ABSTRACT
Greenhouse experiments were conducted consisting of 5 treatments: Metarhizium anisopliae (6.0 x 103 cfu /ml) formulated with Nonylphenol ethoxylate, Metarhizium anisopliae (6.0 x 103 cfu /ml) conidia alone, Nonylphenol ethoxylate alone, compared to a standard pesticide having Indoxacarb 85g/L and Emmamectin benzoate 15g/L. The field experiment consisted of Metarhizium anisopliae (6.0 x 103 cfu /ml) in Nonylphenol ethoxylate, Metarhizium anisopliae (6.0 x 103 cfu /ml) conidia alone, Nonylphenol ethoxylate alone and a standard pesticide Indoxacarb 85g/L with Emmamectin benzoate 15g/L.
Rio grande variety had the largest leaf area in the two seasons but this was comparable with that recorded for M82, Eden, Cal J and Moneymaker. The evaluated tomato varieties retained viable conidia of Metarhizium anisopliae on their leaves but Rio grande variety significantly (p<0.05) retained the most colonies. Adjuvants, Nonylphenol ethoxylate 15% and Tween 80 significantly (p<0.05) increased the radial growth of Metarhizium anisopliae ICIPE 69 and ICIPE 78 isolates, compared to control whereas liquid soap significantly (p<0.05) prevented the radial growth of Metarhizium anisopliae ICIPE 69 and ICIPE 78 isolates at all concentrations when compared to control.
Greenhouse experiments were conducted consisting of 5 treatments: Metarhizium anisopliae (6.0 x 103 cfu /ml) formulated with Nonylphenol ethoxylate, Metarhizium anisopliae (6.0 x 103 cfu/ml) conidia alone, Nonylphenol ethoxylate alone, compared to a standard pesticide having Indoxacarb 85g/L and Emmamectin benzoate 15g/L. The field experiment consisted of Metarhizium anisopliae (6.0 x 103 cfu /ml) in Nonylphenol ethoxylate, Metarhizium anisopliae (6.0 x 103 cfu /ml) conidia alone, Nonylphenol ethoxylate alone and a standard pesticide Indoxacarb 85g/L with Emmamectin benzoate 15g/L.
Rio grande variety had the largest leaf area in the two seasons but this was comparable with that recorded for M82, Eden, Cal J and Moneymaker. The evaluated tomato varieties retained viable conidia of Metarhizium anisopliae on their leaves but Rio grande variety significantly (p<0.05) retained the most colonies. Adjuvants, Nonylphenol ethoxylate 15% and Tween 80 significantly (p<0.05) increased the radial growth of Metarhizium anisopliae ICIPE 69 and ICIPE 78 isolates, compared to control whereas liquid soap significantly (p<0.05) prevented the radial growth of Metarhizium anisopliae ICIPE 69 and ICIPE 78 isolates at all concentrations when compared to control.
The findings of the laboratory assays show that Metarhizium anisopliae significantly (p<0.05) caused mortality to Tuta absoluta larvae. One hundred percent (100% ) mortality of Tuta larvae was achieved within 36 hrs of treatment of larvae treated with Metarhizium anisopliae 1.2 x 106 cfu /ml. Metarhizium anisopliae 1.2 x 103 cfu /ml , 1.2 x 104 cfu /ml and 1.2 x 106 cfu /ml did not differ in effect 60hours after treating the larvae. In the greenhouse experiment, no differences were noticed in the population of larvae in the different treatments except in the 8th week where Metarhizium anisopliae & NPnEO recorded the least mean population of larvae and was significantly (P<0.05) lower than control but comparable with the rest of the treatments. The resultant yield recorded show that the standard pesticide, Indoxacarb 85g/L and Emmamectin Benzoate 15g/L, significantly (p<0.05) had the highest yield compared to control but it was comparable to the second highest yield recorded in Metarhizium anisopliae & NPnEO. Control had the most larval population, most damaged tomatoes and lowest yield recorded which were significantly (p<0.05) different from the treated tomatoes. In the open field, Indoxacarb 85g/L and Emmamectin Benzoate 15g/L had the least mean population recorded which was significantly different (p<0.05) from control but not from Metarhizium anisopliae and Nonylphenol ethoxylate 15% treatment with the second lowest population. The resultant yields were significantly (p<0.05) higher compared to control with the least damage percentage of the fruits in both Indoxacarb 85g/L and Emmamectin Benzoate 15g/L and Metarhizium anisopliae and Nonylphenol ethoxylate 15% treatments. Metarhizium anisopliae can be used to manage T. absoluta under field and greenhouse conditions and that Nonylphenol ethoxylate 15% and Tween 80 as adjuvants can be used to formulate and facilitate distribution of the conidia and enhance growth for fast establishment on the crop.
