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Characterization of the Entomopathogenic Bacterium Photorhadus Luminescens Sonorensis, and Bioactivity of Its Secondary Metabolites

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Characterization of the Entomopathogenic Bacterium Photorhadus Luminescens Sonorensis, and Bioactivity of Its Secondary Metabolites Characterization of the Entomopathogenic Bacterium Photorhadus Luminescens Sonorensis, and Bioactivity of its Secondary Metabolites Item Type text; Electronic Thesis Authors Orozco, Rousel Antonio Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 25/09/2021 03:57:11 Link to Item http://hdl.handle.net/10150/228614 1 CHARACTERIZATION OF THE ENTOMOPATHOGENIC BACTERIUM PHOTORHADUS LUMINESCENS SONORENSIS, AND BIOACTIVITY OF ITS SECONDARY METABOLITES. Rousel Antonio Orozco Copyright © Rousel A Orozco 2012 ________________ A Thesis Submitted to the Faculty of the DEPARTMENT OF ENTOMOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 2012 2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. SIGNED: Rousel. A. Orozco. APPROVAL BY THESIS DIRECTOR _______________________________________ S. Patricia Stock, PhD. Professor of Entomology Date ____May 1st 2012_________________ 3 ACKNOWLEDGEMENTS I want to begin by expressing my infinity gratitude to my mentor, Dr. S. Patricia Stock, whose guidance, support and advice has been invaluable in my journey. Also, I am very grateful to Dr. Itsvan Molnar for his technical advice and for all his encouragement. Thanks also to Dr. Xianchun Li for the insightful discussions, and for your support. I would like to thank my lab mates for all the laughs and candid conversations, especially to Patricia Navarro and John McMullen. I am also thankful to Ming-Min Lee for sharing her knowledge with me, and for her advice conducting phylogenetic analyses. Thank you Jesse, Lindsay, Tan and Tara for sharing successes and frustrations bringing this project to reality. I also want to acknowledge Rhodesia Celoy-Mateo for her technical assistance with the HPLC analysis. I am also grateful to Dr. Helge Bode for conducting the mass spectrometry analysis. A thousand thanks for all your help to Mark, Daniel, Joe, Gilberto in the School of Plant Sciences. Endless love to my family: Rigo, Gloria, Adrian, and of course Dustin. 4 TABLE OF CONTENTS LIST OF FIGURES.........................................................................................................................6 LIST OF TABLES...........................................................................................................................8 ABSTRACT ……………………………………………………………………………………... 9 INTRODUCTION ………………………………………..……………………………………. 10 CHAPTER I: CHARACTERIZATION, AND PHYLOGENETIC RELATIONSHIPS OF PHOTORHABDUS LUMINESCENS SUBP. SONORENSIS (-PROTEOBACTERIA: ENTEROBACTERIACEAE) THE BACTERIAL SYMBIONT OF THE ENTOMPATHOGENIC NEMATODE HETEROROHABDISTIS SONORENSIS…………...... 24 Introduction ………………………………………………………………..………........ 24 Materials and methods …………………………………….............….………………... 25 Results ............................................................................................................................. 29 Description of Photorhabdus luminescens subsp. sonorensis subsp. ….............. 33 CHAPTER II: BIOPROSPECTING OF SECONDARY METABOLITES PRODUCED BY THE ENTOMOPATHOGENIC BACTERIUM PHOTORHABDUS LUMINESCENS SUBSP. SONORENSIS (GAMMA-PROTEOBACTERIA, ENTEROBACTERIACEAE) ……………...54 Introduction....................................................................................................................... 54 Materials and Methods...................................................................................................... 57 Results …………………................................................................................................. 60 Chemical characterization of NP extracts………………………………………. 61 5 Insecticidal activity ……………………………………..……………………… 61 Nematicidal, antimycotic, and antibiotic activity………………………………. 62 Discussion………………………………………………………………………………. 63 CONCLUSIONS ……………………………………………………………………................. 76 REFERENCES……………………………………………………………………………......... 81 6 LIST OF FIGURES INTRODUCTION Figure 1. Heterorhabditis-Photorhabdus life cycle ………………………………………….. 19 Figure 2. Families of secondary metabolites produced by Photorhabdus. sp. .……………… 20 CHAPTER I. Figure 1. Phase variation on NBTA selective media ………………………………………….. 34 Figure 2. Colony morphology on different agar media ...