Effects of the Plant Pathogen

Pseudomonas syringae pathovar

syringae on Vitis vinifera

A Thesis submitted to Charles Sturt University for the degree of Doctor of

Philosophy

Stewart Hall

B.Biotech (Med – Honours), B.ForensicBiotech

Submitted

November 2015

i

Contents

Certificate of Authorship iv

Acknowledgements v

Abstract vii

List of Abbreviations x

Nomenclature xiii

List of Figures xiv

List of Tables xvi

List of Publications xvii

Chapter 1 Review of the Literature 1

1.1 Introduction 1

1.2 Pseudomonas syringae plant diseases 2

1.3 Pseudomonas syringae pv. syringae plant diseases 3

1.3.1 Pseudomonas syringae pv. syringae identification 4

1.3.2 Molecular characterisation 5

1.3.3 Genetic diversity 6

1.3.4 Antibiotic resistance 8

1.4 Pseudomonas syringae pv. syringae infection symptoms 9

1.5 Vitis vinifera infection 9

1.6 Host plant entry 11

1.7 Primary virulence toxins of Pseudomonas syringae pv. syringae 12

1.7.1 Syringolin A 13

1.7.2 Biosynthesis of syringolin A 15

1.7.3 Regulation of syringolin A biosynthesis 16

1.7.4 Syringomycins and syringopeptins 17

1.7.5 Mode of syringomycin action 19

1.7.6 Syringomycin biosynthesis 19

1.7.7 Mode of syringopeptin action 20

1.7.8 Syringopeptin biosynthesis 20

1.7.9 Regulation of syringomycin and syringopeptin biosynthesis 21

1.8 Evolved strategies for evading host defences 22

1.9 Plant-pathogen signalling 24

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1.9.1 General plant defences (PAMP-triggered immunity) 25

1.9.2 General plant defences (effector-triggered immunity) 29

1.9.3 Auxins 30

1.9.4 Gibberellins 31

1.9.5 Abscisic acid 32

1.9.6 Cytokinins 34

1.9.7 Phytoalexins and stilbenes 35

1.9.8 Reactive oxygen species 36

1.9.9 Ethylene 37

1.9.10 Salicylic acid 39

1.9.11 Jasmonic acid 41

1.10 Summary 46

1.11 Research aims and objective 48

Chapter 2 Phylogenetic Relationships of Pseudomonas syringae pv. syringae

Isolates Associated with Bacterial Inflorescence Rot in Grapevine 50

2.1 Introduction 50

2.2 Materials and methods 51

2.3 Results 58

2.4 Discussion 72

2.5 Conclusions 81

2.6 Acknowledgements 82

Chapter 3 Pseudomonas syringae pv. syringae From Cool Climate Australian

Grapevine Vineyards: Insight Into Phenotypes and Virulence

Associated With Bacterial Inflorescence Rot 83

3.1 Introduction 83

3.2 Materials and methods 84

3.3 Results 99

3.4 Discussion 112

3.5 Conclusions 125

3.6 Acknowledgements 126

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Chapter 4 Vitis vinifera Defence Responses to Pseudomonas syringae pv.

syringae 127

4.1 Introduction 127

4.2 Materials and methods 129

4.3 Results 135

4.4 Discussion 151

4.5 Conclusions 160

4.6 Acknowledgements 161

Chapter 5 General Discussion 162

Chapter 6 Literature Cited 171

Appendix 1 DNA Extraction of P. syringae Using Qiagen DNeasy Blood and

Tissue Kit 207

Appendix 2 GenBank Accession Numbers of P. syringae Isolates 208

Appendix 3 Analysis of Molecular Variance Results Using Arlequin Software 209

iv

Certificate of Authorship

I hereby declare that this submission is my own work and to the best of my knowledge

and belief, understand that it contains no material previously published or written by

another person, nor material which to a substantial extent has been accepted for the

award of any other degree or diploma at Charles Sturt University or any other

educational institution, except where due acknowledgement is made in the thesis. Any

contribution made to the research by colleagues with whom I have worked at Charles

Sturt University or elsewhere during my candidature is fully acknowledged. I agree that

this thesis be accessible for the purpose of study and research in accordance with normal

conditions established by the Executive Director, Library Services, Charles Sturt

University or nominee, for the care, loan and reproduction of thesis, subject to

confidentiality provisions as approved by the University.

Signature:

v

Acknowledgements

First and foremost, I would like to thank and acknowledge the support and guidance

from my supervisory team; Dr Melanie Whitelaw-Weckert, Dr Ian Dry and Professor

Chris Blanchard. Your wisdom, support, and motivation have helped from the

beginning to the end of this project. I would like to give Melanie just that little bit extra

as you have always been there to help me and followed me every step of the way.

You’ve not only been a great supervisor, exceptional support and a very good friend.

To family: Mum and Dad for always supporting me and reminding me that I can do

anything if I put my mind to it. My sister, Libby, for listening to me whine on the

phone, coming up for visits (with my beautiful nieces) and bringing your homemade

sausage rolls... they are the best comfort food!

To my friends and fellow PhD students and people showing me their support; Briony

McGrath, Gayle Petersen, Ginger Korosi, Jo Huckel, Subhashini Abeysinghe, Nicola

Wunderlich, Jen Bullock, Jasmine MacDonald, Linda Ovington, Lindsay Greer and

Maame Blay. Our friendship and support for each other will be something I will cherish

for a lifetime. I would also like to personally thank Sandra Savocchia, Chris Scott, Suzie

Rogiers, Andrea Crampton, Robyn Harrington and Ashley Radburn for their various

forms of support thoroughout my candidature.

I would also like to acknowledge the following people for helping me with resources to

complete this study: Dr Roger Shivas and Miss Yu Pei Tan, for the pathovars of

Pseudomonas syringae. Dr Thomas Hill, for understanding the challenges of collecting

isolates and supplying me with some of his. Professor Barbara Furnell, for the

Escherchia coli N99 indicator strain. Dr Michael Priest and Mrs Karren Cowan, for

more isolates from the Plant Pathology Herbarium. Lynne Matthews, Naomi Tidd,

Rujaun Huang, Kirsty White, Natalie Allison and Therese Moon (technical officers at

National Life Sciences Hub, School of Biomedical Sciences and School of Agriculture),

for when I didn’t have something, they sure did and would lend it to me. Angela

Germakow, at the Waite Institute in Adelaide for helping me get started with good and

reproducible RNA extractions. Dr Suren Samuelian, for starting me out with my

knowledge of qPCR. Bev Orchard, for opening my mind to the world of statistics and

helping me to analyse my data. David Gopurenko for his help in analysing and

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understanding AMOVAs. John Harper for showing me support when I needed it during

the tough times and being a great mentor. Without all of these people and their feedback

I don’t know where this would be and I am in debt to you.

I would also like to acknowledge Charles Sturt University and the National Wine and

Grape Industry Centre for funding this project, and the Australian Wine and Grape

Authority (formerly Grape and Wine Research and Development Corporation) for their

extra funding both for research and attending Crush 2012.

Finally, I would like to thank my partner, Alex, and our small family (Bella and Zeus

and the chooks) You have put up with so much over these last few years. You have

been a rock and seen me go to crazy and back. Your dreams are our next adventure

together.

To anyone I have missed, your efforts have and always will be appreciated. Just know

that five minutes after handing in my thesis I will remember your help and support and

the guilt of not having your name here will follow me for years to come.

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Abstract

Vitis vinifera is one of the world’s most economically important fruit crops. Recently,

extensive yield losses in wine grape production, caused by bacterial inflorescence rot

(BIR), have been reported in some cool climate Australian vineyards. This disease is

caused by the bacterium Pseudomonas syringae pv. syringae (P. s. syringae).

Symptoms on grapevine caused by P. s. syringae include the production of leaf spots

with chlorotic haloes, necrotic lesions on petioles and shoots, and necrosis of

inflorescences.

The aims of the current study were to i) comprehensively evaluate the relatedness and

distribution of P. s. syringae isolates from Australian cool climate vineyards, ii)

characterise isolates from grapevine, using traditional biochemical techniques, including

toxin production and host range, iii) determine whether phenotypic and genotypic data

are related to the production of bacterial inflorescence rot or pathogenicity in grapevine

using analysis of molecular variance, and iv) determine the grapevine host defence

response to P. s. syringae infection to better understand how the pathogen may be

manipulating host responses.

Putative P. s. syringae isolates from infected grapevines within a range of Australian

vineyards were identified using LOPAT and RNA polymerase β-subunit (rpoB) gene

sequencing for pathovar allocation. The isolates were then characterised by a

combination of multi-locus sequence typing (MLST) and biochemical tests.

Additionally, the production of syringomycin and syringopeptin was assessed, along

with genotyping for these toxins including identification of the syringolin A

biosynthesis gene (sylC).

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Plant defence responses to pathogenic and non-pathogenic P. s. syringae were

investigated on potted Chardonnay grapevines. Callose deposition was observed by

aniline blue staining under epifluorescence microscopy and quantified using high

intensity pixels from digital photographs. Relative expression of defence gene targets

for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and stilbene synthase were

monitored by semi-quantitative PCR (qPCR) from reverse transcribed RNA.

This study identified eight vineyards in six Australian viticultural regions affected by

P. s. syringae, with symptoms of BIR and/or leaf spot. Bacterial isolates from these

vineyards were grouped by MLST data into two well supported P. s. syringae clades,

each containing a mixture of pathogenic and non-pathogenic grapevine isolates.

Pathogenic P. s. syringae isolates were also obtained from grapevine sucker shoots,

suggesting that sucker shoots may allow ‘overwintering’ of the pathogen.

Pathogenicity was associated with tyrosinase negative phenotype whereas those from

healthy and non-BIR vineyards were tyrosinase positive. The pathogenicity of

P. s. syringae was also found to be associated with syringolin A genotypes (sylC).

Although both pathogenic and non-pathogenic P. s. syringae isolates were able to

induce callose deposition in grapevine leaves, the effect was less for pathogenic

P. s. syringae. Semi-quantitative PCR showed that inoculation of grapevine leaves by

pathogenic P. s. syringae caused increases in the activity of the SA and JA/ET mediated

pathways in potted Chardonnay.

The current study has demonstrated that, in cool climate Australian vineyards,

genetically distinct strain groups of P. s. syringae can be isolated from grapevines

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affected by BIR. Phenotypic and genotypic characterisation suggests that P. s. syringae

isolates that produce syringolin A but lack tyrosinase activity are associated with this

disease. Finally, the defence gene studies provide insight into the grapevine defence

responses to pathogenic P. s. syringae, which may open up knowledge for effective

targeted treatment and effective disease management in affected regions.

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List of Abbreviations

ABA Abscisic acid

ABC ATP-binding cassette

AFS Acid from sucrose

AMOVA Analysis of molecular variance

ANOVA Analysis of variance

Avr Avirulence

BAK1 Brassinosteroid associated kinase 1

BIR Bacterial inflorescence rot

BLS Bacterial leaf spot

BRI1 Brassinosteroid receptor 1

BTH Benzothiadiazole

cDNA Coding DNA

CFU Colony forming units

Chit4C Acidic class IV chitinase

CK Cytokinin

COI1 Coronatine insensitive 1

COR Coronatine

CT Cycle threshold

cv. Cultivar

DAFF Department of Agriculture, Fisheries and Forestry

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

ERF Ethylene response factor

ET Ethylene

ETI Effector triggered immunity

ETR2 Ethylene receptor 2

GA Gibberellic acid/Gibberellins

gapA Glyceraldehyde-3-phosphate dehydrogenase (gene)

GATTa Gelatin, Aesculin, Tyrosinase, Tartaric acid

gltA Citrate synthase (gene; also known as cts)

GLU β-1,3-glucanase

GPLTA Grapevine Pathogenicity Leaf Test Assay

gyrB DNA gyrase B (gene)

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Hop hrp outer protein

hpi Hours post inoculation

HR Hypersensitivity reaction

hrc Hypersensitivity response and conserved (gene)

hrp Hypersensitivity response and pathogenicity (gene)

HTH Helix-turn-helix

IAA Indole-3-acetic acid

INA Ice nucleation activity

JA Jasmonic acid

JA-Ile Jasmonic acid-isoleucine

JAZ Jasmonate-ZIM-domain

KB King’s B agar

LOPAT Levan, Oxidase, Potato soft rot, Arginine dihydrolase, Tobacco

leaf HR

MAMP Microbe-associated molecular pattern

MAPK Mitogen activated protein kinase

MKK Mitogen activated protein kinase kinase

MeJA Methyl jasomonate

MLST Multi-locus sequence typing

MPK4 Mitogen-activate protein kinase 4

NA Nutrient agar

NADPH Nicotinamide adenine dinucleotide phosphate

NHO1 NONHOST1

NJ Neighbour-Joining

NPR1 Non-expression of pathogenesis-related protein 1

NR Nitrate reduction

NRPS Non-ribosomal peptide synthetase

nt Nucleotide

PAL Phenylalanine ammonia lyase

PAMP Pathogen associated molecular pattern

PCR Polymerase chain reaction

PDA Potato dextrose agar

PDF1.2 Plant defensin 1.2

PGIP Polygalacturonase-inhibiting protein

PIN Serine protease inhibitor

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PKS Polyketide synthetase

PR Pathogenesis-related protein

PR1 Pathogenesis-related protein 1 (gene)

PR10 Pathogenesis-related protein 10 (gene)

PRR Pattern recognition receptor

PS Pseudomonas selective agar

PTI PAMP-triggered immunity

pv. Pathovar

qPCR Semi-quantitative polymerase chain reaction

R Resistance

ROS Reactive oxygen species

rpoB RNA polymerase β-subunit (gene)

rpoD Sigma factor 70 (gene)

SA Salicylic acid

SAR Systemic acquired resistance

SDW Sterile deionised water

SEM Standard error of the mean

sp./spp. Species (plural)

STS Stilbene synthase (gene)

syp Syringopeptin (gene)

syr Syringomycin (gene)

T3SS Type III secretion system

TTE Type III effector

UPGMA Unweighted Pair Group Method with Arithmetic Mean

VvJAZ5 V. vinifera JAZ class 5 (gene)

VvTL1 V. vinifera thaumatin-like protein 1 (gene)

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Nomenclature

In this thesis the use of P. syringae strictly refers to all pathovars of P. syringae.

Individual pathovars of P. syringae are indicated by their abbreviated name. For

example, P. s. syringae, P. syringae pv. syringae; P. s. tomato, P. syringae pv. tomato

etc.

DELLA jasmonic acid repressors are referred to in Chapters 1, 4 and 5. These families

of proteins are named after their central amino acid structure and are not abbreviations.