TABLE OF CONTENTS
DECLARATION i
PLAGIARISM DECLARATION ii
DEDICATION iii
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS v
LIST OF TABLES ix
LIST OF ABBREVIATIONS AND ACRONYMS xii
GENERAL ABSTRACT xiii
CHAPTER ONE
INTRODUCTION
1.1 Background Information 1
1.2 Problem Statement 2
1.3 Justification 3
1.4 Objectives of the study 4
1.4.1 Broad objective 4
1.4.2 Specific objectives 4
1.5 Hypotheses 5
CHAPTER TWO: LITERATURE REVIEW
2.1 Economic importance of T. absoluta 6
2.2 Tomato production practices 7
2.4 Biology of Tuta absoluta 8
2.5 Spread of Tuta absoluta 11
2.6 Management of Tuta absoluta 13
2.6.1 Monitoring the pest population 13
2.6.2 Cultural control methods 13
2.6.3 Biological control methods 14
2.6.4 Chemicals used for controlling Tuta absoluta 15
2.6.5 Botanical pesticides used against Tuta absoluta 15
2.6.6 Semiochemicals used to manage Tuta absoluta 16
2.6.7 Integrated pest management strategy for Tuta absoluta 16
2.7 Mode of action of Metarhizium anisopliae 16
2.9 Other Metarhizium species and their uses in agriculture 19
CHAPTER THREE
THE EFFECTS OF REGULAR APLLICATION AND LEAF MORPHOLOGY ON METARHIZIUM ANISOPLIAE CONIDIA RETENTION
3.1 Introduction 22
3.2 Materials and methods 23
3.2. 1 Site description 23
3.2.2 Tomato varieties used in the experiments 23
3.2.3 Inoculation of the leaves with Metarhizium anisopliae conidia 23
3.2.4 Leaf sampling and incubation 23
3.2.5 Leaf area determination 24
3.2.6 Determination of the number of Metarhizium anisopliae colonies 24
3.2.7 Data analysis 24
3.3 Results 25
3.3.1 Effect of tomato varieties and on the growth characteristics of Metarhizium anisopliae 25
3.4 Discussion 29
3.5 Conclusions 30
CHAPTER FOUR
4.1 Introduction
4.2.1 Treatment description 34
4.2.2 Morphological identification of Metarhizium anisopliae cultures and data collection 35
4.2.3 Data analysis 35
4.3 Results 35
4.3.1 Pure culture growth 35
4.3.2 Effect of adjuvants on Metarhizium anisopliae ICIPE 69 radial growth 36
4.4 Discussion 47
4.5 Conclusions 48
CHAPTER FIVE
EFFECT OF METARHIZIUM ANISOPLIAE IN THE MANAGEMENT OF TOMATO LEAF MINER, TUTA ABSOLUTA
ABSTRACT 49
5.1 Introduction 50
5.2 Materials and methods 51
5.2.1 Selection of the study site 51
5.2.2 Entomopathogenic fungi conidia production 51
5.2.3 Tuta absoluta larvae 52
5.2.4 Laboratory bioassays of Metarhizium anisopliae 52
5.2.5 Experimental site description for field and greenhouse experiments 53
5.2.6 Tomato seedlings establishment for greenhouse and field experiment 53
5.2.7 Experimental treatment description for greenhouse experiment 54
5.2.8 Experimental treatment description for the field experiment 54
5.2.9 Data analysis 55
5.3 Results 56
5.4 Discussion 65
5.4.1 Potential of Metarhizium anisopliae in the management of tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) under laboratory conditions 65
5.4.2 Effect of Metarhizium anisopliae as a biological control agent in the management of Tuta absoluta within greenhouse and field conditions 66
CHAPTER SIX
GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
6.1 General discussion 68
6.2 Conclusions 69
6.3 Recommendations 70
Reference 71
LIST OF TABLES
Table 3. 1: Mean tomato leaf area of five selected tomato varieties season one 25
Table 3. 2: Mean number of colony forming units retained on tomato leaves of five selected tomato varieties season one 25
Table 3. 3: Pearson Correlation matrix of the tomato leaf area, Metarhizium anisopliae colony forming unit number, sampling period and tomato variety for season one 27
Table 3. 4: Mean tomato leaf area of five selected tomato varieties in season two 28
Table 3. 5: Mean colony numbers retained on tomato leaves of five selected tomato varieties season two 28
Table 3. 6: Pearson Correlation matrix of the tomato leaf area, Metarhizium anisopliae
