EFFECTIVENESS OF BIO-CONTROL AGENTS IN THE MANAGEMENT OF EARLY BLIGHT OF TOMATOES

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ABSTRACT

Tomato, a major vegetable widely used in Kenya faces a number of production challenges along with diseases like late blight, early blight and bacterial wilt. In this study, bio-control agents (BCAs) which are deemed to be environmentally friendly were used for the management of early blight, a major disease of tomato. BCAs including two Trichoderma isolates coded Tricho 7 and Tricho 10, two Bacillus subtilis isolates coded CA51 and CB12 and Pseudomonas fluorescens (from commercial Bio-cure) were tested for their effectiveness in managing Alternaria solani in vitro. The experiments were carried out in Plant Pathology Laboratory at the Department of Plant Science and Crop Protection, University of Nairobi. The dual culture technique was used. The experimental design was a Completely Randomized Design in five replicates. Diameter of A. solani colony was measured and used to calculate the percent growth inhibition. Means were compared using Fisher’s protected least significant difference (LSD) test at 5%. Tricho 7 and Tricho 10 were the most effective against the radial growth of A. solani with percent growth inhibition of 80.9 and 82.2% for Tricho 7 and Tricho 10 respectively. These were followed by CA51 and CB12 with percent growth inhibition of 56.6 and 54.1% respectively. Pseudomonas fluorescens also hindered A. solani radial growth but with a lower percent growth inhibition of 47.6%. The same BCAs were evaluated for their effectiveness in managing tomato early blight under greenhouse and field conditions. Water and Tower (Metalaxyl 8% and Mancozeb 64%) were used as control and standard check respectively.   Greenhouse evaluations were carried out at Kabete Field Station. The experimental design was a Completely Randomized Design in four replicates. Data were collected on disease and plant parameters. 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 with 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. Field evaluations were carried out at Kabete Field Station and at Kenya Agricultural and Livestock Research Organization (KALRO) Mwea. A Randomized Complete Block Design in triplicate was used. At both experimental sites, on the 90th days 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 BCAs was with 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. BCAs are effective in managing early blight in vitro and under greenhouse and field conditions and minimize the effects of early blight on tomato production.

Key words: bio-control agents, management, tomato and tomato early blight.










TABLE OF CONTENTS
 
DECLARATION i
DEDICATION ii
ACKNOWLEDGEMENTS iii
List of Tables ix
List of Figures xi
List of Plates xii
List of Appendices xiii
LIST OF ABBREVIATIONS xiv
ABSTRACT xvi

CHAPTER ONE: INTRODUCTION
1.1. Background information 1
1.2. Problem statement 3
1.3. Study justification 5
1.4. Objectives 6
1.4.1. Main objective 6
1.4.2. Specific objectives 6
1.5. Hypotheses 6

CHAPTER TWO: LITERATURE REVIEW
2.1. Origin of tomatoes 7
2.2. Botanical description of tomatoes 7
2.3. Requirements for growth of tomato plants 8
2.4. Health benefits of tomatoes 9
2.5. Economic importance of tomatoes 9
2.6. Constraints to tomato production in Kenya 13
2.7. Overview on tomato early blight 14
2.7.1. Description of tomato early blight causative agent 14
2.7.2. Infection of tomato plant by Alternaria solani 15
2.7.3. Symptoms caused by early blight on tomatoes 16
2.7.4. Disease cycle and source of inoculum 17
2.7.5. Environmental conditions favoring tomato early blight 18
2.7.6. Global distribution of tomato early blight 19
2.7.7. Management of tomato early blight 19
2.8. Overview on bio-control agents 21
2.8.1. Description and modes of action for Bacillus subtilis 21
2.8.2. Description and modes of action for Pseudomonas fluorescens 23
2.8.3. Description and modes of action of Trichoderma species 24

