ABSTRACT
The Liriomyza leafminer flies (LMF) are invasive pests that attack several horticultural crops one of which is the common bean Phaseolus vulgaris. Endophytic fungi have been shown to deter performance, oviposition and feeding of these Liriomyza flies in beans. However, the clear infection mechanism or pathways of inoculated fungal endophytes against LMF has not been well established. This study, therefore, investigated the induction of active compounds in the endophytically colonized P. vulgaris plants, as defense mechanism against herbivorous insects. Two fungal isolates Beauveria bassiana (G1LU3) and Hypocrea lixii (F3STI) were used for artificial seeds inoculation. Inoculation was done by soaking P. vulgaris seeds in these fungal conidial suspensions prior to planting. Colonization of the different plant parts was assessed to confirm the endophytic property of the inoculated fungi. Volatiles emitted from endophytically colonized P. vulgaris were collected into adsorbent Super-Q traps and evaluated using gas chromatography combined with mass spectrometry (GC-MS). Liquid extraction was conducted with methanol and dichloromethane solvents followed by chromatographic analysis. Methanol and dichloromethane extracts of endophytically colonized plants were screened for their efficacy against leafminer fly, Liriomyza huidobrensis Blanchard and Fall Armyworm (FAW), Spodoptera frugiperda larvae using leaf dipping method and topical application, respectively. The two isolates successfully colonized the entire host plant (roots, stems, and leaves) with significant variation (F = 19.22, Df = 6, P = 0.01) between fungal isolates and the controls. The results also showed qualitative differences in the volatile profiles between the control P. vulgaris plants, endophytically colonized plants and insect damaged plants. The volatile blend from the control P. vulgaris plants consisted mainly of the following compounds: meta cresol (52) (35%) and para cresol (53) (64%). The most abundant emissions from the insect damaged plants included terpinen- 4-ol (59) (28%) and benzaldehyde dimethyl acetal (61) (21%). The H. lixii inoculated plants consistently released m-cresol (52) (72%) and p-cresol (53) (13%). The most abundant emissions from H. lixii colonized plants with insect damage included benzaldehyde dimethyl acetal (61) (6%), butylated hydroxytoluene (63) (4%) and methyl salicylate (76) (3%). Beauveria bassiana colonized plants released the highest number of volatiles with the most abundant including benzaldehyde dimethyl acetal (61) (6%), butylated hydroxytoluene (63) (5%), ()- γ-bisabolene (86) (4%), methyl salicylate (76) (4%) and 4,8,12-trimethyl-1,3(),7(),11-tridecatetraene (87) (4%). The most abundant volatiles detected from B. bassiana colonized plants with insect damage include benzaldehyde dimethyl acetal (61) (16%) and butylated hydroxytoluene (63) (4%). Qualitative and quantitative differences were also reported between solvent extracted compounds detected from control P. vulgaris plant extracts and fungal extracts. All extracts except the B. bassiana fungal extract contained the hexadecenoic acid methyl ester (92) (18%) and 9,12- octadecanoic acid methyl ester (93) (13%). Among the extracts’ compounds, the most abundant included 9,12,15-octadecatrienoic acid methyl esters (94) (70%) and 1-hydroxy-4- methylanthraquinone (102) (58%). The bioassay results showed significant differences between the liquid crude extracts from endophytically colonized plants and the controls plants with regard to the effects on pupation of 2nd instar LMF larvae (F = 4.33, Df = 6, P = 0.03) and adult Liriomyza flies emergence of LMF pupae (F = 5.4, Df = 6, P = 0.02). The survival of the 1st instar FAW larvae dipped into the methanolic endophytically colonized plant extracts was also significantly reduced (F = 3.7, Df = 8, P = 0.001) as compared to the controls. The extracts of