colony number, sampling period and tomato variety for season two 29
Table 4. 1: Mean radial growth of Metarhizium anisopliae ICIPE 69 in mm as affected by Nonylphenol ethoxylate 15% in run one 37
Table 4. 2:Mean radial growth of Metarhizium anisopliae ICIPE 69 in mm as affected by Nonylphenol ethoxylate 15% in run two 38
Table 4. 3:Mean radial growth of Metarhizium anisopliae ICIPE 69 in mm as affected by Tween 80 in run one 38
Table 4. 4:Mean radial growth of Metarhizium anisopliae ICIPE 69 in mm as affected by tween 80 in run two 39
Table 4. 5:Mean radial growth of Metarhizium anisopliae ICIPE 69 in mm as affected by tween 80 in run two 40
Table 4. 6:Mean radial growth of Metarhizium anisopliae ICIPE 69 in mm as affected by liquid soap in run two 40
Table 4. 7:Mean radial growth of Metarhizium anisopliae ICIPE 78 in mm as affected by Nonylphenol ethoxylate 15% in run one 42
Table 4. 8:Mean radial growth of Metarhizium anisopliae ICIPE 78 in mm as affected by Nonylphenol ethoxylate 15% in run two 42
Table 4. 9:Mean radial growth of Metarhizium anisopliae ICIPE 78 in mm as affected by tween 80 in run one 44
Table 4. 10:Mean radial growth of Metarhizium anisopliae ICIPE 78 in mm as affected by Tween 80 in run two 45
Table 4. 11:Mean radial growth of Metarhizium anisopliae ICIPE 78 in mm as affected by liquid soap in run one 45
Table 4. 12: Mean radial growth of Metarhizium anisopliae ICIPE 78 in mm as affected by liquid soap in run two 46
Table 5. 1: Mean percentage in cumulative mortality of Tuta absoluta larvae inoculated with
Metarhizium anisopliae under laboratory conditions run one 57
Table 5. 2: Mean percentage in cumulative mortality of Tuta absoluta larvae inoculated with
Metarhizium anisopliae under laboratory conditions run two 58
Table 5. 3: Mean number of Tuta absoluta larvae as affected by Metarhizium anisopliae Nonylphenol ethoxylate and Indoxicarb combined with Emamectin Benzoate undergreenhouse conditions (Season one) 60
Table 5. 4: Mean number of Tuta absoluta larvae as affected by Metarhizium anisopliae Nonylphenol ethoxylate and Indoxicarb combined with Emamectin Benzoate under greenhouse conditions (Season two) 61
Table 5. 5: Mean weight of the greenhouse tomato fruits damaged and not damaged by Tuta absoluta following treatment with Metarhizium anisopliae Nonylphenol ethoxylate and Indoxicarb combined with Emamectin Benzoate (Season one) 62
Table 5. 6: Mean weight of the greenhouse tomato fruits damaged and not damaged by Tuta absoluta following treatment with Metarhizium anisopliae Nonylphenol ethoxylate and Indoxicarb combined with Emamectin Benzoate (Season two) 63
Table 5. 7: Mean number of Tuta absoluta larvae affected by Metarhizium anisopliae Nonylphenol ethoxylate and Indoxicarb combined with Emamectin Benzoate under field conditions 64
Table 5. 8: Mean weight of the field tomato fruits damaged and not damaged by Tuta absoluta
as effected by Metarhizium anisopliae Nonylphenol ethoxylate and Indoxicarb combined with Emamectin Benzoate 65
LIST OF FIGURE
Fig 2. 1: Tuta absoluta male genitalia (Visser et al., 2017) 9
Fig 2. 2: Map showing the spread of Tuta absoluta in Africa (Source: Mansour et al., 2018) 12
Fig 2. 3: The host infection pathway of Metarhizium spp (Lovett and Leger, 2015) 17
Fig 4. 1: Image (a) pure culture of Metarhizium anisopliae strain ICIPE 78 image (b) Metarhizium anisopliae strain ICIPE 69 36
Fig 5. 1:Tomato fruit damaged by Tuta absoluta larvae. Photo B; Tomato leaf damaged by Tuta absoluta. Photo C; Plants damaged by Tuta absoluta in a greenhouse 59
LIST OF ABBREVIATIONS AND ACRONYMS
ANOVA: Analysis of Variance
LC50: Lethal Concentration 50
IPM: Integrated Pest Management
ICIPE: International Centre of Insect Physiology and Ecology
CFU: Colony forming unit
