139 Pages
English

On the inheritance and mechanism of baculovirus resistance of the codling moth, Cydia pomonella (L.) [Elektronische Ressource] / Sabine Asser-Kaiser geb. Asser

-

Gain access to the library to view online
Learn more

Description

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

Subjects

Informations

Published by
Published 01 January 2009
Reads 43
Language English
Document size 1 MB


“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.
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
ICONTENTS
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 Insects34
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
IICONTENTS
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 ith 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 Insects60
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
IIICONTENTS
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 ...................................................... 76
6.2.4 Construction of the DNA-BD Fusion vector .......................................... 77
6.2.5 Generation of cDNA libraries of CpS and CpRR1................................. 83
6.2.6 Construction of a GAL4-AD fusion library............................................ 85
6.2.7 Screening................................................................................................86
6.3 Results.............................................................................................................86
6.3.1 Construction of the DNA-BD Fusion vector .......................................... 86
6.3.2 Test of the DNA-BD fusion vector for transcriptional activation .......... 89
6.3.3 Generation of cDNA libraries of CpS and CpRR1 and Construction of a
GAL4-AD fusion library ........................................................................................ 90
6.3.4 Identification of potential hot proteon interaction partners of PE38 ...... 91
IVCONTENTS
6.4 Discussion.......................................................................................................93
7 General discussion..................................................................................................98
References..................................................................................................................... 102
Appendix I (Media and buffers) ................................................................................... 119
A.I.1 Insect semi-artificial diet (500 ml)............................................................ 119
A.I.2 LB Medium (Luria-Bertani Medium) (for 1 litre).................................... 119
A.I.3 SOC Medium............................................................................................120
A.I.4 1X PBS buffer (for 1 litre)........................................................................ 120
A.I.5 10 x TAE buffer (for 1 litre) ..................................................................... 120
A.I.6 6 x DNA loading dye................................................................................ 121
A.I.7 YPD Medium (1 litre)............................................................................... 121
A.I.8 SD Medium (1 litre).................................................................................. 121
A.I.9 10 x Dropout (DO) Solution (1 litre)........................................................ 122
Appendix II (Vectors)................................................................................................... 123
VLIST OF ABBREVIATIONS
LIST OF ABBREVIATIONS

GENERAL
AD activation domain
BD binding domain
BC backcross
BV budded virus
bp base pair
CD cytochalasin D
cDNA complemaentary deoxyribonucleic acid
CL confidence limit
CM codling moth
DNA deoxyribonucleic acid
DNA-AD DNA-activation domain
DNA-BD DNA-binding domain
dscDNA double stranded complementary deoxyribonucleic acid
EPNs entomopathogenic nematodes
FB28 fluorescent brightener 28
ha hectare
hpi hours post infection
Ig immunoglobulin
IPM integrated pest management
IRAC Insecticide resistance Action Committee
kbp kilobase pair
LC median lethal concentration 50
LD median lethal dose 50
LD-PCR long distance polymerase chain reaction
LPS lipopolysaccharides
MCS multiple cloning site
MDT mating disruption techniques
VILIST OF ABBREVIATIONS
min minute
MNPV multiple nucleopolyhedrovirus
mRNA messenger ribonucleic acid
OB occlusion body
ODV occlusion derived virus
ORF open reading frame
ori origin of replication
PBS phosphate buffered saline
PCR polymerase chain reaction
PM peritrophic membrane
PTU phenylthiourea
qPCR quantitative polymerase chain reaction
RFLP restriction fragment length polymorphism
RNA ribonucleic acid
ROS reactive oxygen species
RTA repressed transactivator
SMART Switching Mechanism at 5’ end of RNA Transcript
SNPV single nucleoplyhedrovirus
Trx thioredoxin
TtD time to death
UAS upstream activation sequence
v/v volume per volume
w/v weight per volume
WHO World Health Organisation
Y2H yeast two-hybrid

VIRUSES
AcMNPV Autographa californica MNPV
AgseGV Agrotis segetum GV
AgMNPV Anticarsia gemmatalis MNPV
ArGV Artogeia rapae GV
BmDNV-2 Bombyx mori densovirus type 2
VIILIST OF ABBREVIATIONS
BmNPV Bombyx mori NPV
CpGV Cydia pomonella GV
HzSNPV Helicoverpa zea SNPV
LaolGV Lacanobia oleracea GV
LdMNPV Lymantria dispar MNPV
PlxyGV Plutella xylostella GV
SfMNPV Spodoptera frugiperda MNPV
TnSNPV Trichoplusia ni SNPV
XecnGV Xestia c-nigrum GV
VIII