B. bassiana inoculated plants were the most lethal to FAW larvae with median lethal time (LT50) of 4.42 days. Profiled compounds have previously been identified in plant extracts and volatiles with activities including insect repellence, predator attractant and insecticidal properties. Colonization of the host plant P. vulgaris therefore triggers production of compounds for defense against herbivorous insects including the leafminer and fall armyworm. The compounds deter feeding and oviposition of the pest through insect repellence and predator attraction. The toxic compounds also infect the pest through effects on its physiology. However, the identities of compounds were based on comparisons of mass spectra available from an MS library. Therefore, some of the identifications may be tentative especially for volatiles with trans-cis isomers. This study demonstrated the high potential of endophytic fungi H. lixii and B. bassiana, to induce mainly specific defense compounds in the common bean P. vulgaris that can be used against the Liriomyza leafminers and Fall Armyworm.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
LIST OF APPENDICES x
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF ABBREVIATIONS AND ACRONYMS xiii
CHAPTER ONE: INTRODUCTION
1.1 Background Information 1
1.2 Statement of the Problem 3
1.3 Objectives 4
1.3.1 General Objective 4
1.3.2 Specific Objectives 4
1.4 Justification of the Study 4
CHAPTER TWO: LITERATURE REVIEW
2.1 Host Plant: Common Bean Phaseolus vulgaris 5
2.2 Species Composition of Liriomyza Leafminers 5
2.3 Ecology and Geographical Distribution of Liriomyza Species 6
2.4 Biology of Liriomyza species 6
2.5 Economic Impact of Leafminers 7
2.6 Fall Armyworm 8
2.7 Management Strategies for Leafminers and Fall Armyworm 9
2.7.1 Cultural practices 9
2.7.2 Chemical control 9
2.7.3 Biological control 10
2.8 Fungal Endophytes in Pest Management 11
2.9 Active Compounds Isolated from Endophytic Fungi 13
2.10 Chemical Plant Defense 15
2.10.1 Terpenes for Plant Defense 16
2.10.2 Phenolics For Plant Defense 17
2.10.3 Alkaloids for Plant Defense 19
2.11 Mechanism of Plant Defense Compounds 20
2.11.1 Production of Prussic Acid by Wounded Plants 21
2.11.2 Production of Volatile Mustard Oils 21
2.11.3 Production of Isoprenoids 21
2.11.4 Production of Phenols 22
CHAPTER THREE: MATERIALS AND METHODS
3.1 General 24
3.2 Fungal Cultures and Suspensions Preparation 24
3.3 Plant Inoculation and Endophytes Colonization Assessment 25
3.4 Insect Rearing and Treatment 26
3.5 Collection and Analysis of Volatiles 27
3.6 Solvent Extraction 27
3.7 Chromatography 28
3.8 Gas Chromatography Screening of Plant Extracts 28
3.9 High Pressure Liquid Chromatography Screening of Plant Extracts 29
3.10 Plant Extracts Bioassays 29
3.11 Statistical Analyses 30
CHAPTER FOUR: RESULTS AND DISCUSSION
4.1 Colonization Assessment of Phaseolus vulgaris Plants Inoculated with Fungi 32
4.2 Secondary Metabolites Characterized from Phaseolus vulgaris Plants 33
4.2.1 Control Phaseolus vulgaris Plants Volatiles Analysis 34
4.2.2 Leafminer-Damaged Phaseolus vulgaris Plants Volatiles Analysis 35
4.2.3 Hypocrea lixii Inoculated Phaseolus vulgaris Plants Volatiles Analysis 39
4.2.4 Hypocrea lixii Inoculated Phaseolus vulgaris and Leafminer-Damaged Plants Volatiles Analysis 41
4.2.5 Beauveria bassiana Inoculated Phaseolus vulgaris Plants Volatiles Analysis 45
4.2.6 Leafminer-Damaged Beauveria bassiana Inoculated Phaseolus vulgaris Plants Volatiles Analysis 48
4.3 Thin Layer Chromatography 51
4.4 Metabolic Screening with Gas Chromatography-Flame Ionization Detector 53
4.4.1 Controls Plant Extracts Compared to insect (leafminer)-damaged Plant Extracts . 53
4.4.2 Controls Plant Extracts Compared to Hypocrea lixii F3ST1 Inoculated Plant Extracts 56
4.4.3 Controls Plant Extracts Compared to Beauveria bassiana G1LU3 Inoculated Plant Extracts 58
4.5 Metabolic Screening of Liquid Extracts with Gas Chromatography-Mass Spectrometer 60