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List of Figures

Fig. 1.1 Structure of syringolin A 16

Fig. 1.2 Structure of syringomycin and syringopeptin 18

Fig. 1.3 Virulence factors produced by P. syringae that target aspects of

plant immunity 47

Fig. 2.1 Vineyard symptoms of bacterial inflorescence rot 60

Fig. 2.2 Symptoms of P. s. syringae infection on leaves from potted

grapevines 62

Fig. 2.3 Phylogenetic relationships between Pseudomonas spp. based on

rpoB sequence 70

Fig. 2.4 Phylogenetic relationships between Pseudomonas spp. based on

gapA, gltA, gyrB and rpoD concatenated MLST data 71

Fig. 2.5 Pseudomonas syringae pv. syringae from eight cool climate

Australian winegrape vineyards 73

Fig. 3.1 GATTa characterisation of P. syringae phenotypes 87

Fig. 3.2 Pecto- and proteolytic activity of Pseudomonas spp. 89

Fig. 3.3 Hypersensitivity reaction in tobacco leaves 91

Fig. 3.4 Pathogenicity test on mature lemon 92

Fig. 3.5 Pathogenicity test on detached grapevine leaves 93

Fig. 3.6 Determination of syringomycin and syringopeptin production 95

Fig. 3.7 PCR products amplified with syrB, sylC, sypC and cfl primers 105

Fig. 3.8 Phylogenetic tree of P. s. syringae isolates and distribution of

pathogenicity, antibiotic resistance and toxin phenotypes 107

Fig. 4.1 Lesion development in Chardonnay leaves treated with water, non-

pathogenic and pathogenic P. s. syringae 135

Fig. 4.2 Lesion development in grapevine leaves infected with P. s. syringae 136

Fig. 4.3 Histochemical analysis of Chardonnay leaves infected with

P. s. syringae 138

Fig. 4.4 Effect of inoculation with P. s. syringae on cellular defence

response in grapevine leaves 139

Fig. 4.5 Housekeeping gene expression 142

Fig. 4.6 Transcript accumulation of PR1 145

Fig. 4.7 Transcript accumulation of PR10 146

Fig. 4.8 Transcript accumulation of VvTL1 147

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Fig. 4.9 Transcript accumulation of VvJAZ5 148

Fig. 4.10 Transcript accumulation of ETR2 149

Fig. 4.11 Transcript accumulation of STS 150

xvi

List of Tables

Table 2.1 Primers used for rpoB and MLST 56

Table 2.2 LOPAT identification of P. syringae 61

Table 2.3 Characteristics of isolates of P. s. syringae from Australian

vineyards with symptoms of BIR 64

Table 3.1 Primers used for detection of gyrA, syrB, sylC, sypC and cfl genes 97

Table 3.2 Biochemical and antibiotic reactions of P. s. syringae from

grapevine and other P. syringae 100

Table 3.3 AMOVA between sample populations using MLST data 110

Table 3.4 AMOVA between sample populations using MLST data from BIR

affected grapevine P. s. syringae isolates 111

Table 4.1 Sequence of V. vinifera primers used for qPCR 132

xvii

List of Publications

Hall, S.J. (2012). Vitis vinifera defence system interactions with Pseudomonas syringae

and Botrytis cinerea. Oral presentation at Crush 2012, the Grape and Wine Science

Symposium. Adelaide, Australia

Hall, S.J. & Whitelaw-Weckert, M.A (2013). Pseudomonas syringae pv. syringae.

Australasian Plant Pathology Society Pathogen of the Month – December 2013.

Hall, S.J., Dry, I.B., Blanchard, C.L., & Whitelaw-Weckert, M.A. (2014). Pseudomonas

syringae pv. syringae isolates causing bacterial inflorescence rot and the grapevine

response. Poster presentation at the Australian Society for Biochemistry and Molecular

Biology COMBIO 2014 conference. Canberra, Australia.

Hall, S.J., Dry, I.B., Blanchard, C.L., & Whitelaw-Weckert, M.A. (2016). Phylogenetic

relationships of Pseudomonas syringae pv. syringae isolates associated with bacterial

inflorescence rot in grapevine. Plant Disease, 100(3), 607-616.

1

Chapter 1 Review of the Literature

1.1 Introduction

Pseudomonas syringae van Hall pv. syringae (P. s. syringae) causes extensive

yield losses in wine-grape production in some Australian cool climate viticultural

regions. This pathogen causes Bacterial Inflorescence Rot (BIR), a relatively new

disease to grapevine, characterised by leaf spots, necrotic lesions, and necrosis of

inflorescences in spring leading to loss in crop yields (Hall et al., 2016;

Whitelaw-Weckert et al., 2011).

This review begins by describing existing examples of P. s. syringae plant

infection and the effects this pathogen has on Vitis vinifera hosts. The remainder

of the review investigates evidence that has shaped our understanding of

P. s. syringae-host interactions, and the plant defence response to P. syringae.

The intent is to demonstrate the plant-pathogen interactions, potential host range,

and impact on crop production in Australia of this bacterium, particularly in cool

climate vineyards.

In the past decade, wine grapes (V. vinifera) grown in the Tumbarumba

viticultural region of south eastern New South Wales have been affected by a new

bacterial disease. Grapevine symptoms include leaf spots, necrotic lesions on leaf

blades and shoots, and loss of inflorescences early in the season. The disease,

‘bacterial inflorescence rot’ (BIR), is caused by bacterium Pseudomonas syringae

pv. syringae (P. s. syringae) (Whitelaw-Weckert et al., 2011). The Tumbarumba

region has a ‘cool/moderate’ climate and is prone to spring frosts (Bureau of

2

Meteorology, 2012) so that overhead water sprinkler systems are commonly used

to prevent frost damage. It has been proposed that the promotion of humid foliar

microclimates by the overhead sprinkler systems may be responsible for some of

the grapevine symptoms (Whitelaw-Weckert et al., 2011).

1.2 Pseudomonas syringae plant diseases

Pseudomonas syringae is a phytopathogenic bacterium associated with over 180

species of annual and perennial crops (Prokič et al., 2012; Zhao et al., 2015). By

2010 more than 50 known pathovars of P. syringae had been described (Bultreys

& Kaluzna, 2010; Studholme, 2011; Young, 2010). This large number of

pathovars involving variations in P. syringae symptomatology and host range

provides an exceptional opportunity to study virulence and host specificity (Gašić

et al., 2012; Hwang et al., 2005). Considerable variation in host range may occur

between and within the P. syringae pathovars (Sawada et al., 1999). For example,

P. syringae pv. tomato (P. s. tomato) causes a hypersensitivity response (HR) in

Arabidopsis and tomato, but not bean, whereas P. syringae pv. phaseolicola

(P. s. phaseolicola) can cause HR in bean and Arabidopsis but not in tomato (Feil

et al., 2005).

In Australia, P. syringae has been recorded as the causal agent for bacterial

canker of olive in South Australia (Warcup & Talbot, 1981). In other host

species, Peters et al. (2004) demonstrated the importance of P. syringae pv.

maculicola (P. s. maculicola) in Brassica spp. dating from 1978 in Wagga

Wagga, NSW. Also demonstrated was the variation in pathogenicity that can

3

occur within pathovars (Peters et al., 2004). In Australia, other P. syringae

pathovars such as porri have been associated with bacterial blight in leek (Noble

et al., 2006) and in more recent years pv. actinidiae in kiwifruit (Everett et al.,

2011).

1.3 Pseudomonas syringae pv. syringae plant diseases

Pseudomonas syringae pv. syringae (P. s. syringae) belongs to genomospecies 2

within the P. syringae complex (Baltrus et al., 2011) and is known as a

widespread pathogen of a large number of hosts. Where other pathovars of

P. syringae have narrow host ranges, P. s. syringae is pathogenic to a large

number of horticultural plant hosts world-wide (Bultreys & Kaluzna, 2010)

including mango (Golzar & Cother, 2008), stone fruits (Abbasi et al., 2013),

apple (Mansvelt & Hattingh, 1989), pear (Moragrega et al., 2003), pumpkin

(Balaž et al., 2014), and lychee (Afrose et al., 2014).

In Australia, P. s. syringae causes significant economic damage to non-

viticultural crops. In the 1980s P. s. syringae was reported to be the cause of

bacterial canker in leaves, buds and shoots of apricot, cherry and other stone

fruits across Victoria (Wimalajeewa & Flett, 1985). More recent studies have also

identified P. s. syringae as a problem in other crops across Australia, such as

mango in Western Australia (Golzar & Cother, 2008), wheat in northern and

southern areas of Australia (Murray & Brennan, 2009), field pea in south eastern

Australia (Richardson & Hollaway, 2011), olive in South Australia (Hall et al.,

2003), and fenugreek in Victoria (McCormick & Hollaway, 1999). In mango

4

hosts, P. s. syringae causes necrosis of the stem tip, flowers, buds, leaf tissues

(Golzar & Cother, 2008) and stem cankers (Young, 2008). Bacterial blight

(brown leaf spots) is commonly seen in wheat and field pea hosts (Murray &

Brennan, 2009; Richardson & Hollaway, 2011), necrotic lesions are observed in

olive stems (Hall et al., 2003) and in fenugreek hosts the stems, petioles and

leaves exhibit necrotic lesions (McCormick & Hollaway, 1999). In Australia,

P. s. syringae was originally thought to be a weak pathogen, but it is now classed

as a reportable disease in Northern Territory (Australia, 2013). Although widely

distributed, the presence of P. s. syringae in vineyards is regarded as recent

within the Australian wine industry.

1.3.1 Pseudomonas syringae pv. syringae identification

The P. syringae pathovars are traditionally defined by factors such as host of

isolation, host range (Baltrus et al., 2011), biochemical (Lelliott et al., 1966) and

taxonomic analysis (Young, 2010). Molecular genetic analyses of P. syringae

pathovars has been used extensively to produce a repertoire of knowledge on

microbial pathogenicity and plant defence responses. In addition, genome wide

comparative analysis between pathovars has expanded our understanding of host

specificity and the evolution of this pathogen (Feil et al., 2005). The classification

of P. s. syringae has traditionally been achieved using phenotypic methods such

as biochemical analyses, host range and disease symptoms on the affected host

(Lelliott et al., 1966; O’Brien et al,. 2011; Studholme, 2011; Young, 2010).

Detection of pyoverdin production using fluorescence techniques is generally the

first step in the identification of plant pathogenic Pseudomonas spp. isolated from

a number of different hosts (Bultreys & Kaluzna, 2010; Gilbert et al., 2009;

5

Lelliott & Stead, 1987; Whitelaw-Weckert et al., 2011). Levan, oxidase, potato

soft rot, arginine dihydrolase, and tobacco leaf hypersensitivity reaction (LOPAT)

tests can then be used to identify P. syringae from other Pseudomonas species

(Hall et al., 2016; Lelliott & Stead, 1987). Methods commonly used for pathovar

discrimination of P. syringae strains are the use of gelatin liquefaction, aesculin

hydrolase, tyrosinase activity and tartaric acid utilisation (GATTa) tests (Gašić et

al., 2012; Jones, 1971; Lelliott et al., 1966).

Some phenotypic tests are not always informative, and avirulent P. s. syringae

isolates can produce atypical results, leading to incorrect identification. To

circumvent this, multiple testing on different hosts should be considered to

determine accurate host range and pathogenicity (Bultreys & Kaluzna, 2010;

Gašić et al., 2012). For example, although GATTa tests can be used for

discrimination between the P. syringae pathovars syringae, morsprunorum, and

persicae (Gašić et al., 2012; Whitelaw-Weckert et al., 2011), results for other

pathovars are unknown and there may be an overlap in results within the

P. syringae complex (Gilbert et al., 2009; Vicente & Roberts, 2007). It is

therefore wise to use phenotypic tests in conjunction with some of the more

advanced techniques that have now become more widely available, to increase

the sensitivity and discrimination between pathovars.

1.3.2 Molecular characterisation

Phylogenetic analysis using polymerase chain reaction (PCR) fingerprinting

methods has played an important role in demonstrating hierarchical clustering of

bacterial pathogens, including P. syringae (Clarke et al., 2010; Gardan et al.,

6

1999; Hwang et al., 2005; Sarkar et al., 2006; Sawada et al., 1999; Whitelaw-

Weckert et al., 2011; Yamamoto et al., 2000). Recently, phylogenetic analysis

using multi-locus sequence typing (MLST) has become an integral tool in

bacterial evolution analysis studies. MLST involves the concatenation of a

number of core genome sequences that are ubiquitous among all strains of a

bacterial species, and which are essential for the survival of the organism (Hwang

et al., 2005). These housekeeping genes are chosen on the basis of being less

prone to horizontal gene transfer and providing insight into the evolutionary

history of bacteria (Hacker & Carniel, 2001). This has been demonstrated by

Sarkar and Guttman (2004) who determined the evolutionary history of 60

P. syringae isolates using seven housekeeping genes, such as aconitate hydratase

B (acnB), phosphofructokinase (pkf), phosphoglucoisomerase (pgi), and others.

Hwang et al. (2005) later refined the number of housekeeping genes to four

(citrate synthase, cts also known as gltA; glyceraldehydes-3-phosphate

dehydrogenase, gapA; DNA gyrase B, gyrB; and sigma factor 70, rpoD) in an

effort to obtain similar results at a lower cost. The sampling of diverse genomes

within a phylogenetic framework can reveal general evolutionary trends

indicative of changes in lifestyle (Clarke et al., 2010; Hwang et al., 2005). This

also allows for the identification of genetic changes that differentiate between

populations that have recently undergone host range shifts (Sarkar et al., 2006).

1.3.3 Genetic diversity

High variation among strains of P. s. syringae has been demonstrated using PCR

fingerprinting methods (Scortichini et al., 2003). Studies have also demonstrated

genetic differentiation between P. s. syringae populations from pear/ stone fruit

7

and from other host plants (Little et al., 1998), although these studies analysed

restriction fragment length polymorphism, which produces a genetic fingerprint

that may not produce the discrimination of MLST (Behringer et al., 2011).

Several studies have utilised MLST to detail and clarify whether host range and

P. syringae pathovar can be expressed phylogenetically (Berge, et al., 2014;

Clarke et al., 2010; Hwang et al., 2005; Martín-Sanz et al., 2013; Sarkar et al.,

2006; Sarkar & Guttman, 2004). Analysis by MLST, of P. syringae isolated from

numerous plant hosts, has shown that this bacterium is highly clonal and stable as

a species (Sarkar & Guttman, 2004). Furthermore, the high level of genetic

variation could only weakly predict associations with host species. Interestingly,

pathogenic and non-pathogenic P. syringae can be phylogenetically separated.

Clarke et al. (2010) demonstrated that non-pathogenic P. syringae, lacking the

hypersensitivity response and pathogenicity/hypersensitivity response and

conserved (hrp/hrc) loci (required for virulence) are phylogenetically separate

from pathogenic P. syringae with intact hrp/hrc loci. Studies on P. s. syringae

isolates from pea have also demonstrated genetic diversity, but their degree of

virulence was not associated with the host of isolation (Martín-Sanz et al., 2013).

Sequencing of P. s. syringae genomes across host species has not been deep

enough to uncover trends indicating evolutionary differentiation among groups

(Lindeberg et al., 2009). Numerous plant species play host to P. s. syringae and

this provides extra challenges in determining evolutionary differentiation,

especially in hosts such as grapevine.

Interestingly, when applied to P. s. syringae isolates, MLST produces a general

spread across two phylogenetic clusters (Hall et al., 2016; Hwang et al., 2005;

8

Martín-Sanz et al., 2013; Sarkar & Guttman, 2004), indicating core genetic

diversity within P. s. syringae. This genetic diversity within P. s. syringae may be

a consequence of wide host range and/or evolution (Afrose et al., 2014a; Sarkar

& Guttman, 2004). Both the genetic diversity and the wide plant host range may

cause the classification of P. s. syringae to be problematic. Other P. syringae

pathovars have a narrower host range which can aid in their classification

(Bultreys & Kaluzna, 2010). Analysis of the phenotypic and genetic variability of

about 800 strains of P. syringae has demonstrated a lack of relationship between

phylogeny context and phenotype (Berge et al., 2014). Berge et al. (2014) also

point to the important variability of phenotypic traits used in the LOPAT

identification scheme among strains are closely related phylogenetically or

considered to be the same pathovar. Berge et al. (2014) provides MLST

sequences for a set of reference strains allowing users to insert their own data into

a standardised phylogenic tree.

