“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.....................................................