……………………………………… 35 Figure 3. Best maximum parsimony tree, 16s rDNA ..………………………………………… 36 Figure 4 .Best maximum parsimony tree, dnaN ……....……………………………………..... 37 Figure 5. Best maximum parsimony tree, gtlX …....……………………………………............ 38 Figure 6. Best maximum parsimony trees, gyrB…..…………………………………………… 39 Figure 7. Best maximum parsimony trees, RecA ... ...…………………………………………..40 Figure 8. MP analysis of concatenated matrix …………………………………………………. 41 Figure 9. Bayessian analysis of concatenated matrix .…………………………………………. 42 7 LIST OF FIGURES – Continued CHAPTER II Figure1. HPLC-UV spectra for crude extract of each bacteria train ………………………….. 67 Figure.2. Mass spectra of stilbene ……………………………………………………………. 68 Figure.3. Insecticidal activity of crude extracts……………………………………………….. 69 Figure.4. Nematicidal effect on M. incognita ………………………………………………... 70 Figure.5. Antibacterial activity on P. syringae............................................................................. 71 Figure.6. Antimycotic effect on F. oxysporum……………………………………..………………. 72 Figure. 7. Effect of crude extracts on F. oxysporum …………………….....…………………... 73 8 LIST OF TABLES INTRODUCTION Table 1. Current species of the genus Heterorhabditis ………………………………………… 21 Table. 2. Photorhabdus species and subspecies described up to date …………………………. 22 CHAPTER I Table 1. Primers considered in this study ……………………………………………………... 43 Table 2. Photorhabdus species and isolates considered in this study ……………………….... 44 Table 3. BIOLOG GN2 assays ………………………………………………………………… 51 Table 4. API20 NE assays …...………………………………………………………………... 53 CHAPTER II Table 1. Rf values ..…………………………………………………………………………….. 74 Table 2. Mass of compounds detected by MS analysis ………………………………………... 75 9 ABSTRACT Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with entomopathogenic Heterorhabditis nematodes. Nematodes vector the bacteria from one insect host to another, while the bacterial symbiont produces toxins and secondary metabolites that kill that the insect host. In this study, we characterize the bacterial symbiont of Heterorhabditis sonorensis, recently discovered in the Sonoran desert. Biochemical and molecular methods including sequence data from five genes: 16s rDNA, gyrB, recA, gltX, dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses. We also surveyed for secondary metabolites (SM) produced by this microorganism, considering HPLC and mass spectrometry analyses. SM crude extracts showed activity against the corn ear worm Helicoverpa zea, the root-knot nematode (Meloidogyne incognita), the bacterium Pseudomonas syringae, and the fungus Fusarium oxysporum; and were more toxic that those produced by related species. Results from these studies showed that Photorhabdus l. sonorensis’ secondary metabolites have potent antagonistic activity against these plant pathogens. 10 INTRODUCTION Nematodes are pseudocoleomate roundworms in the phylum Nematoda. They have a worldwide distribution and have colonized a variety of niches in this planet ranging from aquatic to terrestrial ecosystems. Many groups are free-living, however, others are parasites of plants and animal, and are relevant to human and veterinary medicine, agriculture and forestry (Barker, 1994, Kaplan 2004, Morales-Hojas, 2009. It has been estimated that more than 25,000 species of nematodes have been described. However, estimates of their diversity range between 50,000 to one million species (Lambshead, 2003). Among invertebrate parasites, there are 30 nematode families that are associated with insects and other invertebrates (Stock and Hunt, 2005). Seven of these have the potential for being considered as biological control agents. Nematode- insects associations are very primitive and date from prehistoric times. The oldest fossil record dates from the Silurian period, almost 400 million years ago for a mermithid nematode associated to insects (Engel, 2004). Nematode-insect associations occur in many contexts; and can be traced to back to 40 million years ago, as evidenced by fossils found in Baltic amber (Nickle, 1972). Many species of nematodes have a phoretic relationship with insects which carry them on their body from one location to another. This relationship is innocuous to the insect, but advantageous for the nematodes. For example, Bursaphelenchus cocophilus (Aphelenchida: Parasitapheleichidae) and Monochamus beetles (Coleoptera: Cerambycidae) are involved in a commensal association
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