1.3.4 Antibiotic resistance

Individual strains of P. s. syringae also vary in their ability to resist antimicrobial

compounds (Hwang et al., 2005; Sundin et al., 1993). In the past, compounds

such as streptomycin have been used to control P. syringae infection in the field

(Cooksey, 1994; Dye, 1953), but the use of streptomycin is now prohibited in

Australian vineyards (Young, 2008). Pseudomonas spp. are also known to be

highly resistant to a number of antimicrobial compounds (Neu, 1992). Moreover,

Hwang et al. (2005) indicated that P. syringae strains may come into contact with

medically important antibiotics with related resistance genes that may then spread

throughout the environment.

9

1.4 Pseudomonas syringae pv. syringae infection symptoms

It is generally accepted that P. s. syringae attacks the aerial organs of hosts:

leaves, fruit, twigs and branches. On mango P. s. syringae causes leaf symptoms

along the petiole and veins, producing a water-soaked appearance followed by

necrosis (Golzar & Cother, 2008). Other hosts, such as field pea and wheat may

produce characteristic leaf spots that may often first appear water-soaked (Hall et

al., 2002; Hall et al., 2016; Richardson & Hollaway, 2011; Whitelaw-Weckert et

al., 2011). Fruit symptoms are also commonly caused by P. s. syringae in non-

grape crops. Pear flowers can become infected appearing brown, necrotised, and

water-soaked; often abscising before mature fruit can begin to develop (Gilbert et

al., 2010). Wood and stem cankers are also seen in hosts such as mango (Young,

2008), cherry and apricot (Kotan & Şahin, 2002; Wimalajeewa & Flett, 1985),

hazelnut (Kaluzna et al., 2010) and numerous other stone fruit hosts (Abbasi et

al., 2013; Bultreys & Kaluzna, 2010; Gašić et al., 2012). These cankers develop

on branches, trunks and around spurs, branch junctions and wounds (Bultreys &

Kaluzna, 2010). Early infections of stems and branches are often described as

sunken, brown and water-soaked (Hall et al., 2003). These symptoms have been

described across a number of host species and have become the characteristic

symptoms of P. s. syringae infection (Hirano & Upper, 2000; Young, 2010).

1.5 Vitis vinifera infection

The first report of P. syringae in V. vinifera was recorded in Argentina (Klingner

et al., 1976), then in Sardinia (Cugusi et al., 1986) and later Azerbaijan (Samedov

10

et al., 1988). Symptoms described ranged from necrotic lesions on leaf blades,

tendrils, petioles and rachii (Klingner et al., 1976), to bark necrosis (Cugusi et al.,

1986) and general bacteriosis (Samedov et al., 1988). In Australia, Hall et al.

(2002) first described V. vinifera bacterial leaf spot (BLS) and stem lesions

occurring on cv. Verdelho in the Adelaide Hills, South Australia, during wet

spring conditions. The initial leaf symptoms were noted as small dark spots with

yellow haloes. The spots developed into necrotic angular lesions, delineated by

veins, which sometimes coalesced, causing chlorosis and senescence of the leaves

(Hall et al., 2002). Isolates were collected and deposited in the Australian

Collection of Plant Pathogenic Bacteria/NSW Industry and Investment Culture

Collection as DAR73915 and DAR75241. In the following season, further

infections were confirmed, with increased severity, spreading to three other

nearby South Australian cool winegrowing regions and affecting the cultivars

Cabernet Sauvignon, Viognier, Merlot, Sauvignon Blanc and Chardonnay.

Although P. s. syringae was recovered from stem lesions on these vines, there

was no reported effect on inflorescences or loss of crop and P. s. syringae was

considered to be a weak pathogen with little economic impact (Hall et al., 2002).

In more recent years, various cool climate vineyards across the New South Wales

Tumbarumba district have experienced similar BLS symptoms, along with

bacterial inflorescence rot (BIR), leading to a loss of crop yield, in the cultivars

Pinot Noir, Sauvignon Blanc, Riesling and Chardonnay (Whitelaw-Weckert et al.,

2011). Recently, BIR has now also been reported in V. vinifera table grapes in

Iran (Abkhoo, 2015). Although significant losses in crop yield have been

reported by winegrowers in the Tumbarumba region of New South Wales,

11

protocols are not in place to limit or restrict the spread of P. s. syringae across

Australian cool climate viticultural regions. The extent of this particular pathogen

within the Australian V. vinifera industry is unknown. Both the Adelaide Hills

and Tumbarumba districts are cool climate viticultural regions prone to wet

springs. In addition, Tumbarumba vineyards use water sprinklers to prevent foliar

damage from spring frosts. These conditions can promote humid microclimates

which may provide optimal conditions for infection by P. s. syringae (Whitelaw-

Weckert et al., 2011). Previous reports have shown that P. s. syringae commonly

occurs after heavy spring rainfalls and prefers cooler climates, and its spread may

be facilitated by contaminated pruning equipment (Lamichhane et al., 2014), or

by air currents and raindrop formation (Arnold et al., 2011; Morris et al., 2008).

1.6 Host plant entry

Unlike fungi, bacteria are unable to directly penetrate plant cells themselves. To

enter the apoplastic (extracellular or cell wall) spaces, bacteria may enter through

natural openings such as stomata or via wound entry. Some bacteria move using

flagella, structures that enable surface swarming and motility (Taguchi et al.,

2006; Taguchi et al., 2010). Mutations in flagella-related genes have caused

remarkable loss of virulence in P. syringae pv. tabaci (P. s. tabaci) in tobacco

plants (Ichinose et al., 2003).

Swarming of bacteria around open stomata is the most likely method for entry of

P. s. syringae into plants (Melotto et al., 2006; Whitelaw-Weckert et al., 2011).

Mechanisms underlying stomatal entry by P. s. syringae are poorly understood

12

but the production of phytotoxins may be a factor. Coronatine-producing

P. syringae pv. tomato (P. s. tomato) DC3000 can manipulate stomatal immunity

by inhibiting phytohormone induced signalling pathways (Melotto et al., 2006).

Syringolin A produced by P. s. syringae B728a also antagonises stomatal closure

entry in bean (Schellenberg et al., 2010) indicating that entry via open stomata is

a likely source of entry for bacterial pathogens (Schulze-Lefert & Robatzek,

2006). Lamichhane et al. (2014) suggested that leaf scars may also provide

inoculum of P. syringae, allowing for overwintering and subsequent blossom

colonisation. Similarly, in hazelnut the ports of entry used by P. syringae pv.

avellanae are leaf scars and lesions (Scortichini, 2002). Infection on cherry may

also occur through leaf scars during cooler temperatures and wind-driven rains

(Crosse, 1956), allowing for transport of P. syringae from leaf surfaces to leaf

scars (Lamichhane et al., 2014).

1.7 Primary virulence toxins of Pseudomonas syringae pv.

syringae

During the course of interactions between the plant host and P. s. syringae, a

repertoire of virulence-associated compounds is expressed by the bacterium.

These can include phytotoxins, effectors, proteins, antimicrobial compounds, and

pectolytic enzymes. Individual strains of P. s. syringae vary in their ability to

produce these compounds. Most pathovars of P. syringae produce one of four

primary virulence factor toxins: coronatine, tabtoxin, phaseolotoxin or

syringomycin, which contribute to chlorosis or necrosis of plant tissue (Hwang et

al., 2005). These toxins have a number of modes of action in plant cells including

13

proteasome inhibition by syringolin A (Schellenberg et al., 2010); induction of

necrosis by syringomycins and syringopeptins (Duke & Dayan, 2011); arginine

deficiency by phaseolotoxin (Bender et al., 1999); inhibition of glutamine

synthesis by tabtoxin (Thomas et al., 1983); and phytohormone molecular

mimicry by coronatine (Feys et al., 1994; Katsir et al., 2008b).

Virulence factors coronatine, tabtoxin and phaseolotoxin are not produced by

P. s. syringae. However, various P. s. syringae strains produce syringostatins,

syringotoxins, syringomycins, syringopeptins, and syringolins (Bender et al.,

1999; Donadio et al., 2007). The predominant virulence factor produced may

depend on the host plant. For example, syringostatin and syringotoxin are related

lipodepsinonapeptides produced by P. s. syringae strains isolated only from lilac

and citrus hosts, respectively (Ballio et al., 1994a; Fukuchi et al., 1992). In

contrast, syringomycins and syringopeptins are produced by most strains of

P. s. syringae (Bender et al., 1999). Both syringomycins and syringopeptins cause

electrolyte leakage by pore formation within the plasma membrane of host plant

cells (Curnev et al., 2002; Dudnik & Dudler, 2014; Duke & Dayan, 2011).

Although syringomycins are not essential for pathogenicity, their absence in

mutant P. s. syringae attenuates virulence (Xu & Gross, 1988).

1.7.1 Syringolin A

Syringolin A is an important P. s. syringae toxin capable of manipulating host

defences. It belongs to a family of proteasome inhibitors that contain a 12-

membered macrolactam ring (Ramel et al., 2009). Syringolin A causes a

hypersensitive response (HR) in tobacco (Misas-Villamil et al., 2013). Curiously,

14

applications of syringolin increased resistance to Pyricularia oryzae in rice,

despite having no antifungal effect in vitro (Wäspi et al., 1998). More recent

studies have now begun to clarify the role of syringolin A in the manipulation of

plant defence.

The proteasome plays a role regulating the Non-expression of Pathogenesis-

Related Protein 1 (NPR1) transcription factor in salicylic acid (SA) mediated

pathways, and is necessary for stomatal immunity (Spoel et al., 2009; Zeng & He,

2010). Schellenberg et al. (2010) demonstrated that syringolin A greatly reduces

Pathogenesis-Related Protein 1 (PR1) expression in Arabidopsis and counteracts

abscisic acid (ABA)-induced stomatal closure. The proteasome in its

phosphorylated state must turn over NPR1 to activate target genes in the SA-

mediated pathway (Spoel et al., 2009). Proteasome inhibition by syringolin A

maintains stomatal aperture for bacterial invasion in bean (Schellenberg et al.,

2010). Other evidence suggests that syringolin A also acts on SA-mediated

pathways by the suppression of acquired resistance in adjacent tissues (Misas-

Villamil et al., 2013), thereby promoting wound entry.

Crystallographic studies of syringolin A have demonstrated its mode of action

(Clerc et al., 2009; Pirrung et al., 2010). Syringolin A binds to all proteolytically

active sites on the proteasome (Clerc et al., 2009) and forms an irreversible

covalent adduct with the proteasome (Pirrung et al., 2010). In Arabidopsis,

syringolin A inhibited two out of three proteasome catalytic subunits (Misas-

Villamil et al., 2013), resulting in incomplete inhibition.

15

1.7.2 Biosynthesis of syringolin A

Syringolin A is a low-molecular weight molecule synthesised by a series of

peptide synthetases. Its 12-membered ring is formed by 5-methyl-4-amino-2-

hexenoic acid and 3,4-dehydrolysine (Wäspi et al., 1998) connected by a

ureidovaline (Imker et al., 2009; Ramel et al., 2009). The gene cluster sylA-sylE is

required for syringolin A biosynthesis (Fig. 1.1). The gene sylA contains a

conserved helix-turn-helix (HTH) LuxR domain at its C terminus typically found

in transcriptional activators. Disruption of sylA in P. s. syringae was shown to

abolish syringolin A accumulation (Amrein et al., 2004). The gene sylB is a

putative amino acid desaturase, responsible for the introduction of double bonds

into the moiety, such as desaturation of lysine to 3,4-dehydrolysin (Wuest et al.,

2011). Both sylC and sylD may account for the activation and condensation of

two amino acids contained within syringolin A (Amrein et al., 2004; Wuest et al.,

2011). In addition, sylC has been shown to activate amino acid monomers to

construct the ureido linkage required for syringolin activity (Imker et al., 2009).

Experiments with 13

C-labelled syringolin A concluded that syringolin A is

combined by ureidovaline synthesised and incorporated into syringolin A by sylC

gene products (Ramel et al., 2009). It is suggested that cyclisation of the final

macrolactam ring is achieved by sylD gene products (Wuest et al., 2011).

Although little work has been done on the role of sylE in syringolin synthesis in

P. syringae, there are several studies suggesting that it may be involved in the

transport of syringolin (Amrein et al., 2004; Dudler, 2013; Marco et al., 2005).

16

Fig. 1.1. Structure of syringolin A. (A) The sylA-sylE gene cluster is required for syringolin A

biosynthesis. sylA encodes transcription activation (Amrein et al., 2004), sylB encodes putative

amino acid desaturase (Wuest et al., 2011), and sylC and sylD encode non-ribosomal peptide

synthetase (NRPS) and polyketide synthetase (PKS) modules, and sylE encodes efflux transporter

(Amrein et al., 2004; Ramel et al., 2009). (B) Chemical structure of syringolin A, derived from

Donadio et al. (2007).

1.7.3 Regulation of syringolin A biosynthesis

The sylA locus is under the control of the gacS/gacA two-component regulatory

system (Heeb & Haas, 2001; Wäspi et al., 1998). sylA in turn positively regulates

the expression of sylB and sylCDE as a single polycistronic element (Ramel et al.,

2012). This gacS/gacA system is also required for the expression of syringomycin

and syringopeptin toxins, indicating overlapping pathways. Ramel et al. (2012)

have described the molecular events that occur during syringolin A synthesis and

activation. salA, under the control of the gacS/gacA two-component system in

17

planta, encodes a HTH LuxR type transcription factor. Upon detection, salA

indirectly activates sylA promoters to begin synthesis of syringolin A. External

stimuli that initiate transcription are unknown but quorum sensing may initiate

the production of syringolin for enhanced virulence and lesion formation, with

oxygen concentration playing a central role (Ramel et al., 2012).

1.7.4 Syringomycins and syringopeptins

The production of syringomycins and syringopeptins appears to be conserved

among isolates of P. s. syringae. Syringomycins belong to the family of

lipodepsinonapeptides that include syringotoxin, syringostatin and pseudomycin,

whereas syringopeptins belong to the family of lipodesipeptides. Both of these

families in P. s. syringae are believed to be synthesised as part of the non-

ribosomal peptide synthetase (NRPS) system and contain similar structures

consisting of cyclic peptide heads attached to a 3-hydroxy fatty acid tail (Fig. 1.2)

(Di Giorgio et al., 1996; Segre et al., 1989). Furthermore, the formation of

syringomycin and syringopeptin requires multi-enzymatic complexes (Grgurina

et al., 1996). The mode of action of these toxins is also similar in that they target

the plasma membrane for pore formation (Hutchison & Gross, 1997).

Syringomycins and related lipodepsinonapetides are generally produced in most

strains of P. s. syringae that have a wide host range. They contain between 22 or

25 amino acids, depending on the bacterial strain (Bender et al., 1999; Di Giorgio

et al., 1996). Related lipodepsinonapetides such as syringotoxin and syringostatin

are produced in strains that originate from citrus and lilac hosts (Ballio et al.,

18

1994b; Fukuchi et al., 1992), while saprophytic strains are reported to produce

pseudomycin (Ballio et al., 1994a).

