Diversity and Host Interaction of Phthorimaea operculella granulovirus Vom Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des akademischen Grades eines Doctor rerum naturalium (Dr. rer. nat.) Dissertation von Andreas Larem Erstgutachter: Prof. Dr. Johannes A. Jehle Zweitgutachter: Prof. Dr. Gerhard Thiel Darmstadt 2019 Larem, Andreas: Diversity and Host Interaction of Phthorimaea operculella granulovirus Darmstadt, Technische Universität Darmstadt, Jahr der Veröffentlichung der Dissertation auf TUprints: 2019 URN: urn:nbn:de:tuda-tuprints-83998 Tag der mündlichen Prüfung: 18.01.2019 Veröffentlicht unter CC BY-SA 4.0 International https://creativecommons.org/licenses/ “Look deep into nature and you will understand everything better.” - Albert Einstein - Contents LIST OF ABBREVIATIONS ........................................................................................................................................................ IV SUMMARY ............................................................................................................................................................................. VII ZUSAMMENFASSUNG .......................................................................................................................................................... VIII CHAPTER I INTRODUCTION ........................................................................................................................................................................ 1 GELECHIIDAE AND THEIR IMPACT ON GLOBAL FOOD PRODUCTION ...................................................................................................... 1 POTENTIAL PEST CONTROL APPROACHES AGAINST GELECHIIDAE ......................................................................................................... 2 BACULOVIRUSES IN PLANT PROTECTION ......................................................................................................................................... 2 BACULOVIRUSES: GENERAL FEATURES ........................................................................................................................................... 3 BACULOVIRUS INFECTION CYCLE ................................................................................................................................................... 5 BACULOVIRUS COVERT INFECTION AND VIRUS TRANSMISSION............................................................................................................ 6 General Features .............................................................................................................................................................. 7 Discovery and Historic Use of PhopGV ............................................................................................................................. 9 MICROSPORIDIA IN PLANT CONTROL ........................................................................................................................................... 10 MICROSPORIDIA ...................................................................................................................................................................... 10 Nosema .......................................................................................................................................................................... 11 AIM OF THE THESIS ................................................................................................................................................................ 12 CHAPTER II BIOLOGICAL ACTIVITY OF PHOPGV ISOLATES IN TERMS OF MEDIAN LETHAL CONCENTRATION (LC50) AND MEDIAN LETHAL TIME (LT50) IN THE HOST SYSTEM PHTHORIMAEA OPERCULELLA ........................................................................... 13 ABSTRACT .............................................................................................................................................................................. 13 INTRODUCTION ....................................................................................................................................................................... 13 MATERIAL AND METHODS ........................................................................................................................................................ 14 Insect Rearing................................................................................................................................................................. 14 PhopGV Isolates ............................................................................................................................................................. 14 Virus Propagation........................................................................................................................................................... 15 Purification of Occlusion Bodies from Infected Larvae ................................................................................................... 15 OB Quantification ........................................................................................................................................................... 16 Biological Activity of PhopGV Isolates ............................................................................................................................ 16 Statistical Analyses, ToxRat Probit ................................................................................................................................. 17 RESULTS ................................................................................................................................................................................ 17 Estimation of Median Lethal Concentration (LC50) ......................................................................................................... 17 Survival Rate of P. operculella Infected With PhopGV-GR1.1 ........................................................................................ 22 Estimation of Median Lethal Time (LT50) ........................................................................................................................ 24 Virus Caused Mortality of a Different P. operculella Strain from Tunisia ....................................................................... 28 DISCUSSION ........................................................................................................................................................................... 29 CONCLUSION .......................................................................................................................................................................... 30 CHAPTER III ELUCIDATING THE GENETIC DIVERSITY OF PHTHORIMAEA OPERCULELLA GRANULOVIRUS (PHOPGV)............................. 31 ABSTRACT .............................................................................................................................................................................. 31 INTRODUCTION ....................................................................................................................................................................... 32 Insect Rearing................................................................................................................................................................. 33 I PhopGV Isolates ............................................................................................................................................................. 33 Virus Propagation........................................................................................................................................................... 34 Purification of Occlusion Bodies from Infected Larvae ................................................................................................... 34 OB Quantification ........................................................................................................................................................... 35 DNA Extraction ............................................................................................................................................................... 35 Whole Genome Sequencing ........................................................................................................................................... 35 Bioinformatic Analysis of Whole Genome Data ............................................................................................................. 36 CLC Assembly and Bowtie2 Analysis............................................................................................................................... 36 Additional De Novo Assembly ........................................................................................................................................ 36 Identification of Indel Mutations.................................................................................................................................... 37 SNP Detection................................................................................................................................................................. 37 Phylogenetic Analysis ..................................................................................................................................................... 38 PCR Amplification ..........................................................................................................................................................