4.6 High Pressure Liquid Chromatogram Profile Comparisons 65
4.7 Effects of Plant Extracts on Pupation of 2nd Instar Leafminer Larvae 66
4.8 Effects of Plant Extracts on Emergence of Leaf miner Adult Flies 68
4.9 Effects of Plant Extracts on 1st Instar FAW Larvae 70
CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS
Conclusion
Recommendations
REFERENCES 76
APPENDICES 89
Mass spectra of identified compounds 89
LIST OF APPENDICES
Appendix 1: Mass spectrum of meta cresol 89
Appendix 2: Mass spectrum of para cresol 89
Appendix 3: Mass spectrum of phellandrene 90
Appendix 4: Mass spectrum of terpinene 90
Appendix 5: Mass spectrum of sabinene hydrate cis 91
Appendix 6: Mass spectrum of benzaldehyde dimethyl acetal 91
Appendix 7: Mass spectrum of camphor 92
Appendix 8: Mass spectrum of terpinene-4-ol 92
Appendix 9: Mass spectrum of heneicosane 93
Appendix 10: Mass spectrum of caryophyllene 93
Appendix 11: Mass spectrum of butylated hydroxytoluene 94
Appendix 12: Mass spectrum of tetramethyl cyclohexane 94
Appendix 13: Mass spectrum of phenol 95
Appendix 14: Mass spectrum of benzyl alcohol 95
Appendix 15: Mass spectrum of 3-methylanisole 96
Appendix 16: Mass spectrum of ocimene 96
Appendix 17: Mass spectrum of naphthalene 97
Appendix 18: Mass spectrum of methyl salicylate 97
Appendix 19: Mass spectrum of heptadecane 98
Appendix 20: Mass spectrum of tetradecane 98
Appendix 21: Mass spectrum of propyl butanoate 99
Appendix 22: Mass spectrum of cedrene 99
Appendix 23: Mass spectrum of dibutyl phthalate 100
Appendix 24: Mass spectrum of bisabolene 100
LIST OF TABLES
Table 2.1. Colonization of various plants from artificial inoculation of entomopathogenic fungi. 12
Table 4.1. Volatiles identified from Control Phaseolus vulgaris plants 34
Table 4.2. Volatiles identified from Leafminer-damaged Phaseolus vulgaris plants 35
Table 4.3. Volatiles identified from Hypocrea lixii inoculated Phaseolus vulgaris plants 39
Table 4.4. Volatiles identified from Hypocrea lixii inoculated and Leafminer-damaged Phaseolus vulgaris plants 42
Table 4.5. Volatiles identified from Beauveria bassiana inoculated Phaseolus vulgaris plants . 46 Table 4.6. Volatiles identified from Leafminer-damaged Beauveria bassiana inoculated Phaseolus vulgaris plants 49
Table 4.7. Mean pupation of 2nd instar leafminer larvae dipped in Phaseolus vulgaris plant extracts colonized with fungal isolates Hypocrea lixii F3ST1 and Beauveria bassiana G1LU3. 67 Table 4.8. Mean emergence of 2nd instar leaf miner larvae dipped in Phaseolus vulgaris plant extracts colonized with fungal isolates Hypocrea lixii and Beauvaria bassiana (F3ST1 and (G1LU3) 69
Table 4.9. Mean mortality of 1st instar Fall Armyworm larvae dipped in Phaseolus vulgaris plant extracts colonized with fungal isolates Hypocrea lixii F3ST1 and Beauveria bassiana G1LU3. 71 Table 4.10. Median lethal time (LT50) 7 days post treatment of 1st instar FAW larvae dipped in plant extracts 72
LIST OF FIGURES
Figure 2.1. Liriomyza leafminer fly on okra leaf (left), newly hatched larvae (middle), and pupa within tunnel of onion (right) (Source: https://infonet-biovision.org ) 7
Figure 2.2. Damage caused by leafminers (Source: https://infonet-biovision.org/ ) 8
Figure 4.1. Percentage colonization of leaf, stem and root parts of Phaseolus vulgaris plants by endophytic isolates of Hypocrea lixii F3ST1, and Beauveria bassiana G1LU3. Means with the same letter are not significantly different. 32
Figure 4.2. Control Phaseolus vulgaris plants chromatogram 34
Figure 4.3. Insect damaged Phaseolus vulgaris plants chromatogram 36
Figure 4.4. Hypocrea lixii inoculated Phaseolus vulgaris plants chromatogram 40
Figure 4.5. Leafminer-damaged Hypocrea lixii inoculated Phaseolus vulgaris plants chromatogram 42
Figure 4.6. Beauveria bassiana inoculated Phaseolus vulgaris plants chromatogram 46
Figure 4.7. Leafminer-damaged Beauveria bassiana inoculated Phaseolus vulgaris plants chromatogram 49