Fig. 1.2. (A) Chemical structure of syringomycin. (B) Chemical structure of syringopeptin.

19

1.7.5 Mode of syringomycin action

Generally, syringomycins are considered to be one of the major groups of

virulence factors in P. s. syringae, inciting stem disease in monocots and dicots

within temperate growing regions (Scholz-Schroeder et al., 2001). These

compounds also exhibit antifungal activity to similar degrees (Sorensen et al.,

1996). Additionally, these compounds have been previously linked to reduced

stomatal aperture in bean (Di Giorgio et al., 1996; Iacobellis et al., 1992).

Although there are few recent reports on syringomycin and its effects on stomata,

it is generally accepted that its main effect is pore formation on the plasma

membrane (Bender et al., 1999; Hutchison & Gross, 1997; Hwang et al., 2005). It

is thought that syringomycin promotes the passive influx of H+ and Ca

2+ ions

through the transmembrane, leading to acidification of the cytoplasm, resulting in

a calcium related signalling cascade (Hutchison & Gross, 1997).

1.7.6 Syringomycin biosynthesis

Syringomycin biosynthesis, reviewed by Bender et al. (1999), is known to take

place via the NRPS pathway and involves a multi-enzyme complex. More recent

reports have indicated that arginine may play a role in the synthesis of

syringomycins and other lipodepsinonapeptides (Lu et al., 2003). The argA gene

is involved in arginine biosynthesis in Pseudomonas aeroginosa, and shares high

homology with P. s. syringae syrA, one of the genes required for syringomycin

synthesis and pathogenicity (Lu et al., 2003). Other studies have shown that the

chlorination step in syringomycin biosynthesis is important for its biological

activity (Grgurina et al., 1994).

20

1.7.7 Mode of syringopeptin action

Syringopeptins are peptides that elicit necrotic symptoms in plant host tissue.

They are considered to be more potent and forty times more active than the

lipodepsinonapeptides in causing electrolyte leakage (Iacobellis et al., 1992;

Lavermicocca et al., 1997) and induce greater ionic conductance in phospholipid

bilayers than syringomycin (Bensaci et al., 2011). High syringopeptin activity by

P. s. syringae B359 from millet has been demonstrated in several species of host

plants such as carrot (Iacobellis et al., 1992) and immature cherry (Scholz-

Schroeder et al., 2001).

1.7.8 Syringopeptin biosynthesis

Although the biosynthesis of syringopeptin is under control of the same

regulators as syringomycins, syringopeptin has its own set of biosynthetic genes.

Biosynthesis of syringopeptins is under the control of proteins encoded by sypA,

sypB and sypC genes. The role of these peptide synthetases has been detailed

previously (Scholz-Schroeder et al., 2003) and exhibit significant homology to a

family of proteins in the thio-template mechanism of biosynthesis. The same is

also true for the syr genes involved in syringomycin biothsynthesis (Guenzi et al.,

1998; Scholz-Schroeder et al., 2003). In the case of syringopeptin, there is

evidence that syrB1 is essential for syringopeptin synthesis. Mutations in this

gene result in complete inhibition of virulence, whereas mutations in syrA

exhibited only attenuated virulence (Scholz-Schroeder et al., 2001). Interestingly,

the N-terminal of syrA exhibits homology to other peptide synthetases that are

involved in antibiotic, and siderophore synthesis. This may indicate that these

genes encode a number of products to enhance pathogen virulence (Stachelhaus

21

& Marahiel, 1995; Stein & Vater, 1996). The sypC product is predicted to

catalyse the cyclisation of the lactone ring and release syringopeptin from the

synthetase (Scholz-Schroeder et al., 2003).

1.7.9 Regulation of syringomcyin and syringopeptin biosynthesis

Syringomycins, syringopeptins and syringolins share a common pathway for the

regulation of their biosynthesis. Both syringopeptins and syringomycins are

controlled by the SalA regulon (Lu et al., 2002), a regulator under the control of

the GacS/GacA two component pathway (Kitten et al., 1998). The two-

component response regulator, GacA, is considered the master regulator for the

P. s. tomato genes required for the hrp type III effector (TTE) production and

translocation. However, this role for GacA is not reported for P. s. syringae

(Chatterjee et al., 2003). In P. s. syringae, a GacS mutant study has demonstrated

that the GacS/A pathway may be involved in signal transduction of syr-syp

genomic island containing genes for syringomycin and syringopeptin production

(Wang et al., 2006b). This may indicate that the targets of GacS/A are

syringomycin and syringopeptin products instead of effectors of the T3SS. These

two genes (syr-syp) are located within the same cluster (Scholz-Schroeder et al.,

2003) indicating that syringopeptins and syringomycins may be simultaneously

regulated.

PseEF is an ATP-binding cassette (ABC) transporter, located on the boarder of

the syr-syp genomic island in P. s. syringae. Mutations of this gene in

P. s. syringae B372a have demonstrated that it is required for secretion and

expression of syringomycin and syringopeptins (Cho & Kang, 2012). Indeed

22

PseF expression is dramatically increased in bean, whereas mutants deficient in

salA have been shown to not display these increases (Cho & Kang, 2012). This

indicates that the PseEF efflux system is required not only for secretion of these

toxins, but also for expression under the control of salA. Furthermore, control of

salA is under the control of syrF for syringomycin and syringopeptin regulation

(Cho & Kang, 2012; Lu et al., 2002; Wang et al., 2006a) and syringopeptin has

not been detected in syrF and salA mutants (Wang et al., 2006a). Others have also

demonstrated overlapping expression of these genes. Mutations in the syrD

encoding ABC transporter result in strains of P. s. syringae that are defective in

these two phytotoxins (Grgurina et al., 1996).

1.8 Evolved strategies for evading host defences

Effectors are small molecules secreted by pathogens to promote their invasion

and proliferation in host tissue. The evolution of effectors in P. syringae has

developed over time, resulting in complex repertoires of effectors. Recognition

and evasion between pathogen and plant can generate highly polymorphic

repertoires of effectors and resistance (R) proteins, respectively (McHale et al.,

2006). Avirulent (avr) proteins have been identified based on their effector-

triggered immunity (ETI) phenotype conferring virulence in test plants (Keen,

1990). Effectors that are contained within these repertoires are generally

considered essential for virulence, but can be individually dispensable (Cunnac et

al., 2009). Due to this high dispensability, effectors can be easily lost under

natural field conditions in crops exhibiting R-gene protection, and this may lead

to a loss of R-gene resistance (Cunnac et al., 2009). Effector repertoires have been

23

shown to vary in size and composition between pathovars of P. syringae (Buell et

al., 2003; Feil et al., 2005; Joardar et al., 2005). Comparisons between

P. s. syringae, P. s. tomato and P. s. phaseolicola have demonstrated that

approximately 40 effectors are either unique to each pathovar or are only shared

between two (Chang et al., 2005; Vinatzer et al., 2006). This may indicate that

these unique effectors are the main determinants for host range in P. syringae.

Pathogens have evolved strategies to diminish flagella dependent detection by the

plant immune system. Mitogen-activated protein kinases (MAPKs) in

Arabidopsis are involved in a number of plant processes and are activated by

pathogen-associated molecular patterns (PAMPs). The hrp outer protein (Hop)

AI1 inhibits MAPK in Arabidopsis by removing phosphate groups from

phosphothreonine using a unique lyase activity (Zhang et al., 2007a).

Alternatively, HopF from P. syringae inhibits MAPK kinase 5 during natural

infection of Arabidopsis (Wang et al., 2010). This leads to suppression of cell

wall reinforcement and activation of PAMP triggered gene expression. In

Arabidopsis, HopAI1 from P. s. syringae has enhanced disease susceptibility by

suppressing flg22 induced NONHOST1 (NHO1), required for basal resistance on

non-host bacteria (Li et al., 2005; Zhang et al., 2007a). This indicates that PAMP-

mediated signalling is targeted by the HopAI1 effector and may contribute to the

first line of evading the plant defences in P. s. syringae.

Pseudomonas syringae can also evade basal plant immunity by reducing

expression of flagellar genes. Psyr_2711 is a diguanylate cyclase (chp8) in

P. s. syringae B728a (Feil et al., 2005) and is increased during epiphytic growth

24

(Engl et al., 2014). Chp8 (found within the hrp/hrc gene cluster of P. s. tomato)

has been shown to decrease flagellin production, and increase extracellular

polysaccharide production, escalating Arabidopsis susceptibility to infection

(Engl et al., 2014) (Fig. 1.3).

Comparative genomic studies have used P. s. syringae B728a, P. s. tomato

DC3000 and P. syringae pv. phaseolicola (P. s. phaseolicola) for comprehensive

identification of effector genes (Baltrus et al., 2011; Buell et al., 2003; Feil et al.,

2005; Joardar et al., 2005; Lindeberg et al., 2006). Subsequently, these

comparisons have demonstrated that members of P. syringae group II (e.g.

P. s. syringae B728a) contained a markedly lower number of effector repertoires

than other P. syringae groups (Lindeberg et al., 2012). Indeed, less than 26

effectors have been identified in P. syringae group II. Because of the relatively

small number of effectors compared with other P. syringae pathovars, it has been

hypothesised that P. s. syringae may rely on the production of virulence factors

that are non-T3SS based (Baltrus et al., 2011), such as syringomycins and

syringopeptins.

1.9 Plant-pathogen signalling

Signalling that occurs within populations of P. s. syringae prior to infection is

known as quorum sensing. Pseudomonas syringae pv. syringae B728a, a

pathogen of bean, grows to large numbers on leaf surfaces and needs extracellular

signalling to initiate a fundamental change from an epiphytic to a pathogenic

lifestyle (Thakur et al., 2013). Quorum sensing may rely on molecules secreted

25

by the host plant upon pathogen perception. Large quantities of specific sugars

that occur on the leaf tissue may enhance signalling activity of P. s. syringae (Mo

& Gross, 1991). Phytotoxins have been produced by P. s. syringae in vitro when

grown on minimal media containing basic plant signalling molecules (Wäspi et

al., 1998). Other studies have shown that phenolic glycosides such as arbutin,

salicin and aesculin, and sugars such as fructose, which are abundant in many

plant species, exhibit syrB1 inducing activity (Cho & Kang, 2012; Mo & Gross,

1991; Wang et al., 2006b). These molecules increase syringomycin (syr) and

syringopeptin (syp) gene expression for their synthesis (Bensaci et al., 2011).

Such reports lend credence to the supposition that pathogens such as

P. s. syringae have evolved mechanisms for invasion/disease that are activated

upon plant perception. Evidence of grapevine producing these phenolic

glycosides could not be found at the time of writing.

1.9.1 General plant defences (PAMP-triggered immunity)

Plants have evolved a complex immune system to protect themselves from biotic

stressors. The primary defence mechanism involves basal defences that provide

protection against a broad range of pathogens. The basal defence is engaged when

the host plant cell detects molecules conserved within a pathogen species,

pathogen-associated molecular patterns (PAMPs), resulting in PAMP-triggered

immunity (PTI). Certain pathogens have evolved the ability to evade or suppress

PTI through the secretion of small proteins called effectors. Some plant species

have evolved a second line of defence which is activated following recognition of

specific effectors. This secondary response is known as effector-triggered

immunity (ETI). The primary difference between PTI and ETI is the ability to

26

sense infectious-self and non-self, or microbe-mediated modifications of the host,

respectively (Malinovsky et al., 2014). Additionally, these two defence systems

may not be strictly separate and modifications in one aspect of defence can have

consequences for the other pathway.

Pathogen-associated molecular patterns are highly conserved amino acid

sequences easily recognised by eukaryotic cells. The flagellin 22 amino acid

peptide (flg22) is one of the most characterised examples used in basal defence

response in both animal and plants. Plant recognition of bacterial flagella occurs

during both epi- and endophytic growth of P. syringae, triggering calcium influx

into plant cells (Kwaaitaal et al., 2011) and SA-dependent defence mechanisms,

such as stomatal closure (Melotto et al., 2006), induction of pathogenesis-related

(PR) antimicrobial proteins (Navarro et al., 2004), and increased reactive oxygen

species (ROS)(Tanaka et al., 2003). Perception of flg22 also stimulates MAPK

phosphorylation leading to callose deposits (Lu et al., 2011; Luna et al., 2010;

Naito et al., 2007), and ethylene biosynthesis (Liu & Zhang, 2004) into to what is

known as PAMP-triggered immunity (PTI).

Pathogen associated molecular pattern-triggered immunity is mediated by the

ligand surface exposed transmembrane pattern recognition receptors (PRRs)

(Malinovsky et al., 2014). One commonly used example of a PRR is the flagellin

receptor, flagellin sensing 2 (FLS2) which recognises the 22 amino acid peptide

flg22 at the N terminus of bacterial flagellin (Felix et al., 1999). Flagellin receptor

FLS2 is also associated with BAK1, a leucine rich receptor (LRR) kinase

(Chinchilla et al., 2007; Heese et al., 2007). When a PAMP, such as bacterial

27

flg22, is perceived by plant species, early and late responses are activated. These

responses include rapid ion fluxes across the plasma membrane, oxidative burst

(Trdá et al., 2014), and activation of mitogen activated and calcium-dependent

protein kinases (Asai et al., 2002), which begin downstream signalling (Bigeard

et al., 2015; Wu et al., 2014). The PAMP-triggered immunity has been shown to

develop within a few hours of recognition of PAMPs or type III secretion system

(T3SS)-deficient bacteria by plant cells (Cunnac et al., 2009). In Arabidopsis,

flagella-induced receptor kinase (FRK1) is associated with PAMP-responsive

microRNAs (Cunnac et al., 2009). In grapevine, little is known about the

perception of flg22, but recent evidence suggests that a higher level of immunity

is triggered in response to flg22 measured by the expression of PR genes (Trdá et

al., 2014). The evasion of PTI by a pathogen usually requires the microorganism

to secrete a range of effector proteins that can either modulate or suppress PTI

components (Jones & Dangl, 2006).

One of the earliest plant defence responses is the increased influx of H+ and Ca

2+

ions leading to membrane depolarisation (Wendehenne et al., 2002). The

cytosolic increase in Ca2+

concentrations can also act as secondary messenger for

other membrane channels (Blume et al., 2000) and calcium-dependent protein

kinases involved in ethylene signalling (Ludwig et al., 2005). These changes in

cytosolic Ca2+

may also precede stomatal closure (Irving et al., 1992).

Mitogen-activated protein kinases activate WRKY-type transcription factors

(Asai et al., 2002) and lead to the phosphorylation of proteins. Phosphorylation of

flg22-responsive MAPKs may then target respiratory burst oxidase homologue D

28

(RbohD), a NADPH oxidase that mediates oxidative burst (Benschop et al., 2007;

Nühse et al., 2007). During oxidative burst, ROS produced may then function

both as signalling molecules and executioners (Tsuda & Katagiri, 2010). Their

role in PTI is generally considered to be rapid and transient, whereas in ETI-type

responses, ROS production is biphasic with a low first stage before a second

higher more sustained increase (Torres et al., 2006). The early oxidative burst

contributes to cell wall linking and as secondary messengers in plant defence

signalling (Apel & Hirt, 2004).

