UNIVERSIDAD DE LA FRONTERA Facultad De Ingeniería, Ciencias Y Administración Programa De Doctorado En Ciencias De Recursos Naturales
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UNIVERSIDAD DE LA FRONTERA Facultad de Ingeniería, Ciencias y Administración Programa de Doctorado en Ciencias de Recursos Naturales POTENTIAL INSECTICIDE EXTRACTS ISOLATED FROM CESTRUM PARQUI L’ HERIT (SOLANACEAE) FOR CONTROLLING OF HYLURGUS LIGNIPERDA (FABRICIUS) DOCTORAL THESIS IN FULFILLMENT OF THE REQUERIMENTS FOR THE DEGREE DOCTOR OF SCIENCES IN NATURAL RESOURCES CLAUDIA NATALIA HUANQUILEF ARRIAGADA TEMUCO – CHILE 2020 “POTENTIAL INSECTICIDE OBTAINED FROM EXTRACTS ISOLATED FROM CESTRUM PARQUI L’ HERIT (SOLANACEAE) FOR CONTROLLING OF HYLURGUS LIGNIPERDA (FABRICIUS)” Esta tesis fue realizada bajo la supervisión del director de Tesis Dr. ANDRES EDUARDO QUIROZ CORTEZ, perteneciente al Departamento de Ciencias Químicas y Recursos Naturales de la Universidad de La Frontera y es presentada para su revisión por los miembros de la comisión examinadora. CLAUDIA NATALIA HUANQUILEF ARRIAGADA Dr. Andrés Quiroz Cortez Dr. Andrés Quiroz Cortez DIRECTOR DEL PROGRAMA DE Dr. Ana Mutis Tejos DOCTORADO EN CIENCIAS DE RECURSOS NATURALES Dr. Alejandro Urzúa Moll Dr. José Manuel Perez Donoso Dra. Mónica Rubular D. DIRECTORA ACADÉMICA DE POSTGRADO UNIVERSIDAD DE LA FRONTERA Dr. León Bravo Ramirez A mi familia… 4 Acknowledgments First, I would like to thanks to Dr. Andrés Quiroz Cortez, for giving me the chance to conduct my PhD theses and for supporting me in the complicated moments during these years; Dra. Anita Mutis for their constant support during my studies at the doctorade. I am grateful to Dr. Alejandro Urzúa Moll for coaching me in my work and for their generous assistance and hospitality during my stay in Santiago. I would also like to thank my committee, members Dr. Léon Bravo, Dr. José Manuel Perez and Dr Alejandro Úrzua, for all their advice. I thank the members of Ecological Chemistry Laboratory for their collaboration and their friendlydness. My recognition to Dr. Javier Espinoza, Dr Emilio Hormazabal, Dr. Leonardo Bardhele, for their support in the ecological chemistry laboratory, Universidad de La Frontera, Temuco, Chile. Finally, I thank my family and friends who encouraged me all this time. i Summary and outline of this theses The increasing international trade in forest and wood products forests are increasingly being affected for the introduction of exotic wood-boring beetles. Among it most important species present in Chile is found golden-haired bark-beetle, H. ligniperda (Coleoptera: Curculionidae). It was first detected in the mid 1980s and can be present throughut region with P. radiata plantations. Currently, the invasive exotic species cost millions of euros every year from all exporting countries. To cope with these problems the forestry industry has used widely methyl bromide pesticide for the fumigation of forest product export. On the other hand, international environmental organism has banned its use for the next decade becaue it is considered an ozone- depleting substance. Hence, research to find controlling alternative treatments are urgently needed prioritizing the use of safe systems. One auspicious strategy for insect pest control is the use of feeding deterrent compounds due that this may act on the gustatory receptors inhibiting the consumption of food by herbivorous. The focus of the thesis was study pesticidal activity of C. parqui extracts obtained with different polarities of solvents. The thesis is composed of four parts: In Chapter I, detail a review of the literature related on H. ligniperda, antifeedants substances and pesticidal activity of C. parqui leaves for finish with hypothesis and general and specific objectives of the Thesis. In Chapter II of the thesis is related in the obtention the chloroform, ethyl acetate and n-butanol extracts (saponic extract) from C. parqui leaves and then evaluate the extracts on the feeding behavior of H. ligniperda adults, and 5th and 6th instar larvae. In Chapter III detail the analysis and identification of the active sub-fractions from chloroform fraction obtained from C. parqui leaves ethanol extracts using chromatography techniques. The ii identification of active sub-fraction from C. parqui was obtained through antifeedant assays on H. ligniperda. In Chapter IV. Finally, the general conclusions of the work and the future perspective are presented. iii TABLE OF CONTENTS Acknowledgements i Summary of this thesis ii Table of Contents iv List of figures viii List of tables ix CHAPTER 1: General Introduction 1. Natural Insecticides from plants 1 1.1. Direct defense 1 1.1.1. Plant cuticle 2 1.1 2. Spinescence 2 1.1.3. Trichomes 2 1.1.4. Secondary metabolites and plant defense 3 1.2. Indirect defense 6 1.3. Radiata pine plantations in Chile 7 1.4. Red-haired pine bark beetle, Hylurgus ligniperda (Fabricius, 1787) 7 1.5. Systematic and morphological description 8 1.6. Antifeedant