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“On the Inheritance and Mechanism of Baculovirus Resistance of the Codling Moth, Cydia Pomonella (L.)”

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“On the Inheritance and Mechanism of Baculovirus Resistance of the Codling Moth, Cydia Pomonella (L.)” “On the inheritance and mechanism of baculovirus resistance of the codling moth, Cydia pomonella (L.)” Dissertation zur Erlangung des Grades “Doktor der Naturwissenschaften” am Fachbereich Biologie der Johannes Gutenberg-Universität in Mainz Sabine Asser-Kaiser geb. Asser geb. am 03.08.1980 in Karlsruhe Mainz, 2009 Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: 29.04.2010 CONTENTS CONTENTS Contents .............................................................................................................................I List of abbreviations .......................................................................................................VI Summary.........................................................................................................................IX 1 General introduction................................................................................................. 1 1.1 Cydia pomonella as a pest in apple prodution .................................................. 1 1.1.1 Life cycle of codling moth........................................................................ 1 1.1.2 Control of codling moth............................................................................ 2 1.2 Baculoviruses.................................................................................................... 5 1.2.1 Introduction............................................................................................... 5 1.2.2 Structure and Classification...................................................................... 5 1.2.3 Genome and gene expression ................................................................... 7 1.2.4 Cydia pomonella Granulovirus (CpGV) as a member of the genus Betabaculovirus ........................................................................................................ 8 1.2.5 Pathology .................................................................................................. 8 1.2.6 Symptoms of CpGV infection ................................................................ 10 1.3 Resistance ....................................................................................................... 11 1.3.1 Definition of resistance........................................................................... 11 1.3.2 Resistance to baculoviruses .................................................................... 12 1.3.3 Occurrence of CpGV resistant codling moth populations in Europe ..... 14 1.4 Aims of this thesis........................................................................................... 15 2 Resistance of codling moth against Cydia pomonella granulovirus (CpGV) is inherited by a single, dominant gene located on the Z-chromosome ............................. 18 I CONTENTS 2.1 Introduction..................................................................................................... 18 2.2 Material and methods...................................................................................... 19 2.2.1 Virus........................................................................................................ 19 2.2.2 Test insects.............................................................................................. 19 2.2.3 Single pair crosses .................................................................................. 20 2.2.4 Homogenisation of CpR ......................................................................... 20 2.2.5 Bioassays ................................................................................................ 21 2.2.6 Determination of the sex of individuals.................................................. 22 2.3 Results and Discussion................................................................................... 23 3 Sex linkage of CpGV resistance in a heterogeneous field strain of the codling moth Cydia pomonella (L.)...................................................................................................... 32 3.1 Introduction..................................................................................................... 32 3.2 Materials and methods.................................................................................... 34 3.2.1 Insects ..................................................................................................... 34 3.2.2 Virus........................................................................................................ 34 3.2.3 Bioassays ................................................................................................ 34 3.2.4 Single pair crosses .................................................................................. 35 3.3 Results............................................................................................................. 36 3.3.1 Single pair crosses within CpR............................................................... 36 3.3.2 Hybrid crosses between CpS and CpR ................................................... 38 3.3.3 Backcrosses of the F1 generation to CpR............................................... 39 3.4 Discussion....................................................................................................... 41 4 Resistance overcoming character of the Iranian isolate CpGV-I12 and testing on cross resistance to Cry1Ab ............................................................................................. 47 II CONTENTS 4.1 Introduction..................................................................................................... 47 4.2 Material and methods...................................................................................... 49 4.2.1 Test Insects............................................................................................. 49 4.2.2 Virus........................................................................................................ 49 4.2.3 Bioassays with CpGV............................................................................. 49 4.2.4 Bioassays with Cry1Ab protein .............................................................. 50 4.2.5 Statistical analysis................................................................................... 51 4.3 Results............................................................................................................. 51 4.3.1 Bioassays with CpGV-M and CpGV–I12 .............................................. 51 4.3.2 Instar-specific mortality of CpS and CpR............................................... 52 4.3.3 Bioassays with Bacillus thuringiensis Cry1Ab protein .......................... 54 4.4 Discussion....................................................................................................... 54 5 Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication ................................................................................................ 57 5.1 Introduction..................................................................................................... 57 5.2 Materials and methods.................................................................................... 60 5.2.1 Insects ..................................................................................................... 60 5.2.2 Virus........................................................................................................ 61 5.2.3 Bioassays with fluorescent brightener 28 ............................................... 61 5.2.4 Intra-haemocoelar infection.................................................................... 62 5.2.5 Tissue specific virus replication ............................................................. 62 5.2.6 DNA isolation and quantitative PCR...................................................... 63 5.2.7 Haemolymph transfusion........................................................................ 63 5.3 Results............................................................................................................. 63 III CONTENTS 5.3.1 Bioassays with fluorescent brightener 28 ............................................... 63 5.3.2 Intra-haemocoelar infection.................................................................... 64 5.3.3 Replication of CpGV in different tissues................................................ 65 5.3.4 Haemolymph transfusion........................................................................ 67 5.4 Discussion....................................................................................................... 68 6 Investigations on protein-protein interactions between PE38 of CpGV-I12 and host proteins of a CpGV resistant codling moth strain........................................................... 72 6.1 Introduction..................................................................................................... 72 6.1.1 Background and aim of the study ........................................................... 72 6.1.2 Yeast two-hybrid system ........................................................................ 73 6.2 Material and methods...................................................................................... 76 6.2.1 Test insects.............................................................................................. 76 6.2.2 Virus........................................................................................................ 76 6.2.3 Agarose gel electrophoresis of DNA.....................................................
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