molecular basis of verticillium dahliae pathogenesis on potato ahmed farag am el-bebany doctor of

195 Pages·2010·4.12 MB·English

MOLECULAR BASIS OF VERTICILLIUM DAHLIAE
PATHOGENESIS ON POTATO

BY

AHMED FARAG A. M. EL-BEBANY

A Thesis Submitted to the Faculty of Graduate Studies
in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department of Plant Science
University of Manitoba
Winnipeg, Manitoba
Canada

© Copyright by AHMED F.A.M. El-BEBANY 2010
THE UNIVERSITY OF MANITOBA
FACULTY OF GRADUATE STUDIES
*****
COPYRIGHT PERMISSION

Molecular Basis of Verticillium dahliae Pathogenesis on Potato

by

Ahmed Farag A. M. El-Bebany

A Thesis/Practicum submitted to the Faculty of Graduate Studies of The University of
Manitoba in partial fulfillment of the requirements of the degree
of
Doctor of Philosophy

Ahmed F.A.M. El-Bebeany © 2010

Permission has been granted to the Library of The University of Manitoba to lend or sell
copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis
and to lend or sell copies of the film, and to University Microfilm Inc. to publish an
abstract of this thesis/practicum.

This reproduction or copy of this thesis has been made available by authority of the
copyright owner solely for the purpose of private study and research, and may only be
reproduced and copied as permitted by copyright laws or with express written
authorization from the copyright owner.
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ACKNOWLEDGMENTS
I would like to express my deep thanks and sincere appreciation to my advisor Dr.
Fouad Daayf for his exceptional guidance, encouragement, support and valuable
suggestions throughout my Ph.D. program. He is always accessible and open minded to
discuss any idea with enthusiasm.

I would like to express my sincere gratitude and thanks to Dr. Christof Rampitsch
for continuous encouragement and valuable advice during the part of my work in his
laboratory and throughout my program. I would like to express my thanks to Dr. James
Menzies and Dr. Khalid Rashid for their constant encouragement and guidance during
the course of my program. I would like to thank the late Dr. Lakhdar Lamari for his
support, guidance and limitless encouragement. He left us but he will never be forgotten.

I would like to express my sincere appreciation and thanks to Mr. Lorne Adam for
his help and technical assistance. My thanks are due to all of my current and past lab
mates, Zhen Yao, Maria A. Henriquez, Dr. Abdelbasset El Hadrami, Hassna Alkher,
Holly Derksen, Atta Soliman, Ana Venturino, Dr. Erika Wolski, Dr. Mohamed
Badawi, and Dr. Emmanuel Jourdan. I would like also to thank Wayne Mauthe,
Jackie Ching, Travis Banks and Tao Fan in CRC, AAFC, Winnipeg, Manitoba.

I would like to thank the general office and greenhouse staff members and all my
graduate colleagues in the Department of Plant Science, University of Manitoba.

I would like to thank all the members of the Egyptian cultural and educational bureau
in Montreal, Canada. The financial support form the Government of Egypt is greatly
acknowledged and appreciated.

I wish to express my thanks and appreciation from deep of my heart to my father,
mother, my wife Marwa El-Sherbeny, my kids Rahma and Taha, sisters and brother, as
well as to my father-in-law and mother-in-law.

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TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS………………………………………………… iii
LIST OF TABLES…………………………………………………............ viii
LIST OF FIGURES………………………………………………………… ix
ABSTRACT…………………………………………………....................... xiii
FOREWORD………………………………………………........................ xvi
1.0 INTRODUCTION……………………………………………............... 1
2.0 LITERATURE REVIEW……………………………………………... 4
2.1 The Host, Potato, Solanum tuberosum……………………………... 4
2.1.1 Origin and classification……………………………...................... 4
2.1.2 Geographical distribution…………………………………………. 4
2.1.3 Global production……………………………...…………………. 4
2.1.4 Production in Canada and Manitoba……………………………… 5
2.1.5 Potato diseases……………………………………………………. 5
2.2 The Pathogen, Verticillium dahliae……………………………......... 6
2.2.1 Nomenclature and classification…………………………….......... 6
2.2.2 Host range and global distribution………………….…………...... 7
2.2.3 Morphological and molecular characteristics…………………….. 8
2.2.4 Survival structures………………………………………………... 9
2.2.5 Vegetative compatibility groups…………. ……………………… 10
2.2.6 Verticillium dahliae pathogenicity determinants…………………. 11
2.2.6.1 Infectious structures……………………………...…………… 11
2.2.6.2 Production of cell-wall degrading enzymes…………………... 12
2.2.6.3 Production of potential phytotoxins and elicitors…………….. 13
2.2.6.4 Signaling in V. dahliae pathogenicity……………...…………. 15
2.3 Verticillium dahliae-Solanum tuberosum interactions ……..……… 18
2.3.1 Disease cycle and symptoms……………………………...………. 18
2.3.2 Economic significance …………………………...………………. 19
2.3.3 Early dying syndrome …………………………...…….................. 19
2.3.4 Mechanisms of host defense ……………………...……………… 20
2.3.5 Disease management………………………...…………................. 23
2.3.5.1 Cultural practices……………………………...……………… 23
2.3.5.2 Chemical control……………………………...………………. 24
2.3.5.3 Biological control……………………………………...……… 25
2.3.5.4 Host resistance……………………………...……………........ 25
2.3.5.5 Integrated disease management ……………………………… 26
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3.0 ESTABLISHMENT OF A DIFFERENTIAL POTATO-
VERTICILLIUM DAHLIAE PATHOSYSTEM……………………... 27