CV: Correlation of Variation
PDA: Potato Dextrose Agar
TDTA: (3E, 8Z, 11Z)-3,8,11-tetradecatrienyl acetate
EPN: Entomopathogenic Nematodes
IPPC: International Plant Protection Convention
J1: First Juvenile
NPnEO: Nonylphenol ethoxylate
N: Population size
USD: United States Dollar
CHAPTER ONE
INTRODUCTION
1.1 Background Information
Tomato (Solanum lycopersicum L) is horticulture crop valued for its fruit. It can be used as food and as a commercial crop. The tomato plant has botanical characteristics such as fleshy fruit, a sympodial shoot, and compound leaves. It belongs to the large Solanaceae family. Within Kenya, tomato is ranked second among other vegetables in terms of value and production next to irish potato (Sigei et al., 2014). It contributes 14% of the total vegetables produced and 6.72% of the total horticultural crops (Ochilo et al., 2019).
The Food and Agriculture Organization (FAO) reported in 2021 global land area under tomato cultivation was 5.03 million hectares which produced 180.8 million kilos (FAO, 2021). The leading tomato producer in the world is China producing 62.9 million kilos of the total worldwide production( FAO, 2021). It is followed by India, the United States of America, Turkey, and Egypt ( FAO, 2021). A land area of 1.6 million ha is used to cultivate tomatoes in Africa. These yielded 21.7 metric tons of tomatoes in 2019. The top tomato producer in Africa is Egypt with an average production of 6.8 million kilos in 2019 followed by Nigeria, Tunisia, and Morocco. Kenya only produces 0.2 % of the tomatoes produced globally (FAO, 2021).
Production of the tomatoes can be done either in an open field or under greenhouse conditions. Production under field conditions accounts for 95%, while greenhouse production contributes 5% of the total tomato produced in the country. Kenya is ranked sixth among the tomato-producing countries in Africa. The total production is estimated to be 397,007 tones. Kirinyaga, Kajiado, and Taita Taveta are the major tomato-producing counties in Kenya (Geofreyet al., 2014). Abiotic factors, pest and diseases are the major group of constrains that affect tomato production (Ochilo et al, 2019). The main abiotic constrains facing tomato production are water availability and soil fertility(Karuku et al., 2017).
1.2 Problem Statement
Tomato (Solanum lycopersicum L) is a popular vegetable in Kenya (Sigei et al., 2014). Tomato is grown for income generation and used for food (Ochilo et al., 2019). However productivity of tomato is affected by pests and diseases (Ochilo et al, 2019). Tuta absoluta the main pest affecting tomato production. It can cause up to 100% yield loss (Assinapol, 2020). Tuta absoluta is an invasive pest from South America (Tropea et al., 2012). Direct losses of tomato yield can be incurred through further reduction in production rate when the pest gains entry into a new area (Venkatramanan et al., 2019). Even though some management measures have been developed, the cryptic nature of Tuta absoluta makes it challenging to manage in places where the pest has been reported (Biondi et al., 2018). The pest is reportedly causing indirect effects and has affected farmers by increasing cost of production (Hill et al., 2019). In Kenya, the impact of Tuta absoluta and other invasive pests on the well-being of people is primarily felt in the rural area, where there are people depend mainly on agriculture (Shackleton et al., 2019).
Small scale farmers prefer the use of chemical pesticides for its management. This choice, although feasible, is threatened by the ability of Tuta absoluta to develop chemicals resistance (Peris et al., 2018). The Brazilian population of Tuta absoluta has shown resistance to abamectin, cartap, and permethrin (Siqueira et al., 2000).