CHAPTER THREE: EFFECTS OF BIO-CONTROL AGENTS ON RADIAL GROWTH OF Alternaria solani
3.1. Abstract 27
3.2. Introduction 28
3.3. Materials and methods 29
3.3.1. Isolation and identification of Alternaria solani 29
3.3.2. Inoculum preparation 30
3.3.3. Raising of tomato seedlings 30
3.3.4. Pathogenicity test of Alternaria solani 30
3.3.5. Isolation and identification of Trichoderma isolates 31
3.3.6. Isolation of Pseudomonas fluorescens from commercial formulation 31
3.3.7. In vitro activity of bio-control agents against Alternaria solani 31
3.3.8. Data analysis 33
3.4. Results 34
3.4.1. Isolation and identification of Alternaria solani 34
3.4.2. Pathogenicity test for Alternaria solani 34
3.4.3. Isolation and identification of Trichoderma isolates 35
3.4.4. Activity of bio-control agents against Alternaria solani 36
3.4.4.1. Preliminary screening of Bacillus isolates against Alternaria solani 36
3.4.4.2. In vitro activity of Bacillus subtilis isolates selected from preliminary screening and
Pseudomonas fluorescens against Alternaria solani 37
3.4.4.3. Preliminary screening of Trichoderma isolates against Alternaria solani 38
3.4.4.4. In vitro activity of Trichoderma isolates selected from preliminary screening against
Alternaria solani 39
3.5. Discussion 41

CHAPTER FOUR: EFFECTIVENESS OF BIO-CONTROL AGENTS IN THE MANAGEMENT OF TOMATO EARLY BLIGHT IN THE FIELD AND THE GREENHOUSE
4.1. Abstract 45
4.2. Introduction 46
4.3. Material and methods 47
4.3.1. Description of the study sites 47
4.3.2. Sampling protocol for field evaluations 48
4.3.3. Production of culture filtrates from bio-control agents 48
4.3.3.1. Production of cultures filtrates from Bacillus subtilis isolates 48
4.3.3.2. Production of culture filtrates from Trichoderma isolates 49
4.3.4. Application of the treatments for greenhouse and field experiments 50
4.3.5. Field evaluation of bio-control agents 50
4.3.6. Greenhouse evaluation of bio-control agents 52
4.3.7. Assessment of plant growth parameters 53
4.3.8. Data analysis 53
4.4. Results 54
4.4.1. Effects of bio-control agents on tomato early blight percent disease incidence 54
4.4.2. Effects of bio-control agents on tomato early blight percent disease severity 57
4.4.3. Effects of bio-control agents on tomato early blight percent disease index 60
4.4.4. Effects of bio-control agents on tomato early blight area under disease progress curve 63
4.4.5. Effects of bio-control agents on tomato plant growth parameters 66
4.4.6. Effects of bio-control agents on the quantity of marketable tomato fruits 67
4.4.7. Correlations between tomato early blight disease parameters and plant growth parameters 69
4.4.8. Site interactions between Kabete Field Station and KALRO Mwea for tomato early blight
disease parameters and tomato plant growth parameters 73
4.5. Discussion 74

CHAPTER FIVE: GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1. General discussion 80
5.2. Conclusion 82
5.3. Recommendations 83
CHAPTER SIX: REFERENCES 84
APPENDICES 106





 
List of Tables

Table 2.1: Tomato production from the major tomato producing countries in the world 10
Table 2.2: Tomato production from the major tomato producing countries in the East African Community 11
Table 3.1: In vitro activity of Bacillus subtilis isolates selected from preliminary screening and Pseudomonas fluorescens against Alternaria solani 38
Table 3.2: In vitro activity of Trichoderma isolates selected from preliminary screening against Alternaria solani 40
Table 4.1: Effects of bio-control agents on tomato early blight percent disease incidence at Kabete Field Station 54
Table 4.2: Effects of bio-control agents on tomato early blight percent disease incidence at KALRO Mwea 55
Table 4.3: Effects of bio-control agents on tomato early blight percent disease incidence in the greenhouse 56
Table 4.4: Effects of bio-control agents on tomato early blight percent disease index at Kabete Field Station 61
Table 4.5: Effects of bio-control agents on tomato early blight percent disease index at KALRO Mwea. 62
Table 4.6: Effects of bio-control agents on tomato early blight percent disease index in the greenhouse 63
Table 4.7: Effects of bio-control agents on quantity of marketable tomato fruits (Tons/hectare) at Kabete Field Station 67 
Table 4.8: Effects of bio-control agents on the quantity of marketable tomato fruits (Tons/hectare) at KALRO Mwea 68
Table 4.9: Effects of bio-control agents on quantity of marketable tomato fruits (Kg/Plant) in the greenhouse 69
Table 4.10: Correlations between tomato early blight parameters and plant growth parameters at Kabete Field Station 70
Table 4.11: Correlations between tomato early blight parameters and plant growth parameters at KALRO Mwea 72