Figure 4.8. Comparison TLC plates for solvent system 30% ethyl acetate in n-hexane. Plates observed with the naked eye (A), plates exposed to visualization agent iodine (B) and plates observed under UV light (C) and plates screened with sulfuric acid (D). C – control plant extracts, IG1 – Beauveria bassiana G1LU3 inoculated plant extracts, IF3 – Hypocrea lixii F3ST1 inoculated plant extracts, Fs – Fungal extract (Beauveria bassiana), F3 – Fungal extract (Hypocrea lixii). 52
LIST OF ABBREVIATIONS AND ACRONYMS
a.s.l Above sea level
CDCl3 Deuterated trichloro methane EPF Entomopathogenic Fungi
FSNWG Food Security & Nutrition Working Group FAW Fall Armyworm
GDP Gross Domestic Product
HPLC High Pressure Liquid Chromatography
ICIPE International Centre of Insect Physiology and Ecology KNBS Kenya National Bureau of Statistics
LMF Leafminer flies
LT50 Median lethal time
MS Mass Spectroscopy
PTLC Preparative Thin Layer Chromatography UNEP United Nations Environment Programme
CHAPTER ONE
INTRODUCTION
1.1 Background Information
Ornamental crops and vegetables production contribute significantly to the economic development in Africa, mostly as a result of high financial returns and employment creation (Ekesi et al., 2016). The agricultural sector in Kenya is the largest contributing up to 24% of the economy (UNEP, 2018). The horticultural division is among the rapidly growing sectors in Kenya with exports growing by 29.3% from $28.7 billion in 2017 to $37.1 billion in the 2018 Gross Domestic Product (GDP) review (KNBS, 2018). In the efforts to improve horticultural productions, it has been observed that the productivity of crop systems in Kenya is constrained by factors including outbreaks and high prevalence of pests and diseases (Mangeni et al., 2014). The Liriomyza leafminer flies (LMF) are polyphagous invasive pest species that attack common bean crops in Kenya. Leafminer larvae mines under the leaf surface creating winding trails on the foliage; and a heavy infestation causes degradation of plant tissues and eventual death (Gathage et al., 2016). Three key Liriomyza species, L. sativae (Blanchard), L. trifolii (Burgess) and L. huidobrensis (Blanchard) have been identified in Kenya, with L. huidobrensis (Blanchard) being identified as the most devastating and most predominant. Crop losses due to LMF range between (20%-100%) depending on the affected crops or regions (Chabi-Olaye et al., 2013). The quarantine status of leafminers has also caused export restrictions to international markets and as a result, loss of revenue. To control leafminer attacks, farmers rely heavily on the application of chemical insecticides which are harmful not only to humans but also to the environment. Problems associated with chemical insecticides also include elimination of the natural enemies of the pest, and they also speed up the development of resistance to these chemical interventions (Johnson et al., 1980). In recent years, research has shifted to exploration of sustainable alternative methods such as the usage of parasitoids and inundative use of entomopathogenic fungi (EPF) (Migiro et al., 2010). The use of fungal endophytes has also been demonstrated to be effective against LMF with Phaseolus vulgaris and Vicia faba as host plants (Akutse et al., 2013; Gathage et al,. 