subtances in plants 10 1.7. Fedding deterrent activity of herba plants from Solanaceae extracts 11 1.8. Plants extracs from C. parqui as potential sources of pesticidal agents 13 1.9. Antifeedant activity on insect the Coleoptera order 14 iv 1.10. Gustatory system in Coleoptera (beetles) 14 1.11. Hypothesis 22 1.12. General objective 22 1.13. Specific objectives 22 1.14. Reference 23 CHAPTER 2: 2. Introduction 41 2.1. Materials and Methods 43 2.1.1. Plant Material 43 2.1.2. Insects 44 2.1.3. C. parqui extracts obtention 44 2.1.4. No-choice feeding bioassays 45 2.1.5. Statistical analysis 46 2.2. Results 47 2.2.1. C. parqui extracts 47 2.2.1.1. Feeding bioassays of unsexed adults individual 47 2.2.1.2. Feeding bioassays of adult male and female 49 2.2.1.3. Feeding bioassays of larvae 51 2.3. Discussion 53 2.5. Conclusions 55 v 2.6. Acknowledgents 56 2.7. References 57 CHAPTER 3: 3. Introduction 68 3.1. Materials and methods 69 3.1.1. Plant material 69 3.1.2. Insects 69 3.1.3. Extraction and isolation 70 3.1.4. No-choice feeding (antifeedant) assays 72 3.1.5. Larvicidal feeding behavior assay 72 3.1.6. GC/MS analysis 73 3.1.7. Statistical analysis 74 3.2. Results 75 3.2.1.Antifeedant effect of active sub-fractions from chloroform fraction obtained 75 of C. parqui leaves ethanol extract. 3.2.2. Larvicidal activity of active sub-fractions from chloroform fraction 76 3.2.3. Identification of major compounds of active sub-fractions from chloroform 77 fraction by GC/MS analysis 3.3. Disscussion 79 3.4. Conclusions 83 vi 3.5. Acknowledgments 83 3.6. Reference 84 CHAPTER 4 4. General discussion 91 4.1. Conclusions 95 4.2. Future projections 96 4.3. References 97 vii List of figures Figure 1.1. H. ligniperda (Fabricius, 1787). 6 Figure 1.2. Damage caused by H. ligniperda in seedling plantation of P. radiata images obtained from Ciesla (1993). Figure 1.3 H. ligniperda (Fabricius, 1787), head. 18 Figure 2.1. Weight loss/gain (%) of H. ligniperda adults fed with artificial diet 44 supplemented with A) chloroform extract, B) ethyl acetate extract, and C) saponins extract. Figure 2.2. Weight loss/gain (%) of male and female of H. ligniperda fed with 46 artificial diet supplemented with (A) chloroform extract, (B) ethyl acetate extract, and (C) saponin extract. Figure 2.3. Weight loss /gain (%) of H. ligniperda larvae fed with artificial diet 48 supplemented with A) chloroform extract, B) ethyl acetate extract, and C) saponins extract. Figure 3.1. Schematic presentation of the extraction and fractionation from 68 C. parqui leaves Figure 3.2. Wheight loss/gain (%) male adults of H. ligniperda fed with artificial 72 diet supplemented with A) fractions 4, 6 and 8; B) fractions 10, 13, 15 and 17; C) fractions 18 and 24. Figure 3.3. Effect of standard commercial n-alkane compounds and F4, F10 and 73 F18 sub-fraction from chloroform fraction on the development of H. ligniperda. viii List of tables Table 1.1. Secondary metabolites with diet insecticidal activity 9 Table 1.2. Most important chemical insecticides based on their characteristic Table 1.3. Example of studies that supporting the feeding deterrent activity of several Solanaceae plants on insects Table 1.4. Example s of studies that supporting the antifeedant activity on insect of 12 the Coleoptera order. Table 2.1. Concentrations of the tested extract solutions and concentration of each 42 extract in diet using in the bioassay. Table 3.1. GC/MS analyses of active sub-fraction obtained from chloroform 75 fraction. ix CHAPTER 1 General Introduction 1. Natural insecticides from plants Plants and insects, have co-evolved successfully overcome the various environmental pressures to which they have been subjected and have in turn developed a complex network of interactions between them to avoid each other defense systems (War et al. 2012; Wielkopolan and Obrępalska-Stęplowska 2016). Plant use various systemic responses to counteract attack by herbivorous insects including the development of morphological structures, secondary metabolites and proteins, that exhibits detrimental toxic, repellent and anti-nutricional effects on insects (Usha Rani and Jythsna 2011; War et al. 2012). In turn, insect respond to plant through of diverse and elaborated mechanisms of resistance, such as toxin avoidance, target- site alteration, and detoxification that are enconded in the genomeof the insect. In addition, insects are associated with microbes that can modify plant defense responses and plants are associated with endophytes microorganism, which may elicit an insecticidal activity (Wielkopolan and Obrępalska-Stęplowska 2016). Thus, it is very important to consider the multiple factors that involve the interaction between plant and insects. They are a series of directly or indirectly mechanisms that affect the plant-insect interactions that are produced of manner constitutive or induced by plant through which they acquire a series of adaptive advantages (Hanley et al. 2007; war et al 2012. 1.1.