It is generally considered that callose deposition creates a physical barrier (Liu et

al., 2015). Callose deposition is an early response that hinders pathogen invasion

of healthy tissue (Ellinger et al., 2013). In stomata, callose deposition has been

hypothesised to limit nutrient exchange and prevent the invasion of healthy

stomata (Kortekamp et al., 1997; Toffolatti et al., 2012). Callose deposition and

the formation of papillae (callose-containing cell-wall appositions) are markers of

PTI response after treatment with PAMPs in response to pathogen attack (Liu et

al., 2015; Voigt, 2014). Papillae were shown to play an active role in resistance to

pathogen penetration in Arabidopsis (Ellinger et al., 2013). In addition, callose

deposition around stomata may prevent infection of healthy stomata (Kortekamp

et al., 1997; Toffolatti et al., 2012). In grapevine, callose has been shown to

provide a physical barrier, limiting further penetration into host cells and

preventing further pathogen growth (Liu et al., 2015). Callose deposition also

develops on stomata when grapevines are infected with downy mildew pathogen

Plasmopara viticola (Liu et al. 2015; Gindro et al. 2003) and P. s. syringae

29

(Whitelaw-Weckert et al., 2011). However, there has been much controversy

regarding the role of callose within the plant defence response (Luna et al., 2010).

1.9.2 General plant defences (effector-triggered immunity)

Induced plant responses may not occur until pathogen challenge is established

later in infection (Van Loon, 1997). Pathogen induced plant responses are thought

to arise in the tissue surrounding the initial infection site, through the action of

signalling molecules such as salicylic acid (SA), jasmonic acid (JA) and/or

ethylene (ET). It is generally accepted that SA signalling is up-regulated in

response to pathogens with a biotrophic lifestyle, whereas JA/ET signalling is

upregulated in response to necrotrophic pathogens, UV damage, and wounding

(Glazebrook, 2005; Koornneef & Pieterse, 2008). However these phytohormones

also play roles in plant growth, senescence and numerous other developmental

processes. The final outcome of these in defence responses can be greatly

influenced by the composition, timing and concentration of the hormones

produced (De Vos et al., 2005; Koornneef & Pieterse, 2008; Leon-Reyes et al.,

2010). The mechanisms regulating these pathways, although extensively studied

in Arabidopsis, are poorly understood in grapevine.

Effector-triggered immunity (ETI) has been described as an accelerated and

amplified PTI response, resulting in disease resistance and usually a

hypersensitive reaction (HR) (Greenberg & Yao, 2004; Thilmony et al., 2006).

Effector-triggered immunity occurs when a pathogen can bypass or evade basal

immunity, such as PTI. It is generally accepted that ETI is a result of a resistance

(R) gene recognising a corresponding virulence promoting protein delivered by an

30

invading pathogen (Jones & Dangl, 2006). These R genes mostly encode

nucleotide binding leucine-rich repeat (NB-LRR) disease resistant proteins

(Collier & Moffett, 2009; Meyers et al., 2003) that recognise specific effectors

(Avr’s). The recognised Avr with its corresponding R gene/NB-LRR protein

imitates strong ETI signalling (Ellis et al., 2007; Tsuda & Katagiri, 2010). This

activation of specific NB-LRRs often results in a network of cross-talk between

response pathways maintained by SA and jasmonic acid (JA) (Glazebrook, 2005).

This may then allow for the differentiation between pathogens with a

necrotrophic lifestyle (those that gain nutrients from dead tissue) from pathogens

with a biotrophic lifestyle (those that gain nutrients from living tissue) (Jones &

Dangl, 2006).

1.9.3 Auxins

Auxins are involved in just about every aspect of plant development. Transgenic

Arabidopsis constitutively expressing the P. syringae type III effector (TTE)

AvrRpt2 from P. s. tomato DC3000, produces phenotypes of altered auxin

signalling characterised by gravitropism (growth movement) defects, less

pronounced apical hooks and cotyledons that open to a greater extent than control

seedlings (Chen et al., 2007), confirming their role in plant development.

Furthermore, auxins induce the suppression of SA biosynthesis and signalling

(Chen et al., 2004; Navarro et al., 2006; Robert-Seilaniantz et al., 2007; Wang et

al., 2007), up-regulate ethylene (ET) (Abel et al., 1995), and stimulate cell wall

changes to increase permeability (Chen et al., 2007). P. s. tomato DC3000

carrying AvrRpt elevates auxin signalling in susceptible Arabidopsis and

promotes virulence (Chen et al., 2007). Production of auxins by micro-organisms

31

may provide a desirable avenue for manipulation of SA-mediated plant defences.

Production of auxins is a common trait in P. syringae pathovars but the quantity

produced varies (Glickmann et al., 1998).

Indole-3-acetic acid (IAA) is the primary auxin produced in plant tissue infected

with P. s. tomato DC3000 (O'Donnell et al., 2003). Most pathovars of P. syringae

do not produce IAA, but strong production is observed in those that do

(Glickmann et al., 1998). P. syringae may also increase IAA in plant tissues

without direct production by the bacterium. P. s. syringae B728a contains an

arylacetonitrilase capable of hydrolysing plant indole-3-acetonitrile to IAA

(Howden et al., 2009). Such induction of plant IAA promotes stomatal opening

(Irving et al., 1992). Although auxin production by pathogenic bacteria is a useful

tool for manipulation of plant defences, other effectors have also been shown to

lead to perturbations in defence signalling by auxin modulation.

1.9.4 Gibberellins

Gibberellins (GA) such as gibberellic acid promote gene expression by relieving

the restraint imposed on DELLA growth repressor proteins (Robert-Seilaniantz et

al., 2010; Sun & Gubler, 2004). The stabilisation of DELLAs has been shown to

contribute to flg22 induced growth inhibition in Arabidopsis (Navarro et al.,

2008) demonstrating that DELLA function is also involved in plant defences.

Furthermore, GAs repress SA and promote susceptibility to virulent P. s. tomato

DC3000 (Navarro et al., 2008).

32

Proteasome degradation prevents the induction of GA metabolism (Frigerio et al.,

2006). Proteasome inhibition by syringolin A-producing P. syringae may also act

on GA and auxin pathways. Additionally, auxins promote GA-mediated DELLA

degradations by expression of GA metabolism genes (Frigerio et al., 2006; Fu &

Harberd, 2003). Attenuation of auxins may also delay GA-mediated DELLA

stabilisation (Fu & Harberd, 2003), reducing the concentration of DELLAs and

their growth restraining effects (Frigerio et al., 2006). This suggests that

crossover between the auxin, DELLA and GA pathways increases targets for

pathogen manipulation perhaps to promote susceptibility. Because the

mechanisms behind GAs, SA suppression and P. s. syringae are not clearly

understood, there are opportunities and targets for future investigations on

pathogen-phytohormone interactions.

1.9.5 Abscisic acid

Abscisic acid (ABA) controls a number of physiological processes in plants

including grape berry ripening (Böttcher et al., 2013) and abiotic stress tolerance

(Ton et al., 2009). The inactive glucose ester conjugate of ABA is highly mobile

and activated when it reaches its target tissue (Ton et al., 2009). Once at its target

tissue, the ABA responses may be separated into three phases depending on the

nature of the invasion (i.e whether it is bacterial or fungal etc). Phase one results

in stomatal closure (Melotto et al., 2006; Sun et al., 2014), phase two leads to

callose deposition (Ton & Mauch-Mani, 2004) and phase three is late defences

such as SA/JA signalling (Adie et al., 2007).

33

ABA also plays an important role in plant defence signalling. Infection of

Arabidopsis with P. s. tomato has been shown to lead to an increase in ABA

levels (de Torres-Zabala et al., 2007). Some have proposed that HopAM1 from

P. s. tomato manipulates ABA responses to enhance virulence (Goel et al., 2008).

In a report by de Torres-Zabala et al. (2007), P. s. tomato was found to secrete

effectors that target ABA signalling.

The accumulation of ABA by P. s. tomato in Arabidopsis resulted in the

suppression of many genes in the SA/phenylpropanoid biosynthesis plant defence

pathway (Mohr & Cahill, 2007). Earlier studies showed that ABA enhanced the

susceptibility of P. s. tomato in Arabidopsis (Mohr & Cahill, 2003) and

suppressed SA-dependent signalling (Audenaert et al., 2002). Others have

demonstrated that ABA suppressed the induction of SA-mediated system

acquired resistance (SAR) (Yasuda et al., 2008). Furthermore, endogenous

applications of benzothiadiazole (BTH; a SA analogue) reversed ABA

accumulation induced by abiotic stress (Yasuda et al., 2008).

Negative regulation between ABA and JA/ET pathways, reported by Anderson et

al. (2004), also provides important insights into the plant-pathogen interactions

that may result in enhanced susceptibility. Exogenous application of ABA has

resulted in suppression of JA-mediated genes (Adie et al., 2007; Anderson et al.,

2004). Taken together, these studies demonstrate that elevated ABA may increase

susceptibility to P. syringae (by negatively regulating either the JA/ET or SA

defence pathways) and that some pathogens may target ABA signalling for

invasion.

34

1.9.6 Cytokinins

Plant cytokinins (CKs) are phytohormones derived from adenine. They are

involved in the regulation of root and shoot growth and leaf longevity (Robert-

Seilaniantz et al., 2010) and in inducing plant resistance to mainly hemi-

biotrophic pathogens (Argueso et al., 2012; Choi et al., 2010; Großkinsky et al.,

2014). Cytokinins may indirectly inactivate GA to promote DELLA stabilisation

through induction of GA2 oxidase (Jasinski et al., 2005). Plant CKs are

recognised by histidine kinases similar to the gacS/gacA two-component system

in bacteria (Müller & Sheen, 2007; To & Kieber, 2008) and have been correlated

with increased resistance to P. s. tomato, SA biosynthesis, and PR1 expression in

Arabidopsis (Choi et al., 2010). Furthermore, others have demonstrated that

treatment with kinetin (a CK) suppresses symptom development in tobacco

infected with P. s. tabaci (Großkinsky et al., 2014).

Mutations in plant SA biosynthesis genes have negative consequences on PR1

expression but have no effect on CKs. Interestingly, over-expression of CK

oxidase/dehydrogenase genes increases SA signalling (Igari et al., 2008),

indicating that they may play roles upstream of SA-mediated defence signalling.

This has been substantiated by Naseem et al. (2013) who showed that CK

promoted SA-mediated plant immunity (Naseem et al., 2013). Interactions

between SA signalling and CKs have been described with CKs up-regulating

plant defences by SA-dependent responses, which, in turn, then inhibit CK

signalling (Argueso et al., 2012; Choi et al., 2010).

35

In contrast to the effects of plant CKs inducing plant resistance to pathogenic

bacteria, CKs produced by P. savastoni and other gall-forming bacteria contribute

to their virulence (Kennelly et al., 2007). The P. s. tomato DC3000 effector

HopQ1 has been shown to interfere with cytokinin signalling in Arabidopsis

(Hann et al., 2014). These responses suggest that CK signalling may lead to

suppression of FLS2 accumulation and thus, PTI-mediated signalling (Hann et

al., 2014). Studies undertaken by others suggested that plant CKs degrade ABA

to phaseic acid rather than inhibiting its synthesis (Cowan et al., 1999;

Großkinsky et al., 2014). This suggests that CKs may antagonise ABA-mediated

stomatal closure (Tanaka et al., 2006) allowing for pathogen entry via open

stomata.

1.9.7 Phytoalexins and stilbenes

Phytoalexins are antimicrobial compounds that accumulate after inoculation with

a plant pathogen. An important class of phytoalexins in V. vinifera, stilbenes

(Timperio et al., 2012), are constitutively expressed (Jeandet et al., 2002) but are

significantly increased by pathogen challenge. In recent literature, it has been

shown that cultivar-specific phytoalexin responses can occur. Indeed, Cabernet

Sauvignon has been demonstrated to express higher levels of stilbene, trans-

resveratrol, than Merlot following fungal challenge (Timperio et al., 2012).

Infiltration of V. vinifera leaves with P. syringae pv. pisi (P. s. pisi) causes

induction of stilbene synthase (STS), followed by accumulation of resveratrol

(Robert et al., 2001). Similarly, Botrytis cinerea and Plasmopara viticola

treatments strongly induced STS in V. vinifera leaves (Chong et al., 2008; Le

Henanff et al., 2009). These studies indicate that phytoalexins and stilbene

36

expression may be a common trait across pathogenic microbe studies in

V. vinifera plants.

1.9.8 Reactive oxygen species

Reactive oxygen species (ROS) are small molecules, generated in response to

eukaryotic cell stress, that play diverse roles as intracellular messengers (Finkel,

1998). Several studies have demonstrated the effectiveness of bacterial flg22

peptide in increasing ROS production in plant leaf tissue (Chang & Nick, 2012;

Felix et al., 1999; Torres, 2010). Nicotinamide adenine dinucleotide phosphate

(NADPH) oxidases are one source of ROS generation during plant-pathogen

interactions (Mammarella et al., 2014; Torres et al., 2002; Wu et al., 2014). In

Arabidopsis, mutations in reactive oxygen intermediates exhibit reduced

programmed cell death capacity when infected with avirulent P. s. tomato

DC3000 (Torres et al., 2002) showing that ROS play a major role in PTI-

mediated immune responses and HR, an induced localised necrosis of the site of

infection to limit the spread and multiplication of invading pathogens (Jakobek &

Lindgren, 1993; Klement, 1963). ROS have been suggested to not only kill the

pathogen directly but mediate pH changes and ion fluxes, such as Ca2+

, that may

lead to the specific signalling cascades in plants (Jabs et al., 1997; Torres, 2010).

Plant ROS may also be enhanced by phytohormone signalling pathways (Torres,

2010). Accumulation of DELLA promotes JA signalling and DELLA deficiency

increases SA signalling (Robert-Seilaniantz et al., 2010). The induction of

DELLAs and JA signalling promotes expression of the genes involved in ROS

detoxification, thereby reducing ROS levels (Achard et al., 2008;

37

Sasaki‐Sekimoto et al., 2005). Alternatively, early SA signalling in tobacco

potentiates the production of H2O2 in response compatible P. syringae

interactions (Mur et al., 2000). Others have hypothesised that the action of ROS

production enhances SA signalling (Chen et al., 2011). In the manner of ROS

production and phytohormone signalling, a biphasic oxidative burst is commonly

observed. This biphasic oxidative burst may allow for increased plant signalling

for augmented resistance to P. s. tabaci (Mur et al., 2005). This may explain

biphasic oxidative bursts that have been observed in plants in response to

pathogenic P. syringae interactions (Pham & Desikan, 2012).

1.9.9 Ethylene

Ethylene (ET) is a gaseous hormone in higher plants involved in plant

senescence, flowering, fruit ripening, leaf expansion, abscission of various organs

and other developmental and growth processes (Belhadj et al., 2008; Chang &

Shockey, 1999; Wang et al., 2002; Yoo et al., 2009). The role of ET in plant

development has been demonstrated in plants with mutated ET pathways

producing stunted phenotypes (Shapiro & Zhang, 2001). Regulated by other

hormones such as auxin and gibberellins for its synthesis, ET is enhanced during

periods of wounding or pathogen attack (Yoo et al., 2009) and early plant

defences (Ludwig et al., 2005). Emerging evidence has suggested that ET

sensitivity differs in different plant tissues and in response to endogenous and

environmental signals (Alonso & Stepanova, 2004; Yoo et al., 2009), but its

diverse and complex functions are still unclear. The role of ET in plant defence

has become controversial, with some authors indicating that ET plays a role with

JA in the resistance to necrotrophic pathogens, whereas others have indicated that

38

ET-induced senescence promotes necrotrophic infection (Govrin et al., 2006;

Thomma et al., 1999).