2016). Similar systemic endophytic properties were also reported on thrips (Muvea et al., 2014) and bean stem maggot (Mutune et al., 2016). However, the clear infection mechanism or pathways of inoculated fungal endophytes against insect pests has not been well established. Metabolic interactions of fungal endophytes and the host plants stimulate the synthesis of bioactive secondary compounds such as alkaloids and phenols that are toxic to the pest at larval stages (Zhao et al., 2012). Secondary metabolites that have been characterized from fungal extracts such as pyrrolizidine alkaloids, and indole derivatives have been associated with insecticidal activity on arthropods (Kumar et al., 2008). The alkaloid ergotamine (1) caused reduced larval weight and leaf area consumption of the Fall Armyworm (FAW) Spodoptera frugiperda (Lepidoptera: Noctuidae), a newly invasive pest in Africa and Asia (Wink et al., 2018, Akutse et al., 2019, 2020). An indole derivative, 6-isoprenyl indole-3-carboxylic acid (2) from the endophyte Collectotrichum species showed growth inhibitions against plant fungi (Kumar et al., 2008). Endophytic fungi are therefore prospective inducers of defense compounds in host plants that can be exploited in maintaining healthy crops (Puniani et al., 2010). This study therefore investigated the induction of active defense compounds in endophytically colonized P. vulgaris plants and tested their toxicity against L. huidobrensis (Blanchard) and the newly invasive Fall Armyworm (FAW).
1.2 Statement of the Problem
Horticultural crops production in Kenya is extremely strained by attacks of the invasive Liriomyza species and other arthropod pests. Up to 100% losses have been recorded on beans due to leafminer damage (Chabi-Olaye et al., 2013). The quarantine status of Liriomyza species has resulted in export restrictions and rejections at international inspections therefore causing further losses of lucrative markets and revenue. In addition to LMF, FAW is another notorious invasive pest that is affecting food security in Africa and Asia. Attempts to manage these insects via the usage of synthetic insecticides are heavily afflicted by elimination of natural enemies and development of pest resistance (Johnson et al., 1980). Campaigns on food security and safety, health issues and environmental protections have put pressure on agricultural producers to search for alternative pest control approaches. The use of biopesticides has been promoted and the application of entomopathogenic fungi and fungal endophytes as biocontrol management has been proven to be efficient on various pest species. However, there is still a need to understand and establish the systemic infection mechanism of the endophytically colonized host plant against these herbivorous pests. This study, thus, aimed to investigate the induction of defense compounds from endophytically colonized P. vulgaris plant and assess their in vitro efficacy on LMF and the newly emerging Fall Armyworm larvae.
1.3 Objectives
1.3.1 General Objective
To assess the induction of secondary metabolites by endophytic fungi in the common bean Phaseolus vulgaris for sustainable management of Liriomyza leafminer and Fall Armyworm.
1.3.2 Specific Objectives
i. To evaluate the colonization of P. vulgaris host plant by the endophytic fungi H. lixii F3STI and B. bassiana GILU3 through seeds inoculation
ii. To characterize the active secondary metabolite compounds identified from endophytically colonized P. vulgaris plant
iii. To assess the insecticidal effects of active extracts on LMF and FAW larvae in vitro.
1.4 Justification of the Study
The outcomes of this study will enable us not only to identify and characterize the secondary metabolites, but also to establish and understand the systemic infection mechanism of an endophytically colonized P. vulgaris plant against the pests. It will also improve the adoption and application techniques of fungal endophytes in pest management programs. Bioactive compounds from an endophytically colonized plant that were profiled could be used as potential pest control components against other key agricultural and livestock pests. This study would therefore allow/ facilitate adaptation of microbial insecticides that are environmentally friendly, while reducing the abusive use of synthetic pesticides with maximum residue limits and eventually open more lucrative markets to horticultural crops in the country.
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