3.1 Abstract……...……………………...…………………………........... 27
3.2 Introduction…………………...………………………………........... 28
3.3 Materials and Methods…………...……………….………………… 30
3.3.1 Verticillium dahliae isolates………..…………………..………… 30
3.3.2 Potato cultivars……………………………………………………. 31
3.3.3 Inoculum preparation, inoculation procedure,
and experimental design………………………………………….. 31
3.3.4 Disease assessment and statistical analysis……………………….. 32
3.4 Results and Discussion………...………………...…………………... 33
3.4.1 Microscopic analysis, growth, and sporulation of V. dahliae
isolates…………………………………………………………….. 33
3.4.2 Pathogenicity evaluation of the V. dahliae isolates………………. 36

4.0 TRANSCRIPTOMIC ANALYSIS TO IDENTIFY CANDIDATE
PATHOGENICITY-RELATED GENES IN VERTICILLIUM
DAHLIAE AFTER ELICITATION WITH POTATO ROOT
EXTRACTS………………………............... …………………………. 41
4.1 Abstract……………………...………………………………….......... 41
4.2 Introduction………………………………………………………….. 42
4.3 Materials and Methods……………………………………………… 45
4.3.1 Verticillium dahliae isolates…………………………….………… 45
4.3.2 Potato root extract preparation and elicitation treatments………... 45
4.3.3 Total RNA extraction and mRNA isolation………………………. 46
4.3.4 Analysis of differentially expressed transcripts in V. dahliae
isolates using SH/cDNA-AFLP …………………………........... 47
4.3.5 Experimental design and data analysis………………………….... 48
4.4 Results……………………………………………………………….. 50
4.4.1 Differential expression of root extract-elicited transcripts
in V. dahliae and their classification……………………………. 50
4.4.2 Amplification of the differentially expressed transcripts and
functional assignment……………………………………….….. 53
4.5 Discussion……………………………………………………………. 59

5.0 COMPARATIVE PROTEOMIC ANALYSIS OF THE
PHYTOPATHOGENIC SOILBORNE FUNGUS VERTICILLIUM
DAHLIAE REVEALS POTENTIAL PATHOGENICITY
FACTORS IN AGGRESSIVE ISOLATES………………………...... 65

5.1 Abstract………………………….…………………………................ 65
5.2 Introduction…………………………………………………….......... 66
5.3 Materials and Methods……………………………………………… 68
5.3.1 Fungal isolates and culture conditions……………………………. 68
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5.3.2 Protein extraction and quantification……………………………... 69
5.3.3 2-DE (IEF/SDS-PAGE) and gel analysis………………………… 70
5.3.4 In-gel digestion…………………………………………………… 71
5.3.5 Mass spectrometry and database searches………………………... 71
5.4 Results and Discussion………………………………………...…..... 72
5.4.1 Biological differences and 2-DE…………………………….......... 72
5.4.2 MS analysis and database search…………………………………. 73
5.4.3 Differential proteins in the highly aggressive V. dahliae isolate…. 78
5.4.4 Differential proteins in the weakly aggressive V. dahliae isolate… 83
5.5 Concluding Remarks………………………………………………... 84

6.0 ISOLATION, CLONING AND COMPARATIVE SEQUENCE
ANALYSIS OF STRESS RESPONSE REGULATOR A,
ISOCHORISMATASE HYDROLASE AND TETRAHYDROXY-
NAPHTHALENE REDUCTASE GENES FROM THE HIGHLY
AND WEAKLY AGGRESSIVE VERTICILLIUM DAHLIAE
ISOLATES……………………………………………….……………. 86

6.1 Abstract………………………………………………………………. 86
6.2 Introduction………………………………………………………….. 87
6.3 Materials and Methods……………………………………………… 89
6.3.1 Verticillium dahliae isolates………………….………………… 89
6.3.2 Genomic DNA extraction………………………………………… 90
6.3.3 PCR amplification ………………………………………………... 90
6.3.4 Cloning, sequencing and comparative sequence analysis………... 91
6.4 Results and Discussion……………………………………………… 94
6.4.1 Stress response regulator A gene…………………………………. 96
6.4.2 Isochorismatase hydrolase gene…………………………………... 101
6.4.3 Tetrahydroxynaphthalene reductase gene………………………… 103