Expression of PR proteins in V. vinifera is increased in response to ET treatment.

During ethylene-releasing ethephon treatment of Cabernet Sauvignon, PR

proteins including acidic class IV chitinases (Chit4C), serine protease inhibitor

(PIN), polygalacturonase-inhibiting protein (PGIP) and β-1,3-glucanases (GLU)

were up-regulated rapidly and powdery mildew growth was decreased (Belhadj et

al., 2008). Interestingly, PGIPs are known to interact with fungal

endogalactouronases to inhibit their enzymatic activity (De Lorenzo et al., 2001).

Botrytis cinerea infection in Chardonnay has also been shown to up-regulate

PGIPs (Bézier et al., 2002). Others have also shown an increase in chitinase

activity on V. vinifera cv. Sultana in response to ethephon treatments (Jacobs et

al., 1999).

It is generally accepted that ET and JA pathways cooperate against necrotrophic

pathogens, and may antagonise SA meditated pathways. Concomitant activation

of JA and ET pathways has been demonstrated by the induction of plant defensin

1.2 (PDF1.2) in Arabidopsis (Penninckx et al., 1998). Similarly, protein analysis

in V. vinifera has demonstrated overlap between JA and ET in response to

powdery mildew (Yao et al., 2012), indicating that these two pathways may exert

their effects synergistically during the plant defence response.

In plants, ethylene receptors, located in the endoplasmic reticulum, share

sequence similarity with bacterial two-component histidine kinase (Alonso &

39

Stepanova, 2004). This may allow bacteria to manipulate plant defences to

establish infection or promote a hospitable environment within the plant.

Alternatively, virulent P. syringae are able to activate transcription of ET

response factor genes (ERF) in a phytotoxin- and TTE-dependent manner (He et

al., 2004). The promotion of JA and ET-signalling pathways may then open up an

important avenue for P. syringae parasitism in host plants.

1.9.10 Salicylic acid

Salicylic acid (SA) is a small phenolic compound that plays a major role in the

regulation of systemic acquired resistance (SAR) in plants and resistance to

biotrophic pathogens that require living plant tissue for their nutrients. The

activation of SA-dependent pathways usually results in the expression of genes

encoding PR proteins, such as PR1, PR2 and PR10 (Leon-Reyes et al., 2010b;

Ziadi et al., 2001), which can lead to increased resistance and may have

antimicrobial properties.

Salicylic acid signalling is believed to mediate the resistance to biotrophic

pathogens such as Erysiphe orontii and P. syringae (Thomma et al., 2001). Early

experiments on P. s. syringae in cucumber demonstrated that single point

inoculation can result in SAR (Rasmussen et al., 1991). Others have also shown

that subsequent inoculation of distant leaves in previously infected Arabidopsis

produce SAR and increased SA (Summermatter et al., 1995).

Salicylic acid-mediated resistance involves the expression of several proteins

including PR1, PR2, PR5 and PR10. Pathogenesis-Related Protein 1 (PR1) has

40

been established as a prominent marker for the activation of the SA pathway (Cao

et al., 1998). Pathogenesis-Related Protein 2 (PR2) has also been shown to be

responsive to P. s. tomato infection and SA treatment in Arabidposis (Wathugala

et al., 2012). Pre-treatment of V. vinifera leaves with benzothiadiazole (BTH; a

SA analogue) induces a range of PR proteins enhancing resistance to biotrophic

pathogens (Dufour et al., 2012). Furthermore, differential PR protein expression

was observed in V. vinifera leaves after infection with P. viticola or Erysiphe

necator (Dufour et al., 2012), indicating a “fine-tuned” response to different

stressors, producing a unique response in the host.

One of the most well studied defence related proteins in plants; PR1, is known to

be enhanced by SA-mediated responses (Chong et al., 2008; Li et al., 2011;

Wielgoss & Kortekamp, 2006). Transgenic expression of V. vinifera PR1 in

tobacco revealed that basic PR1 is capable of conferring resistance to P. s. tabaci

(Li et al., 2011). Pathogenesis-related 10 is also considered important in SA-

mediated responses. In a V. vinifera cv. Riesling study, Kortekamp et al. (2006)

reported that PR10 was strongly up-regulated in response to pathogen P. viticola

infection.

In addition to the general antagonism of JA and SA signalling, interactions within

other arms of the defence system also occur. SA is suppressed by applications of

ABA (Kusajima et al., 2010). P. syringae AvrPtoB modification of ABA

signalling in planta has been reported (de Torres-Zabala et al., 2007).

Interestingly, this may indicate that some pathovars of P. syringae can incite

defence pathway perturbations, resulting in disease from compromised SA-

41

signalling mediated by ABA (Mohr & Cahill, 2007). Abscisic acid mediated

suppression of SA is described as representing a control mechanism for plants to

prioritise their response of abiotic stress over biotic stress (Mohr & Cahill, 2007).

The ABA suppression of SA creates an ingenious method of manipulation by

P. syringae carrying AvrPtoB to overcome biotic stress responses by host plants.

DELLAs have also been shown to promote susceptibility to biotrophs by altering

the strength of SA signalling via down-regulation during P. s. tomato DC3000

infection (Navarro et al., 2008). An early auxin-responsive plant gene, GH3.5,

has been shown to act as a bifunctional modulator of SA and auxin signalling

during P. syringae infection of Arabidopsis (Zhang et al., 2007b). Indeed the SA

pathway can be amplified by the induction of GH3.5 (Zhang et al., 2007b).

However, as discussed earlier, some P. syringae pathovars can produce auxins,

which may then repress SA-mediated signalling. Therefore, pathogens such as

P. syringae may engage a number of effectors, proteins or phytotoxins (such as

avrPtoB, coronatine, auxins and ET) to suppress SA-mediated defences and

promote disease susceptibility.

1.9.11 Jasmonic acid

Believed to be a central regulator in the resistance against necrotrophic

pathogens, jasmonic acid and other jasmonates are also involved in UV damage,

wounding and many aspects of necrotrophic plant pathogen interactions.

Jasmonic acid is an oxylipin derived from the oxidation of linolenic acid (Vick &

Zimmerman, 1984) and is abundant in the cellular membranes of higher plants

(Gfeller et al., 2010). Many biologically active forms of jasmonates are known

42

and these mediate the expression of a wide range of genes in response to

wounding or pathogen attack that can include proteinase inhibitors, antifungal

proteins, PDF1.2, Thi2.1 and VSP2 (Leon-Reyes et al., 2010a; Mur et al., 2006).

Furthermore, upon necrotrophic pathogen detection, some groups have

demonstrated that JA can be rapidly detected in distal leaves (Glauser et al.,

2008). These responses within the V. vinifera, however, are poorly understood

and require a more thorough knowledge for effective treatments in susceptible

crops.

Jasmonic acid activation requires conjugation to an amino acid, such as isoleucine

(Ile) or conjugation of a methyl (Me) group. Methyl-jasmonic acid (MeJA)

promotes stomatal closure by producing alkalinisation in the cytosol of guard

cells and ROS production (Gehring et al., 1997; Suhita et al., 2004) and is known

to diffuse through membranes (Seo et al., 2001). Important transcription factors

that have been implicated in JA-signalling pathways include MYC2 and ORCA3

(Koornneef & Pieterse, 2008; Reuveni, 1998). Indeed it has been demonstrated

that JA and ABA are dependent on MYC2 upon wounding (Anderson et al.,

2004; Lorenzo et al., 2004). ERF1-dependent gene induction is activated by a

combination of JA and ET during pathogen attack (Anderson et al., 2004). This

transcription could be dependent upon signal input from the pathogen. In a recent

study, V. vinifera MYC2 was identified through mRNA analysis after MeJA

treatment (I. Dry, CSIRO PI, personal communication).

In Vitis studies, thaumatin-like 1 (VvTL1) has been used as an indicator of JA-

mediated defence responses. Although widely reported to play a role in berry

43

ripening, VvTL1 is also induced in V. vinifera upon methyl-JA treatment (I. Dry,

personal communication). The role of VvTL1 during biotrophic pathogen attack in

V. vinifera is unknown at this stage, but its role in fungal pathogen attack has

been reported. Thaumatin-like proteins are up-regulation in grapevine leaf in

response to the V. vinifera pathogens, Erysiphe necator and Phomopsis viticola

(Jacobs et al., 1999; Monteiro et al., 2003). The role of VvTL1 during fungal

infections has been established in several V. vinifera studies. The VvTL1 protein

significantly inhibited spore germination and hyphal growth of Elsinoe ampelina

on Chardonnay (Jayasankar et al., 2003). Additionally, thaumatin extracted from

grape also demonstrate antifungal activity against E. necator, Phomopsis viticola

and B. cinerea in vitro (Monteiro et al., 2003).

Induction of JA-mediated pathways in host plants can be achieved by some

pathovars of P. syringae. Under laboratory conditions, P. s. tomato DC3000

primarily induces SA in tobacco (Liu et al., 2013). Salicylic acid induction may

lead to plant resistance against pathogens such as P. syringae, but P. syringae is

still able to cause disease in spite of increased SA accumulation. One factor that

may allow for this is the phytohormone coronatine, a molecular mimic of JA that

allows for the up-regulation of JA-mediated pathways and thereby causes induced

sensitivity within the host plant to the pathogen.

Others have found that non-coronatine producing pathovars of P. syringae may

produce other compounds to promote JA-mediated pathways. Indeed, recently

P. s. tabaci was shown to produce HopX1 (Gimenez-Ibanez et al., 2014), which is

believed to promote JA by degradation of jasmonate-ZIM domain (JAZ)-

44

repressor proteins. JAZ proteins act by blocking JA-signalling. Upon degradation

via proteasome or DELLA proteins (Sheard et al., 2010; Thines et al., 2007), JA

signalling is increased while SA-mediated defences are down regulated. Others

have demonstrated that AvrB indirectly disrupts JA signalling by interfering with

MPK4 involved in Arabidopsis JA pathway (Cui et al., 2010). These studies

indicate that P. syringae carrying avrB may be able to up-regulate JA-mediated

pathways, thereby increasing plant susceptibility to this pathogen. Although

information on the JA up-regulation by P. s. syringae is limited, one report

indicated that SA-mediated pathways were disrupted by nuclear localisation of

syringolin A (Misas-Villamil et al., 2013). NPR1 requires the nuclear proteasome

for transcriptional activation for SA-signalling (Kolodziejek et al., 2011; Spoel et

al., 2009). Therefore, P. s. syringae carrying sylA may inhibit SA-mediated

signalling, rather than targeting JA up-regulation.

Jasmonic acid regulation is partially controlled by the JAZ proteins. Abiotic stress

and/or JA treatment rapidly triggers expression of JAZ repressors (Zhang et al.,

2012). In several studies JAZ proteins are reported to be negative regulator of JA

signalling in Arabidopsis (Demianski et al., 2012; Thines et al., 2007), and are

enhanced during SA-mediated gene expression (Van der Does et al., 2013). This

negative feedback loop enables the replenishment of JAZ repressors to dampen

the JA response (Katsir et al., 2008a). Transcriptional analysis of JAZ genes in

response to hormone, herbivory, environmental conditions and P. syringae

infection has demonstrated differential expression of JAZ (Demianski et al.,

2012; Zhang et al., 2012). Pseudomonas syringae pv. tomato DC3000 is capable

of inducing a subset of JAZ proteins in early Arabidopsis infection (Demianski et

45

al., 2012). In this case, coronatine producing pathovars of P. syringae, such as

P. s. tomato induce JA signalling (Glazebrook, 2005) and in Arabidopsis that may

account for upregulation of JAZ genes during infection (Demianski et al., 2012).

Beside P. s. tabaci carrying the HopX1 effector (Gimenez-Ibanez et al., 2014), to

date there has been no study found that illustrates P. s. syringae inducing JAZ

expression.

46

1.10 Summary

P. s. syringae is a heterogeneous plant pathogen that produces symptoms

of water-soaked lesions and dark leaf spots, necrosis and abscission of

flowers/fruit and stem/branch cankers in various hosts.

P. syringae has been problematic for various crops throughout Australia,

including Field pea, mango, maize, and now grapevine.

Important pathogenicity factors of P. s. syringae include: syringomycin

and syringopeptin that produce pores in the plasma membrane of host

plants, and syringolin A that may counteract ABA stomatal immunity and

suppress SA-mediated defence pathways (Fig. 1.3).

JA/ET and SA are important phytohormone plant defence pathways and

are considered antagonistic to each other. Also, other important plant

hormones include GAs, CKs, DELLAs and ABA. The role of these

hormones in grapevine defence responses to P. s. syringae is largely

unknown.

Pathogens, such as P. syringae, can produce their own compounds,

hormones and/or hormone analogues (such as CKs, ET, auxins, and

coronatine) that can modify or manipulate plant defence responses (Fig.

1.3).

47

Fig. 1.3 Virulence factor produced in P. syringae that target various aspects of plant immunity.

Chp8 is a diguanylate cyclase that decreases flagellin production (Engl et al., 2014). HopAI1

suppresses NHO1 and PAMP-mediated signalling (Li et al., 2005; Zhang et al., 2007a). AvrE1

reduces lesion formation (Badel et al., 2006). Syringopeptins induce necrosis and electrolyte

leakage (Duke & Dayan, 2011), whereas syringomycin incites pore formation in plasma

membrane (Bender et al., 1999). AvrB is known to interfere with JA-mediate MPK4 signalling

(Cui et al., 2010), HopX1 attaches to JAZ JA-repressors to activate JA signalling (Gimenez-

Ibanez et al., 2014), ET can be produced by some P. syringae (Weingart & Volksch, 1997) and

coronatine (COR) is produced by some pathovars of P. syringae (Hwang et al., 2005). These

effectors/toxins increase JA-mediated signalling in host plants thereby promoting plant

susceptibility. Syringolin inhibits the proteasome in SA signalling (Misas-Villamil et al., 2013)

and ABA-mediated stomatal closure (Schellenberg et al., 2010) whereas HopAM1 increases ABA

signalling (Goel et al., 2008), possibly to inhibit SA-mediated responses (Mohr & Cahill, 2007)

and increase host susceptibility (Audenaert et al., 2002). HopQ1 activates CK signalling which

may then lead to reduced FLS2 accumulation for PTI responses (Hann et al., 2014). AvrRpt2

increases auxin signalling (Chen et al., 2007). Auxins and IAA can be produced by some

P. syringae (O'Donnell et al., 2003) or induce plants to increase IAA by hydrolysing plant indole-

3-acetonitrile to IAA (Howden et al., 2009).

48

1.11 Research aims and objectives

The objectives of the first part of this project were to (i) use phylogenetic and

molecular techniques to identify P. s. syringae isolates collected from diseased

grapevines with symptoms of BIR and BLS (ii) use MLST to compare pathogenic

and non-pathogenic P. s. syringae isolates from the same regions and from other

hosts and (iii) investigate the relationships between the genetic patterns and the

virulence of the isolates from grape and other host species.