 
List of Figures

Figure 2.1: Tomato production trends in Kenya (FAOSTAT, 2019) 12
Figure 3.1: Mean diameter of Alternaria solani in the presence of Bacillus isolates 37
Figure 3.2: Mean Alternaria solani diameter in the presence of Trichoderma isolates 39
Figure 4.1: Effects of bio-control agents on tomato early blight disease progress curve at Kabete Field Station 58
Figure 4.2: Effects of bio-control agents on tomato early blight disease progress curve at KALRO Mwea 59
Figure 4.3: Effects of bio-control agents on tomato early blight disease progress curve in the greenhouse 60
Figure 4.4: Effects of bio-control agents on tomato early blight area under disease progress curve at Kabete Field Station 64
Figure 4.5: Effects of bio-control agents on tomato early blight area under disease   progress curve at KALRO Mwea 65
Figure 4.6: Effects of bio-control agents on tomato early blight area under disease progress curve in the greenhouse 66





 
List of Plates
Plate 2.1: Conidia of Alternaria solani (Kemmitt, 2002) 15
Plate 2.2: Tomato foliage affected by early blight (Kemmitt, 2002). 17
Plate 3.1: Morphological and cultural characteristics of isolated Alternaria solani 34
Plate 3.2: Early blight symptoms on infected tomato leaf and healthy tomato leaf 35
Plate 3.3: Morphological and cultural characteristics of Trichoderma isolates 36
Plate 3.4: Effects of bacterial antagonists on the radial growth of Alternaria solani 38
Plate 3.5: Effects of Trichoderma isolates on the radial growth of Alternaria solani 40
 




List of Appendices
Appendix 1: Analysis of variance for the percent disease incidence at Kabete Field Station 106
Appendix 2: Analysis of variance for the percent disease incidence at KALRO Mwea 106
Appendix 3: Analysis of variance for the percent disease incidence in the greenhouse 106
Appendix 4: Analysis of variance for the percent disease severity at Kabete Field Station 107
Appendix 5: Analysis of variance for the percent disease severity at KALRO Mwea 107
Appendix 6: Analysis of variance for the percent disease severity in the greenhouse 107
Appendix 7: Analysis of variance for the percent disease index at Kabete Field Station 108
Appendix 8: Analysis of variance for the percent disease index at KALRO Mwea 108
Appendix 9: Analysis of variance for the percent disease index in the greenhouse 108
Appendix 10: Analysis of variance for site interaction for area under disease progress curve 109
Appendix 11: Analysis of variance for site interaction for the percent disease incidence 109
Appendix 12: Analysis of variance for site interaction for the quantity of marketable fruits 109






 
LIST OF ABBREVIATIONS

% - Percent
µl - Microliter
µm   - Micrometer
0C - Degrees Celsius
AEZ II  - Agro-ecological zone II
AEZ III - Agro-ecological zone III
ANOVA  - Analysis of Variance
AUDPC  - Area Under Disease Progress Curve
Cm  - Centimeter
RCBD - Randomized Complete Block Design
CRD - Completely Randomized Design
FAOSTAT - Food and Agriculture Organization Statistics
Ha Hectare
HCDA - Horticultural Crop Development Authority
KALRO  - Kenya Agriculture 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
M - Meter
Ml - Milliliter
Mm - Millimeter
PDA - Potato Dextrose Agar
USA - United States of America
 