7.0 PROFILING OF SECONDARY METABOLITES IN
POTATO AFTER INOCULATION WITH HIGHLY VS
WEAKLY AGGRESSIVE ISOLATES OF VERTICILLIUM
DAHLIAE ………………………………….…………….…….……… 107
7.1 Abstract…………………………...………………………….............. 107
7.2 Introduction…………………………………………………….......... 108
7.3 Materials and Methods………………………….…………………... 110
7.3.1 Potato, V. dahliae and inoculation procedure…………………….. 110
7.3.2 Phenolics extraction and high performance liquid 111
chromatography (HPLC) analysis………………………………...
7.3.3 Statistical analysis………………………………………………... 113
7.4 Results and Discussion…………………………………………….... 113
7.4.1 Secondary metabolites identified in potato roots after
inoculation with V. dahliae isolates………………………………. 114

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7.4.2 Secondary metabolites identified in potato stems
after inoculation with V. dahliae isolates………………………... 118
7.4.3 Secondary metabolites identified in potato leaves
after inoculation with V. dahliae isolates…………………………. 124
7.5 Concluding Remarks……………………………………………….. 124

8.0 GENERAL DISCUSSION AND CONCLUSIONS……………..…... 127
8.1 Recommendations for Future Research………………...…………… 132
9.0 LITERATURE CITED………………………………….....…………. 133
10.0 APPENDICES……………………………………….....…………….. 154

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LIST OF TABLES
Table Page
3.1. Disease assessment of Verticillium wilt on Kennebec and Ranger Russet
potato cultivars after 7 weeks of inoculation with isolates Vd1396-9, Vs04-
28, Vs06-13, and Vs06-14 of V. dahliae…………………………………….. 39

4.1. Sequences of the AFLP primers………………..……………………………. . 49

4.2. Primer combinations used for the SH/cDNA-AFLP ………………..………… 49
4.3. BLAST search against NCBI GenBank database of Verticillium dahliae
transcripts differentially expressed in isolates Vd1396-9 (highly-aggressive)
and Vs06-14 (weakly-aggressive) in response to elicitation with root
extracts from the susceptible potato cultivar Kennebec and the moderately
resistant Ranger Russet………………………………………………………. 57

4.4. BLAST search against the Broad Institute of MIT and Harvard genome
database of V. dahliae transcripts differentially expressed in isolates
Vd1396-9 (highly-aggressive) and Vs06-14 (weakly-aggressive) in response
to elicitation with root extracts from the susceptible potato cultivar
Kennebec and the moderately resistant Ranger Russet……………………… 60

5.1. Identification of differentially expressed proteins in Vd1396-9, highly
aggressive, and Vs06-14, weakly aggressive, V. dahliae isolates by LC-ESI-
MS/MS and MASCOT……………………………………………………….. 75

6.1. Sequences of primers used for amplification of stress response regulator A
(VdSrrA), isochorismatase hydrolase (VdIsoch) and tetrahydroxyna-
phthalene reductase (VdThnr) genes from Vd1396-9 (highly aggressive)
and Vs06-14 (weakly aggressive) V. dahliae isolates………………………... 92

6.2. PCR conditions used for amplification of stress response regulator A
(VdSrrA), isochorismatase hydrolase (VdIsoch) and tetrahydroxynaphthalene
reductase (VdThnr) genes from Vd1396-9 (highly aggressive) and Vs06-14
(weakly aggressive) V. dahliae isolates……………………………………… 93

7.1. List of secondary metabolites putatively identified in different parts of potato
cv. Kennebec after inoculation with Vd1396-9 (highly aggressive) and
Vs06-14 (weakly aggressive) V. dahliae isolates……………………………. 115

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LIST OF FIGURES
Figure Page
3.1. Verticillium dahliae microsclerotia formation on PDA by isolates Vd1396-9,
the highly aggressive isolate after 3 weeks and Vs06-14, the weakly
aggressive isolate after 2 months. Microscopic views of Vd1396-9 and
Vs06-14 microsclerotia are at 125 X and 400 X …………………….………. 34

3.2. Fungal biomass and conidiospore counts of the Vd1396-9, the highly
aggressive and Vs06-14, the weakly aggressive V. dahliae isolates………… 35