Because of the high heterogeneity and common phenotypic characteristics of

P. s. syringae, misidentification can easily occur. The aim of the second part of

this research was to make a comparative study of the biochemical phenotypic

characteristics of P. s. syringae from grapevine affected by BIR or BLS.

Additionally, molecular-based PCR techniques were used for phylogenetic

analysis and an evolutionary study of toxin production and BIR symptoms.

How certain pathogens are able to evade or suppress basal defence pathways in

plants has been well reported and characterised in many plant-pathogen

relationships (Chisholm et al., 2006; Engl et al., 2014; Zhang et al., 2007).

Currently, information to date is not useful for understanding disease

epidemiology or managing P. s. syringae disease in grapevine, therefore a more

detailed description of pathogen diversity may be necessary. In the final part of

this research, the aim is to characterise grapevine-specific defence responses to

pathogenic and non-pathogenic P. s. syringae.

49

Chapter 2 reports the studies on the phylogenetic relationships of P. s. syringae

isolates from vineyards in different regions of Australia, including those that are

affected by BIR or BLS and P. s. syringae from other host plants. Chapter 3

examines the biochemistry and molecular biology of the grape P. s. syringae used

in Chapter 2, finding associations between various factors using Arlequin’s

analysis of molecular variance. Chapter 4 studies the defence gene expression

patterns and callose deposition produced by grapevine hosts in response to

pathogenic and non-pathogenic P. s. syringae. Finally Chapter 5 briefly discusses

and findings and conclusion of the current study.

50

Chapter 2 Phylogenetic Relationships of Pseudomonas

syringae pv. syringae Isolates Associated with Bacterial

Inflorescence Rot in Grapevine

(Accepted paper: Plant Disease, 100(3), 607-616)

2.1 Introduction

Pseudomonas syringae pv. syringae (P. s. syringae) causes extensive yield losses

in winegrape production in some Australian cool climate vineyards. Putative

P. s. syringae isolates from infected grapevines within a range of vineyards were

genotyped using the RNA polymerase β-subunit (rpoB), and multilocus sequence

typing (MLST) using primers for glyceraldehyde-3-phosphate dehydrogenase

(gapA), citrate synthase (gltA), DNA gyrase B (gyrB) and Sigma factor 70

(rpoD). The isolates were also evaluated for pathogenicity by inoculation of

detached grapevine leaves. The isolates were grouped by MLST data into two

well supported clades, each containing a mixture of pathogenic and non-

pathogenic grapevine isolates, indicating that P. s. syringae in Australian

vineyards is genetically diverse. Each clade also contained P. s. syringae from

non-grape hosts pathogenic to grapevine, demonstrating a lack of host specificity

and possible potential for cross-infection of grape and other horticultural crops.

Furthermore, the isolation of pathogenic P. s. syringae isolates from grapevine

sucker shoots suggest that sucker shoots may allow overwintering of the

pathogen. Protective measures against P. s. syringae may need to be

reconsidered, due to its easy dispersal through pruning equipment.

51

2.2 Materials and methods

Four vineyards affected by BIR, three in the Riverina region (Tumbarumba, New

South Wales) and one in the Southern Tablelands (Murrumbateman, New South

Wales), were sampled between September and October in 2006, 2011, and 2013.

In addition, grapevine samples were obtained from BIR- affected vineyards in the

Coonawarra (South Australia) and Piper’s River (Tasmania). Samples were also

collected from a vineyard with bacterial leaf spot (BLS), but no bacterial

inflorescence rot (BIR) in the Macedon Ranges region (Hanging Rock, Victoria),

and from apparently healthy grapevines in Victorian vineyards: Glenlofty in the

Pyrenees and Hallston in Gippsland. Leaves, shoots, and rachii were collected

with ethanol-sterilised equipment, placed into Zip-Lock polyethylene bags and

kept at 4°C until bacterial isolation.

Bacterial isolates from culture collections. Pseudomonas syringae pv.

morsprunorum (DAR33419) and P. s. syringae (DAR72042 and DAR73915)

were obtained from the New South Wales Industry and Investment Culture

Collection (Orange, Australia). P. syringae isolates BRIP34823, BRIP38670,

BRIP34831, BRIP34899, BRIP38817, BRIP34832, BRIP34805, BRIP38811 and

BRIP34803 were obtained from the Department of Agriculture, Fisheries and

Forestry (DAFF), Queensland. P. syringae isolates DAR82449, DAR82450,

DAR82451, DAR82432, and DAR8453 were obtained from Dr. Thomas Hill,

Colorado State University, USA.

52

Isolation of Pseudomonas syringae. P. syringae was isolated from leaves, shoots

and inflorescences of grapevines with apparent BIR symptoms. Plant tissues were

rinsed with tap water, surface-sterilised with sodium hypochlorite solution (1%

available chloride) for 3 mins and then rinsed in 3 washes of sterile deionised

water (SDW). The sterilised tissue was then aseptically cut into approximately

5mm x 5mm pieces, placed on Pseudomonas selective CCF agar (PS: Oxoid,

Australia) and incubated in darkness at 25°C for up to 3 days. Pseudomonas

colonies were then subcultured onto nutrient agar (NA) and PS agar to isolate

pure colonies. Pure colonies were tested for Gram stain, fluorescence under UV

light (354 nm) and oxidase production (Lelliott & Stead, 1987).

Identification of P. syringae. Motile Gram-negative, fluorescent, oxidase

negative rod-shaped bacteria were selected and maintained on King’s B (KB)

agar containing 20 g/L peptone, 1.5 g/L MgSO4•7H2O, 1.5 g/L K2HPO4, 10 mL

glycerol, 15 g/L Agar bacteriological No. 1 (Oxoid) at 25°C in darkness. Isolates

were tested under the LOPAT testing scheme for Levan and Oxidase reaction,

Potato soft rot, Arginine dihydrolyase activity, and Tobacco leaf hypersensitivity

response (Lelliott & Stead, 1987). Pure P. syringae cultures were stored at -80°C

in nutrient broth containing 30% (v/v) glycerol. Tests involving 2-keto gluconate

production, nitrate reduction and production of acid from sucrose were also done

as previously described by Lelliott and Stead (1987). Pathovars P. syringae pv.

syringae (P. s. syringae, from grapevine, cowpea and stonefruit), P. syringae pv.

maculicola (P. s. maculicola), P. syringae pv. striafaciens (P. s. striafaciens),

P. syringae pv. phaseolicola (P. s. phaseolicola), P. syringae pv. morsprunorum

(P. s. morsprunorum), P. syringae pv. mori (P. s. mori), and P. syringae pv.

53

tabaci (P. s. tabaci) were also used to confirm LOPAT identification of

P. syringae among pathovars.

Koch’s Postulates. A representative strain of P. s. syringae (DAR82161) from

Tumbarumba was tested for its ability to cause disease (spreading necrotic leaf

lesions) on leaves on four live potted grapevines (cvv. Chardonnay and Shiraz).

Prior to inoculation, two leaves per plant were removed and checked for the

absence of P. s. syringae infection in asymptomatic/healthy-looking plants by

surface disinfecting with sodium hypochlorite (1% available chloride) for 1 min,

macerating with a mortar and pestle with 5-10 mL sterile phosphate buffered

saline (pH 7), serially diluting, spreading (100 μL) over PS agar and incubating at

25°C in darkness. After demonstrating the absence of endogenous P. s. syringae

in the potted V. vinifera cvv. Chardonnay and Shiraz plants, the leaves were

spray-inoculated with a fine mist suspension (1x108 CFU/mL) of P. s. syringae

isolate DAR82161 (obtained originally from a necrotic grapevine rachis,

Tumbarumba) in SDW until visible run off. The plants were then enclosed in

clear plastic bags to maintain humidity at >99%, and arranged in randomised

complete blocks in a glasshouse maintained at 25/15ºC day/night. Potted vines

were watered to field capacity twice weekly. Disease development was monitored

over 21 days, and leaf samples collected at the end of the experiment at which

time leaves were surface sterilised, plated onto PS agar and incubated under the

same conditions as described above.

Grapevine pathogenicity leaf test assay (GPLTA). The GPLTA was carried out

as described by Cohen et al. (1999) with minor changes. Briefly, healthy leaves

54

were detached from V. vinifera cv. Chardonnay plants (grown under greenhouse

conditions) and were surface-sterilised in 1% available chloride solution

containing 100µL/L TWEEN 80 detergent (Sigma, Australia) for 3 min, followed

by four washes in sterile distilled water. Leaf discs (12 mm diameter) were

aseptically cut with a ‘number 8’ cork borer (12 mm diameter) and placed abaxial

side up on 1% agar. For inoculations, 50 µL drops of test bacteria were spot-

inoculated onto leaf discs at concentrations of approximately 1x108

CFU/mL, and

incubated at 25°C for up to 7 days in a moist sealed bag at relative humidity of

>99% in an Intellus Control System Incubator (Percival, USA). Isolates were

considered pathogenic to grapevine if typical necrotic symptoms (i.e. appearance

of brown lesions at the point of inoculation) appeared within 2 days and observed

over 7 days.

RNA polymerase β-subunit gene (rpoB) analysis. Forty-eight hour cultures

grown on KB agar in darkness were used for DNA extraction, using a DNeasy

Blood and Tissue Kit (Qiagen, Australia) following the manufacturer’s

instructions (Appendix 1). RNA polymerase β-subunit gene (rpoB) was used for

pathovar identification by PCR. The primers used for rpoB (Table 3.1) have been

previously shown to amplify a 1247 base pair fragment from Pseudomonas spp.

(Tayeb et al., 2005). PCR reactions (25 L) were carried out using a BioRad

C1000 Thermal Cycler with GoTaq Green® polymerase (Promega, Australia)

according to the manufacturer’s instructions with a final primer concentration of

0.3 µM and approximately 100 ng template DNA. Cycle conditions were: 94°C

for 3 min then 40 cycles of 94°C for 45 s 55°C for 1 min, and 72°C for 90 s, with

a final extension step of 72°C for 10 min.

55

Multi-locus sequence typing. The four housekeeping genes used for MLST were

gapA, encoding glyceraldehyde-3-phosphate dehydrogenase; gltA, encoding

citrate synthase (also known as cts); gyrB, encoding DNA gyrase B; and rpoD,

encoding Sigma factor 70. The MLST protocol was done essentially as described

by Hwang et al. (2005). PCR was done as described above with the following

cycle conditions: 94°C for 2 min, 60°C for 1 min and 72°C for 1 min for 36

cycles) using primers outlined in Table 3.1.

56

Ta

ble

2.1

Pri

mer

s u

sed

for

rpo

B a

nd

ML

ST

Au

tho

rs

Tay

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., 2

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200

5

1. F

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str

and

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(+

), r

ever

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CR

pri

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(p

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s)

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Pat

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var

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Seq

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TG

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TT

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CS

GA

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51

3s

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36

4s

rpo

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222

ps

57

PCR products were purified using a PCR Purification Kit (Qiagen) following the

manufacturer’s instructions and quantified on a 1% agarose gel stained with

ethidium bromide. The purified products were sequenced by the Australian

Genome Research Facility. Sequence data were analysed using MEGA5 software

and aligned using CLUSTAL W. A BLAST search was done on trimmed rpoB

sequences to confirm identity and to determine the pathovar. P. syringae pv.

morsprunorum (DAR33419) and P. s. syringae (DAR72042) were also used in

this study as negative and positive controls, respectively, and Pseudomonas fragi

ST128 (isolated from a grapevine, Hanging Rock, Victoria) was used as the

outgroup. Separate phylogenetic trees were generated using Neighbour-Joining

(NJ; rpoB and MLST data) and Unweighted Pair Group Method with Arithmetic

Mean (UPGMA; rpoB data) with Jukes-Cantor corrected distances (Jukes and

Cantor, 1969; Kumar et al., 2004) and statistical confidence for sequence groups

determined using a bootstrap test with 1000 pseudoreplicates (Felsenstein, 1985).

Accession Numbers. Sequence data obtained in this study were deposited into

GenBank (Appendix 2).

58

2.3 Results

To investigate the genetic diversity of P. s. syringae isolates present in Australian

vineyards affected by BIR, isolates were collected from symptomatic diseased

grapevines (Fig. 2.1). Symptoms of grapevine BIR/BLS included leaf spots with

yellow chlorotic haloes (Fig. 2.1A), necrotic lesions on shoots (Fig. 2.1B),

bacterial ooze and abscised flowers in inflorescences (Fig. 2.1C), and death of

inflorescences (Fig. 2.1D). Abscission of the necrotic inflorescences occurred in

most cases, resulting in full loss of grape bunches. Once established in a vineyard,

the severity of these symptoms generally progressed over following seasons.

Pseudomonas syringae was isolated from all surface-sterilised diseased grapevine

tissues with BIR and BLS symptoms. Bacterial ooze emerging from the diseased

plant tissue and from surfaced-sterilised plant tissue on PS agar consisted of pure

cultures of motile oxidase negative, fluorescent Gram negative bacilli, 0.5-1.0µm

wide and 2.0-3.0µm long. Colonies produced yellow pigment on PS and KB agar

that fluoresced blue under UV light (354 nm) (data not shown). These features are

consistent with those of P. syringae.

The P. syringae isolates were then further characterised using the LOPAT testing

regime which enables the separation of plant saprophytic pseudomonads from

pathogenic pseudomonads (Lelliott & Stead, 1987). All the P. syringae isolates

were positive for levan type colonies on sucrose agar but were negative for

oxidase reaction, potato soft rot and arginine dihydrolyase activity (Lelliott et al.,

1966; Lelliott & Stead, 1987). Non-pathogenic grapevine P. s. syringae isolates

59

(DAR82449, DAR82450, DAR82451 and DAR82452) were unable to cause

tobacco leaf HR (Table 2.2).

Further characterisation involved tests for 2-keto gluconate production, nitrate

reduction, and acid production from sucrose. The results indicate that most

isolates of P. syringae were unable to produce 2-keto gluconate from gluconate or

to reduce nitrate, but were positive for acid production from sucrose. In contrast,

one isolate (BRIP34823, P. s. syringae from cowpea) was able to produce 2-keto

gluconate from gluconate and to reduce nitrate. These results further verified the

identification of most of the isolates as LOPAT group 1a P. syringae from

grapevine (Lelliott & Stead, 1987). Interestingly, DAR82445, DAR82447,

DAR82443 and DAR82162, isolated from grapevine sucker shoots (latent buds

that sprout from the crown, the basal region of the trunk slightly below and above

the soil level, of the grapevine trunk) in BIR-affected Tumbarumba vineyards had

identical results for LOPAT, 2-keto gluconate production, nitrate reduction, and

acid production from sucrose as the P. s. syringae pathogens isolated from

infected rachi in the same vineyards (Table 2.2).

60

Fig. 2.1. (A) Grapevine cv. Riesling with symptoms of dark leaf spots with yellow chlorotic

haloes, Tumbarumba, New South Wales. (B) Riesling petiole with longitudinal necrotic lesions

(Tumbarumba, New Sotth Wales). (C) Riesling inflorescence affected by BIR; bacterial ooze

(arrows) and abscised flowers (Tumbarumba, New South Wales). (D) Necrotic rachis on Cabernet

Sauvignon (from field grapevine, Coonawarra, South Australia). Images supplied by L Quirk (C)

Department of Primary Industries New South Wales and N Scarlett (Dec’d), Rathbone Wine

Group, Melbourne, Victoria. (D).

