
4.3.3. Design and set up of the greenhouse experiment 51
4.3.4. Design and set up of the field experiments 52
4.3.5. Application of the treatments 52
4.3.6. Assessment of early blight 53
4.3.7. Assessment of tomato yield 54
4.3.8. Data analysis 54
4.4. Results 55
4.4.1. Percent disease incidence for early blight in tomato plants treated with the various antagonists 55
4.4.2. Percent disease severity for early blight in tomato plants treated with the various antagonists 57
4.4.3. Percent disease index for early blight in tomato plants treated with the various antagonists 59
4.4.4. Area under disease progress curve for early blight in tomato plants treated with the various antagonists 61
4.4.5. Fruit yield for tomato plants treated with the various antagonists 62
4.4.6. Correlations between tomato early blight parameters and tomato yield 64
4.5. Discussion 65
CHAPTER FIVE: GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1. General discussion 70
5.2. Conclusion 72
5.3. Recommendations 73
REFERENCES 74
APPENDICES 99
LIST OF TABLES
Table 2.1: Tomato production from the major tomato producing countries in the East African Community 7
Table 2.2: Tomato production from the major tomato producing countries in Central Africa 8
Table 2.3: Major microbial based pesticides used to manage plant diseases in Kenya 28
Table 3.1: Isolation frequency and morphological features of Trichoderma isolates 37
Table 3.2: Mean diameter of Alternaria solani colony in the presence of Bacillus subtilis
isolates and mean diameter of Alternaria solani colony in the control 40
Table 3.3: Mean diameter of Alternaria solani colony in the presence of Trichoderma isolates and mean diameter of Alternaria solani colony in the control 42
Table 4.1: Percent disease incidence for early blight in tomato plants treated with the various antagonists 56
Table 4.2: Percent disease severity for early blight in tomato plants treated with the various antagonists 58
Table 4.3: Percent disease index for early blight in tomato plants treated with the various antagonists 60
Table 4.4: Fruit yield for tomato plants treated with the various antagonists 63
Table 4.5: Correlations between tomato early blight parameters and tomato yield at both experimental sites 64
LIST OF FIGURES
Figure 3.1: Mean radius of Alternaria solani colony in the presence of Bacillus isolates 38
Figure 3.2: Mean radius of Alternaria solani colony in the presence of Trichoderma
isolates 41
Figure 4.1: Area under disease progress curve for early blight in tomato plants sprayed with the antagonist culture filtrates 62
LIST OF PLATES
Plate 2.1: Conidia of Alternaria solani adopted from Kemmitt (2002) 15
Plate 2.2: Early blight symptoms on tomato leaf and fruit 17
Plate 3.1: Morphological characteristics of isolated Alternaria solani 35
Plate 3.2: Early blight symptoms on infected tomato leaf (A) and healthy tomato leaf (B) 36
Plate 3.3: Morphological and cultural characteristics of Trichoderma isolates 37
Plate 3.4: Alternaria solani colony in the presence of Bacillus subtilis isolates and
Pseudomonas fluorescens and Alternaria solani colony in the control 39
Plate 3.5: Alternaria solani colony in the presence of Trichoderma spp. and Alternaria solani colony in the control 42
LIST OF APPENDICES
Appendix 1: Analysis of variance for the percent disease incidence 99
Appendix 2: Analysis of variance for the percent disease severity 100
Appendix 3: Analysis of variance for the percent disease index 101
Appendix 4: Temperature readings during field experimental period in 2019 102
LIST OF ABBREVIATIONS
µl Microliter
µm Micrometer
AEZ II Agro-ecological zone II
AEZ III Agro-ecological zone III
ANOVA Analysis of Variance
AUDPC Area Under Disease Progress Curve
BS Bacillus subtilis
Cfu Colony-forming unit
Cm Centimeter
CRD Completely Randomized Design
DRC Democratic Republic of the Congo
FAOSTAT Food and Agricultural Organization Statistics
Ha Hectare