Similarly, some Tuta absoluta populations in Greece also exhibited resistance to diamide pesticides (Roditakis et al., 2015). In Argentina Tuta absoluta populations have shown resistance to deltamethrin because of pesticide selection pressure (Lietti et al., 2005). The resistance experienced in some geographical regions can be dispersed into new areas through pest movement from a part that has resistant populations to new places (Campos et al., 2015). To minimize resistance, other management practices like the use of entomopathogenic fungi should be developed for integrated pest management. Studies have confirmed that pesticides can be associated with the cause of various diseases like cancer, leukemia, and asthma. The integration of entomopathogenic fungi like Metarhizium anisopliae in the management practice will help in reducing exposure to pesticides.
1.3 Justification
Tomato (Solanum lycopersicum L) is a significant vegetable in Kenya (Sigei et al., 2014). Tomato is cultuvated as a cash crop and used for food (Ochilo et al., 2019). Tomato consumption reduces the risk of having some diseases like cancer (Salehi et al., 2019).More than 30 % of tomato farmers in Kenya have reported the effects of Tuta absoluta on their farms (Ochilo et al., 2019 ). The pest has a very rapid dispersal mechanism as it can drift with the help of wind spreading to new areas (Tonnang et al., 2015). This type of dispersal renders quarantine measures ineffective. The pest also has a very high reproductive capacity allowing it to quickly build up populations beyond the economic threshold level within a short period (Tropea et al., 2012). Trading of infested tomato fruits has also aided the fast spread of the pest. The ability of the problem to survive and adapt to changes in the ecological conditions and feed on multiple crop hosts, including weeds, make it difficult to control (Illakwahhi et al., 2017). Yield losses inflicted by Tuta absoluta are up to 100% when the conditions are conducive for the pest, and proper management methods are not implemented (Assinapol, 2020). Presence of the pest causes diversion of capital from meeting production costs to putting in place management strategies; it also raises tomato production costs through increased exposure to chemical pesticides due to the increased amount of pesticides needed to manage the pest (Aigbedion-Atalor et al., 2019). Efficacy of entomopathogenic fungi like Metarhizium anisopliae is not widely tested. Although, the fungus is reported to attack both eggs and the larvae of the Tuta absoluta (Tadele and Emana, 2017). Metarhizium anisopliae has also been shown to effectively manage diamond back moth (Plutella xylostella) (Shehzad et al., 2021). More studies need to be done in Kenya to confirm the potential of Metarhizium anisopliae in management of Tuta absoluta. Other Metarhizium species have also been successfully isolated from other Lepidopteran pests like larvae of Denrolimus species. Therefore, further studies need to be done to confirm the efficacy of M. dendrolimatilis in the management of Dendrolimus and other related pests (Chen et al., 2017). Studies have been done to determine the effect of leaf growth on the retention and distribution of Metarhizium anisopliae conidia on plant leaves (Inyang et al., 1998). This study aims to precisely evaluate the effect of tomato leave growth on the retention of Metarhizium anisopliae conidia. This study seeks to assess the effect of Metarhizium anisopliae in the management of Tuta absoluta to reduce losses associated with the pest. The findings will contribute to the available options for managing the pest. Determination of the influence of adjuvants on the growth of Metarhizium anisopliae will also inform the best formulations possible for effective Tuta management.
1.4 Objectives of the study
1.4.1 Broad objective
To contribute to sustainable management of Tuta absoluta through use of environmentally friendly biopesticides for improved tomato productivity.
1.4.2 Specific objectives
The specific objectives of the study were:
I. To determine the effect of tomato varieties on the regular application of Metarhizium anisopliae conidia.
II. To assess the effect of adjuvants on Metarhizium anisopliae growth.
III. To evaluate the efficacy of Metarhizium anisopliae in managing Tuta absoluta infesting tomato.
1.5 Hypotheses
I. Regular application of Metarhizium anisopliae is not affected by tomato leaf morphology.
II. Metarhizium anisopliae colonies growth is not affected by selected adjuvants, Nonylphenol ethoxylate 15%, Tween 80 and liquid.
III. The application of Metarhizium anisopliae is not effective in managing of Tuta absoluta.
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