3.3. Disease severity of V. dahliae Vd1396-9, Vs04-28, Vs06-13, and Vs06-14
isolates on Kennebec and Ranger Russet potato cultivars…………………… 37
4.1. Polyacrylamide gel electrophoresis (PAGE) of SH-cDNA-AFLP (primers
combinations 9 to 12) to amplify differentially expressed transcripts in V.
dahliae isolates Vs06-14 (weakly-aggressive) and Vd1396-9 (highly-
aggressive) in response to elicitation with root extracts from susceptible
potato cultivar Kennebec and moderately resistant cultivar Ranger Russet…. 52

4.2. Numbers and distribution of the 185 sequenced differentially expressed
transcripts in two V. dahliae isolates Vd1396-9 and Vs06-14, highly-and
weakly-aggressive, respectively, in response to elicitation with root extracts
from Ranger Russet and Kennebec, moderately resistant- and susceptible
potato cultivar, respectively………………………………………………….. 54
4.3. PCR re-amplification, after recovery from the PAGE of differentially
expressed transcripts in two V. dahliae isolates Vd1396-9 (highly-
aggressive) and Vs06-14 (highly-aggressive) in response to elicitation with
root extracts from susceptible potato cultivar Kennebec and moderately
resistant Ranger Russet………………………………………………………. 56

5.1. 2-D gels of proteins extracted from V. dahliae isolates Vd1396-9, the highly
aggressive, and Vs06-14, the weakly aggressive. Differentially expressed
proteins, indicated by arrows, were subjected to digestion and LC-ESI-
MS/MS analysis……………………………………………………………… 74

5.2. V. dahliae isolates Vd1396-9, highly aggressive, and Vs06-14, weakly
aggressive, on PDA medium, and magnification of their 2-D gels showing
spot 18, and MS2 spectrum and deduced amino acid sequence of the
peptide(I16 to K30) tentatively identified as tetrahydroxynaphthalene
reductase……………………………………………………………………… 82

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6.1. PCR amplification of stress response regulator A (VdSrrA), isochorismatase
hydrolase (VdIsoch), and tetrahydroxynaphthalene reductase (VdThnr) genes
from the genomic DNA of V. dahliae isolates Vd1396-9 (highly aggressive)
and Vs06-14 (weakly aggressive)……………………………………………. 95

6.2. Multiple alignment of the full length sequence of stress response regulator A
gene from the highly aggressive V. dahliae Vd1396-9 (SrrA-9), the weakly
aggressive isolate Vs06-14 (SrrA-14), and the Broad Institute genome
database of isolate VdLs.17 (VDAG_02250)………………………………... 99

6.3. Multiple alignment of the full length sequence of isochorismatase hydrolase
gene from the highly aggressive V. dahliae Vd1396-9 (Isoch-9), the weakly
aggressive isolate Vs06-14 (Isoch-14), and the Broad Institute genome
database of isolate VdLs.17 (VDAG_05103)………………………………... 102

6.4. Multiple alignment of the full length sequence of tetrahydroxynaphthalene
reductase gene from the highly aggressive V. dahliae Vd1396-9 (Thnr-9),
the weakly aggressive isolate Vs06-14 (Thnr-14), and the Broad Institute
genome database of isolate VdLs.17 (VDAG_03665)………………………. 105

7.1. Chlorogenic acid content (μg.g-1 fresh weight) in roots of potato cv.
Kennebec un-wounded un-inoculated, wounded un-inoculated control
plants, inoculated with the weakly (Vs06-14) or the highly (Vd1396-9)
aggressive V. dahliae isolates at 1, 3, 7 and 14 days after inoculation………. 116

7.2. Caffeic acid content (μg.g-1 fresh weight) in roots of potato cv. Kennebec un-
wounded un-inoculated, wounded un-inoculated control plants, inoculated
with the weakly (Vs06-14) or the highly (Vd1396-9) aggressive V. dahliae
isolates at 1, 3, 7 and 14 days after inoculation……………………………… 117

7.3. Ferulic acid content (μg.g-1 fresh weight) in roots of potato cv. Kennebec un-
wounded un-inoculated, wounded un-inoculated control plants, inoculated
with the weakly (Vs06-14) or the highly (Vd1396-9) aggressive V. dahliae
isolates at 1, 3, 7 and 14 days after inoculation……………………………… 119

7.4. Caffeic acid content (μg.g-1 fresh weight) in stems of potato cv. Kennebec un-
wounded un-inoculated, wounded un-inoculated control plants, inoculated
with the weakly (Vs06-14) or the highly (Vd1396-9) aggressive V. dahliae
isolates at 1, 3, 7 and 14 days after inoculation……………………………… 120

7.5. Rutin content (μg.g-1 fresh weight) in stems of potato cv. Kennebec un-
wounded un-inoculated, wounded un-inoculated control plants, inoculated
with the weakly (Vs06-14) or the highly (Vd1396-9) aggressive V. dahliae
isolates at 1, 3, 7 and 14 days after inoculation……………………………… 122

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