HCDA Horticultural Crop Development Authority
IBM International Business Machines
IPM Integrated Pest Management
KALRO Kenya Agricultural and Livestock Research Organization
KEPHIS Kenya Plant Health Inspectorate Service
Kg Kilogram
KHCP Kenya Horticulture Competitiveness Project
LM4 Lower Midland 4
LSD Least Significant Difference
Ltd Limited
M Meter
Ml Milliliter
Mm Millimeter
No Number
PCPB Pest Control Products Board
PDA Potato Dextrose Agar
PDI Percent disease index
PDS Percent disease severity
PGI Percent growth inhibition
RCBD Randomized Complete Block Design
Rpm Revolutions per minute
SPS Specialized Products and Services
SPSS Statistical Package for the Social Sciences
USA United States of America
W/w Weight for weight
WP Wettable powder
CHAPTER ONE
GENERAL INTRODUCTION
1.1. Background information
Tomato (Solanum lycopersicum L.) is a major vegetable grown worldwide (Monte et al., 2013). It originated in the western South America, specifically in Peru, Bolivia and Ecuador (Anonymous, 2016). In the 16th and 20th centuries, colonial settlers introduced tomato in Europe and in East Africa respectively (Wener, 2000). Currently, the vegetable is being grown in basically all countries (Abd-El-Kareem et al., 2006). Tomato fruits can be used fresh in salads, prepared as vegetable, or in processed form as tomato paste, tomato sauce, Ketchup and juice. Tomato fruits are beneficial to healthy diet as they contain sufficient amounts of vitamins A, B and C. Additionally, they have significant amounts of potassium, ion and phosphorus (Masinde at al., 2011).
Tomatoes are among the most important and commonly grown horticultural vegetables in Kenya and in other parts of East Africa (Sigei et al., 2014). However, production of tomato fruits is hindered by numerous problems including physiological disorders mainly resulting from water and nutrient stresses, pests and diseases (KALRO, 2005; Mizubuti et al., 2007; Goufo et al., 2008). As an example, temperature and humidity fluctuations during long rain and short rain seasons are conducive for the development of a number of pathogens and the related diseases resulting in lower tomato yield (Engindeniz and Ozturk, 2013). Insect pests including cotton bollworms, whiteflies, melon thrips and tomato leaf miners among others, significantly contribute to tomato yield losses (Engindeniz and Ozturk, 2013; Islam et al., 2013). Diseases such as bacterial canker, bacterial spots, bacterial wilt, Fusarium wilt, early and late blights, root knot nematodes, tomato spotted virus and yellow leaf curl virus among others are major constraints in tomato production (Goufo et al., 2008; Noling, 2013; Sutanu and Chakrabartty, 2014). Early and late blights are the commonest fungal constraints in tomato production (Hou and Huang, 2006). When these fungi infect tomato leaves, they exhibit symptoms which can rapidly spread on entire leaf blades in conducive environments (Xie et al., 2015). Tomato early blight is most commonly managed through application of a limited number of chemical compounds due to withdrawal of some effective fungicides reported to have detrimental effects on the environment and on human health (Singh et al., 2011).
1.2. Problem statement
Early blight is a common disease threatening the production of tomato fruits all over the world and can cause significant yield losses when it is not managed (Adhikari et al., 2017). This may result in malnutrition given that tomato is an important source of nutrients and vitamins A, B and C (Giovanelli and Paradise, 2002; Masinde et al., 2011). Deficiency in vitamins is associated with several health problems (Bouis, 2003; Grosso et al. 2013; Brescoll and Daveluy, 2015). Tomato is a high value vegetable in Kenya and is a source of livelihood for numerous families (Sigei et al., 2014). Therefore, any threat to tomato production can lead to hunger and poverty among people who depend on the production of tomatoes for their livelihoods.