 
CHAPTER ONE: INTRODUCTION

1.1. Background information

Tomato (Solanum lycopersicum L.) a major vegetable grown worldwide (Monte et al., 2013), 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 enclose 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) (KALRO, 2005), pests and diseases (Mizubuti et al., 2007; Goufo et al., 2008). As an example, temperature and humidity fluctuations in 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 along with; cotton bollworms, whiteflies, melon thrips and tomato leaf miners (Engindeniz and Ozturk, 2013; Islam et al., 2013), significantly contribute to yield losses. 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 gardens (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 by application of a limited number of chemical compounds as a result of the withdrawal of some effective fungicides reported to have detrimental effects on the environment and on human health (Singh et al., 2011). Due to lack of suitable tomato germplasm, only a few varieties of tomato have been reported to be tolerant to early blight and can be integrated in the management of the disease (Sikora et al. 2007; Davies and Spiegel, 2011). Cultural practices are often involved in the management of early blight. These include,   sanitation, rotation of tomatoes with non-host crops for at least two to three years, use of pathogen-free seeds and transplants, proper irrigation strategies and maintenance of plant vigor via adequate application of nitrogen and phosphorus fertilizers (Chaerani and Voorrips, 2006; Li, 2012). Treatment of infected seeds with hot water at 50oC for 25 minutes can prevent seedborne infections (Neils et al., 2015). Control of early blight can also be achieved through use of plant extracts including; turmeric, garlic, lemon, ginger among others (Lengai, 2016).

Fluorescent Pseudomonas species, Bacillus species, Streptomyces species and Trichoderma species have been reported to have varied activities which hinder growth and development of many plant pathogens (Alabouvette et al., 2006). These BCAs are friendly to the environment and have minimal effects on non-target organisms, including humans, animals and host plants. The mechanisms of action through which BCAs protect plants from pathogen attack are numerous and differ from one BCA to another (Alabouvette et al., 2006). For instance, some BCAs are associated with the production of active extracellular compounds including siderophores which acts through biological suppression of several soil borne plant pathogens (Alabouvette et al., 2006). In some cases, BCAs have been associated with activities which trigger systemic resistance of host plants against pathogens. Trichoderma species have been reported to induce localized and/or systematic resistance to diseases through excretion of secondary metabolites that promote plant growth. These include; ethylene or terpenoid and phytoalexins among others (Alabouvette et al., 2006).


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 increased food insecurity 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.

Synthetic chemicals are being intensively applied by most farmers to lower the intensity of early blight and the accompanying crop losses given that tomato cultivars which are resistant to early blight, are of low agronomic or commercial quality (Yadav and Dabbas, 2012). In addition, the prolonged survival of early blight pathogen in the soil (Foolad et al., 2008) together with the limitation of lands for cultivation (Karuku et al., 2017), have made the rotation strategy not feasible. Furthermore, because of high demand of tomato in the country and pathogen resistance, farmers increase the rate of chemical application (Waiganjo et al., 2006) and often do not observe the required pre-harvest intervals leading to not only increased chemical residues on the produce but also increased production cost (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 due to misuse or abuse in their usage (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, reducing the 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 (Rhoda et al., 2006) but also to interceptions of produce with excessive chemical residues at the export market (Wandati, 2014). Therefore, 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 BCAs (Mamgain et al., 2013). Most BCAs are biodegradable, friendly to the environment and have minimal effects on humans and non- targeted organisms, including host plants and 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 BCAs along with locally available formulations of microorganisms are known to be effective in the managing early blight (Zhao et al., 2008). These include species of Trichoderma, Pseudomonas, Bacillus and Streptomyces genera among others. These microorganisms differ in their efficacy in managing tomato early blight (Tapwal et al., 2015).


1.3. Study justification

Integration of BCAs 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 BCAs in the management of early blight in the field. This study contributed to a better understanding of the effectiveness of Bacillus isolates, Pseudomonas fluorescens and Trichoderma isolates in managing tomato early blight in vitro and under greenhouse and field conditions.

Integration of effective BCAs in early blight management will contribute to a sustainable production of tomatoes through reduction of the dependence on synthetic 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 given that BCAs are biodegradable and leave no residues on the produce (Gupta et al., 2014). This will result in reduced interceptions of tomato produce at the export market. Integration of BCAs in the management of tomato early blight will also contribute to a better conservation of the environment given that BCAs do not pollute the environment as they are biodegradable. BCAs isolated 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 BCAs in managing early blight for sustainable production of tomatoes.

1.4.2. Specific objectives

To evaluate the antagonistic effects of BCAs namely; Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens on in vitro growth of A. solani.

To evaluate the effectiveness of BCAs (Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens) in managing tomato early blight under field and greenhouse conditions.

1.5. Hypotheses

BCAs namely; Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens have significant antagonistic effects on in vitro growth of A. solani.

BCAs (Trichoderma spp., Bacillus spp. and Pseudomonas fluorescens) are effective in managing early blight of tomatoes under field and greenhouse conditions.
 

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