Tomato cultivars which are resistant to early blight, are of low agronomic or commercial quality. Synthetic chemicals are thereby intensively applied by most farmers to lower the intensity of early blight and the accompanying crop losses (Yadav and Dabbas, 2012). In addition, the rotation strategy is limited by the prolonged survival of A. solani in the soil and scarcity of land for cultivation (Foolad et al., 2008; Karuku et al., 2017). Given the high demand for tomato in Kenya and pathogen resistance, farmers increase the rate of chemical application. The required pre-harvest intervals are often not observed. This leads to increased chemical residues in the produce and increased production costs (Waiganjo et al., 2006; Fabro and Varca, 2011). Regular application of synthetic chemicals has detrimental effects on the environment and on human health (Engindeniz and Ozturk, 2013; Bhattacharjee and Dey, 2014). Moreover, regular application of chemicals enhances the development of new fungal biotypes which may be resistant to chemical compounds (Rojo et al., 2007). Since the introduction of systemic fungicides globally in the early 1970s, farmers are increasingly confronted with pathogen resistance to the available chemical compounds. This is often due to misuse of synthetic chemicals (Sutanu and Chakrabartty, 2014). Synthetic chemicals also kill non-target organisms including pollinating insects (Rhoda et al., 2006; Nderitu et al., 2007). Consequently, quality assurance standards are being implemented to minimize detrimental effects of farming operations to the environment and humans. These entail reduced use of chemical inputs to ensure safety to workers, consumers as well as safe guarding animal welfare (Rhoda et al., 2006; Foolad et al., 2008). These concerns have led not only to restrictions or complete banning of some chemical compounds but also to interceptions of produce with excessive chemical residues at the export market (Rhoda et al., 2006; Wandati, 2014).
1.3. Justification of the study
Sustainable production of tomatoes inevitably requires the development of plant disease management strategies which are friendly to the environment and have minimal negative effects on humans (Mamgain et al., 2013). Control of plant diseases using biological means and breeding for resistance are one of the most promising plant disease management approaches (Alabouvette et al., 2006). Breeding for resistance strategy has not been successful in managing early blight given that tomato varieties which are tolerant to early blight do not perform well in terms of agronomic traits (Foolad et al., 2002; Yadav and Dabbas, 2012).
Extended use of agrochemicals for early blight management can be avoided through the integration of microbial antagonists (Mamgain et al., 2013). These are deemed to be biodegradable, friendly to the environment and have minimal effects on humans and non- targeted organisms including beneficial insects (Alabouvette et al., 2006). Antagonistic microorganisms minimize the effects of plant diseases either from microbial interactions directed against plant pathogens or from an indirect action which triggers host plant pathogen resistance (Alabouvette et al., 2006). Several antagonists along with locally available formulations of microorganisms are known to be effective in managing early blight disease (Zhao et al., 2008). These include species of Trichoderma, Bacillus, Pseudomonas and Streptomyces genera among others. These microorganisms differ in their efficacy in managing tomato early blight (Tapwal et al., 2015). Integration of microbial antagonists in the management of early blight demands a better understanding of their effectiveness (Ngoc, 2013). Moreover, only a few studies have been conducted to evaluate the efficacy of microbial antagonists in the management of early blight in the field. Trichoderma, Bacillus and Pseudomonas antagonists grow rapidly, have long shelf-life at room temperature and can be mass produced at lower costs. They are also deemed to be compatible with several fungicides (Pertot et al., 2015). In contrast, several other microbials including Streptomyces species have been reported to be less efficient and not compatible with many crop plants (Pertot et al., 2015). Integration of effective Trichoderma isolates, Bacillus isolates and Pseudomonas fluorescens in early blight management will contribute to a sustainable production of tomatoes through reduction of the dependence on chemicals (Mizubuti et al., 2007; Engindeniz and Ozturk, 2013).
This will help farmers to minimize the losses caused by tomato early blight and still meet the quality standards which require the agriculture products to be safe for consumers (Gupta et al., 2014). This will result in reduced interceptions of tomato produce at the export market. Consumers will have access to tomato fruits that are free from chemical residues. Integration of selected antagonists in the management of tomato early blight will also contribute to a better conservation of the environment given that selected antagonists do not pollute the environment as they are biodegradable. Trichoderma spp. and Bacillus spp. used in this study are beneficial to biopesticide processing companies and biopesticide resellers.
1.4. Objectives
1.4.1. Main objective
The main objective of this study is to integrate Trichoderma spp., Bacillus spp. and
Pseudomonas fluorescens in managing early blight for sustainable production of tomatoes.
1.4.2. Specific objectives
1. To evaluate the antagonistic effects of Trichoderma spp., Bacillus spp. and
Pseudomonas fluorescens on growth of Alternaria solani under in vitro conditions.
2. To evaluate the effectiveness of Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens in managing early blight and in increasing tomato yield.
1.5. Hypotheses
1. Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens have significant antagonistic effects on in vitro growth of A. solani.
2. Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens are effective in managing early blight and in increasing tomato yield.
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