61

Table 2.2. LOPAT identification of Pseudomonas syringae.

Isolate Lev Ox Pot Arg Tob 2KG NR AFS Pathovar 1

DAR33419 + - - - + - - + morsprunorum

DAR72042 + - - - + - - + syringae

DAR73915 + - - - + - - + syringae

DAR77819 + - - - + - - + syringae

DAR77820 + - - - + - - + syringae

DAR82159 + - - - + - - + syringae

DAR82160 + - - - + - - + syringae

DAR82161 + - - - + - - + syringae

DAR82162 + - - - + - - + syringae

DAR82165 + - - - + - - + syringae

DAR82166 + - - - + - - + syringae

DAR82169 + - - - + - - + syringae

DAR82170 + - - - + - - + syringae

DAR82171 + - - - + - - + syringae

DAR82440 + - - - + - - + syringae

DAR82441 + - - - + - - + syringae

DAR82442 + - - - + - - + syringae

DAR82443 + - - - + - - + syringae

DAR82444 + - - - + - - + syringae

DAR82445 + - - - + - - + syringae

DAR82446 + - - - + - - + syringae

DAR82447 + - - - + - - + syringae

DAR82448 + - - - + - - + syringae

DAR82449 + - - - - - - + syringae

DAR82450 + - - - - - - + syringae

DAR82451 + - - - - - - + syringae

DAR82452 + - - - - - - + syringae

DAR82453 + - - - + - - + syringae

BRIP34803 + - - - + - - + tabaci

BRIP34805 + - - - + - - + mori

BRIP34823 + - - - + + + + syringae

BRIP34831 + - - - + - - + syringae

BRIP34832 + - - - + - - + striafaciens

BRIP34899 + - - - + - - + syringae

BRIP38670 + - - - + - - + syringae

BRIP38811 + - - - + - - + phaseolicola

BRIP38817 + - - - + - - + maculicola

ST128 + - - - - P. fragi 1 Pathovar as determined from rpoB sequencing. Lev = levan like colonies, Ox = oxidase reaction, Pot = potato rot assay,

Arg = arginine dihydrolase activity, Tob = tobacco leaf hypersensitivity reaction, 2KG = 2-keto gluconate production from

gluconate, NR = nitrate reduction, AFS = acid from sucrose

62

To confirm that P. s. syringae was responsible for the disease development on

grapevine, Koch’s Postulates were tested by inoculating leaves of potted

V. vinifera cv. Chardonnay and Shiraz plants with a representative strain of

P. s. syringae (DAR82161) and maintaining the leaves under humid conditions.

Leaf lesions developed similarly to those observed in vineyard infected material,

with dark necrotic spots with yellow chlorotic haloes appearing on the leaves

within 48 h post inoculation (Fig. 2.2A). These lesions progressed along the veins

to non-inoculated regions (Fig. 2.2A), until the leaves senesced (Fig. 2.2B). After

three weeks post-inoculation, tissue samples were collected and P. s. syringae

recovered from the leaves as described above. No P. syringae was isolated from

the non-inoculated plants, thus confirming Koch’s postulates.

Fig. 2.2. Symptoms of P. s. syringae infection on leaves from potted grapevines used to test

Koch’s Postulates. (A) Potted Shiraz leaf showing dark bacterial leaf spots with yellow chlorotic

haloes 48 hours post spray inoculation. (B) Leaf senescence on potted Chardonnay grapevine 4

days post spray inoculation with P. s. syringae.

63

The remainder of the P. syringae isolates collected were also assessed for their

ability to cause necrosis on detached grapevine leaf discs using the Grapevine

Pathogenicity Leaf Test Assay (GPLTA). All P. s. syringae isolates obtained

from grapevines displaying BIR or BLS produced necrosis in the GPLTA

whereas grapevine isolates from healthy vineyards (i.e. DAR82449, DAR82450,

DAR82451 and DAR82452) did not. Interestingly, some P. s. syringae isolates

from non-grape hosts: e.g. DAR72042 from apple leaf spot, Batlow, NSW;

BRIP34823 isolated from cowpeas showing leaf necrosis; BRIP38670,

BRIP38817 and BRIP34899 from stone fruit trees with canker, gave positive

GPLTA results. Of the non-syringae pathovars, P. s. maculicola (BRIP38817

from diseased cabbage); P. s. striafaciens (BRIP34832 isolated from oats with

leaf spot); and P. s. tabaci (BRIP34803 from soybeans with leaf spot) gave

positive GPLTA results; whereas BRIP38811 (P. s. phaseolicola isolated from

beans with leaf spot); BRIP34805 (pv. mori from white mulberry) and

DAR33419 (P. s. morsprunorum from a wild cherry leaf) were negative for

GPLTA but were positive for the tobacco leaf hypersensitivity reaction (Table

2.3).

64

Ta

ble

2.3

. C

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late

s o

f P

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iso

n o

f P

. s.

syr

ing

ae

rpo

B

seq

uen

ces

wit

h t

hat

of

the

know

n T

um

bar

um

ba

bac

teri

al i

nfl

ore

scen

ce r

ot

iso

late

, D

AR

82

44

8.

Nu

mb

er o

f r

po

B n

t

dif

feren

t fr

om

DA

R8

24

48

(o

ut

of

42

6 n

t)

4

0

3

3

3

1

1

7

4

4

a Po

siti

ve

(+)

or

neg

ativ

e (-

) fo

r nec

rosi

s on

lea

f dis

c, G

rapev

ine

Pat

hog

enic

ity

Lea

f T

est

Ass

ay.

BIR

= B

acte

rial

in

flo

resc

ence

ro

t af

fect

ed v

iney

ard

. B

LS

= b

acte

rial

lea

f sp

ot

affe

cted

vin

eyar

d.

ND

= v

iney

ard

no

t

affe

cted

by

lea

f sp

ot

or

bac

teri

al i

nfl

ore

scen

ce r

ot.

C =

can

ker

. P

ss:

P.

s. s

yrin

ga

e; P

ma:

P.

s. m

acu

lico

la;

Pst

r: P

. s.

str

iafa

cien

s; P

sm:

P.

s. m

ors

pru

no

rum

; P

mo

: P

. s.

mo

ri;

Php

: P

. s.

pha

seoli

cola

; P

stab

: P

. s.

tab

aci

. n

t =

nu

cleo

tide

GL

PT

Aa

+

+

+

+

+

+

+

+

+

+

Ho

st o

rig

in

Bat

low

, B

LS

, 1

99

7

Ad

elai

de

Hil

ls,

BL

S,

20

00

.

Tu

mb

aru

mb

a, B

IR,

200

6.

Tu

mb

aru

mb

a, B

IR,

200

6.

Tu

mb

aru

mb

a, B

IR,

201

1.

Tu

mb

aru

mb

a, B

IR,

201

1.

Tu

mb

aru

mb

a, B

IR,

201

1.

Tu

mb

aru

mb

a, B

IR,

201

1.

Tu

mb

aru

mb

a, B

IR,

201

1.

Tu

mb

aru

mb

a, B

IR,

201

1.

Ho

st s

ym

pto

m

leaf

sp

ot

leaf

sp

ot

nec

roti

c ra

chis

shri

vel

led

ber

ry

shri

vel

led

ber

ry

can

e n

ecro

tic

lesi

on

nec

roti

c ra

chis

suck

er s

ho

ot

nec

roti

c le

sio

n

sho

ot

nec

roti

c le

sio

n

leaf

sp

ot

Ho

st

Ma

lus

x d

om

esti

ca (

app

le)

V.

vin

ifer

a c

v u

nsp

ecif

ied

V.

vin

ifer

a c

v S

auv

ign

on B

lan

c

V.

vin

ifer

a c

v S

auv

ign

on B

lan

c

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

Iso

late

Pss

DA

R7

20

42

Pss

DA

R7

39

15

Pss

DA

R7

78

19

Pss

DA

R7

78

20

Pss

DA

R8

21

59

Pss

DA

R8

21

60

Pss

DA

R8

21

61

Pss

DA

R8

21

62

Pss

DA

R8

21

65

Pss

DA

R8

21

66

65

Ta

ble

2.3

. C

har

acte

rist

ics

of

iso

late

s o

f P

. s.

syr

ing

ae

fro

m A

ust

rali

an v

iney

ard

s w

ith

sy

mp

tom

s o

f b

acte

rial

in

flo

resc

ence

ro

t. C

om

par

iso

n o

f P

. s.

syr

ing

ae

rpo

B

seq

uen

ces

wit

h t

hat

of

the

know

n T

um

bar

um

ba

bac

teri

al i

nfl

ore

scen

ce r

ot

iso

late

, D

AR

82

44

8.

Co

nti

nu

ed

Nu

mb

er o

f r

po

B n

t

dif

feren

t fr

om

DA

R8

24

48

(o

ut

of

42

6 n

t)

4

5

0

6

4

7

5

0

4

0

a Po

siti

ve

(+)

or

neg

ativ

e (-

) fo

r nec

rosi

s on

lea

f dis

c, G

rapev

ine

Pat

hog

enic

ity

Lea

f T

est

Ass

ay.

BIR

= B

acte

rial

in

flo

resc

ence

ro

t af

fect

ed v

iney

ard

. B

LS

= b

acte

rial

lea

f sp

ot

affe

cted

vin

eyar

d.

ND

= v

iney

ard

no

t

affe

cted

by

lea

f sp

ot

or

bac

teri

al i

nfl

ore

scen

ce r

ot.

C =

can

ker

. P

ss:

P.

s. s

yrin

ga

e; P

ma:

P.

s. m

acu

lico

la;

Pst

r: P

. s.

str

iafa

cien

s; P

sm:

P.

s. m

ors

pru

no

rum

; P

mo

: P

. s.

mo

ri;

Php

: P

. s.

pha

seoli

cola

; P

stab

: P

. s.

tab

aci

. n

t =

nu

cleo

tide

GL

PT

Aa

+

+

+

+

+

+

+

+

+

+

Ho

st o

rig

in

Tu

mb

aru

mb

a, B

IR,

200

6.

Tu

mb

aru

mb

a, B

IR,

200

6.

Tu

mb

aru

mb

a, B

IR,

201

1.

Mu

rru

mb

atem

an,

BIR

, 2

01

3.

Han

gin

g R

ock

, B

LS

, 2

01

3.

Piper’s R

iver, BIR

, 2014.

Tu

mb

aru

mb

a, B

IR,

201

3.

Tu

mb

aru

mb

a, B

IR,

201

3.

Tu

mb

aru

mb

a, B

IR,

201

3.

Tu

mb

aru

mb

a, B

IR,

201

3.

Ho

st s

ym

pto

m

nec

roti

c ra

chis

sho

ot

nec

roti

c le

sio

n

can

e n

ecro

tic

lesi

on

shri

vel

led

ber

ry

leaf

sp

ot

nec

roti

c ra

chis

suck

er c

ane

nec

roti

c le

sio

n

suck

er s

ho

ot

nec

roti

c le

sio

n

leaf

sp

ot

suck

er s

ho

ot

nec

roti

c ra

chis

Ho

st

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v R

iesl

ing

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v C

har

do

nn

ay

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

V.

vin

ifer

a c

v P

ino

t N

oir

Iso

late

Pss

DA

R8

21

69

Pss

DA

R8

21

70

Pss

DA

R8

21

71

Pss

DA

R8

24

40

Pss

DA

R8

24

41

Pss

DA

R8

24

42

Pss

DA

R8

24

43

Pss

DA

R8

24

44

Pss

DA

R8

24

45

Pss

DA

R8

24

46

66

Ta

ble

2.3

. C

har

acte

rist

ics

of

iso

late

s o

f P

. s.

syr

ing

ae

from

Au

stra

lian

vin

eyar

ds

wit

h s

ym

pto

ms

of

bac

teri

al i

nfl

ore

scen

ce r

ot.

Co

mp

aris

on

of

P. s.

syr

ing

ae

rpo

B

seq

uen

ces

wit

h t

hat

of

the

know

n T

um

bar

um

ba

bac

teri

al i

nfl

ore

scen

ce r

ot

iso

late

, D

AR

82

44

8.

Co

nti

nu

ed

Nu

mb

er o

f r

po

B n

t

dif

feren

t fr

om

DA

R8

24

48

(o

ut

of

42

6 n

t)

4 7

7

3

7

9

4

7

7

a Po

siti

ve

(+)

or

neg

ativ

e (-

) fo

r n

ecro

sis

on

lea

f dis

c, G

rap

evin

e P

atho

gen

icit

y L

eaf

Tes

t A

ssay

. B

IR =

Bac

teri

al i

nfl

ore

scen

ce r

ot

affe

cted

vin

eyar

d.

BL

S =

bac

teri

al l

eaf

spo

t af

fect

ed v

iney

ard

. N

D =

vin

eyar

d

not

affe

cted

by

lea

f sp

ot

or

bac

teri

al i

nfl

ore

scen

ce r

ot.

C =

can

ker

. P

ss:

P.

s. s

yrin

ga

e; P

ma:

P.

s. m

acu

lico

la;

Pst

r: P

. s.

str

iafa

cien

s; P

sm:

P.

s. m

ors

pru

no

rum

; P

mo:

P.

s. m

ori

; P

hp

: P

. s.

pha

seoli

cola

; P

stab

:

P.

s. t

abaci

. nt

= n

ucl

eoti

de

GL

PT

Aa

+

+

- - - - +

+

- +

Ho

st o

rig

in

Tu

mb

aru

mb

a, B

IR,

201

3.

Tu

mb

aru

mb

a, B

IR,

201

3.

Gle

nlo

fty

, N

D,

20

07

.

Gle

nlo

fty

, N

D,

20

07

.

Hal

lsto

n,

ND

, 2

00

7.

Hal

lsto

n,

ND

, 2

00

7.

Co

on

awar

ra,

BIR

, 2

010

.

To

ow

oo

mb

a, B

LS

, 1

97

1.

Sta

nth

orp

e, C

, 19

81

.

No

t sp

ecif

ied

(V

IC),

C,

197

1.

Ho

st s

ym

pto

m

suck

er s

ho

ot

nec

roti

c ra

chis

nec

roti

c ra

chis

sho

ot

lesi

on

sho

ot

lesi

on

hea

lth

y s

ho

ot

hea

lth

y s

ho

ot

nec

roti

c ra

chis

leaf

sp

ot

can

ker

can

ker

Ho

st

V.

vin

ifer

a cv

Pin

ot

No

ir

V.

vin

ifer

a cv

Pin

ot

No

ir

V.

vin

ifer

a cv

fro

st a

ffec

ted

Ch

ard

on

nay

V.

vin

ifer

a cv

fro

st a

ffec

ted

Ch

ard

on

nay

V.

vin

ifer

a cv

Ch

ard

on

nay

V.

vin

ifer

a cv

Ch

ard

on

nay

V.

vin

ifer

a cv

Cab

ern

et S

auv

ign

on

Vig

na

un

gu

icu

lata

(c

ow

pea

)

Pru

nu

s am

eric

ana,

(A

mer

ican

plu

m)

Pru

nu

s p

ersi

ca (

pea

ch)

I

sola

te

Pss

DA

R8

24

47

Pss

DA

R8

24

48

Pss

DA

R8

24

49

Pss

DA

R8

24

50

Pss

DA

R8

24

51

Pss

DA

R8

24

52

Pss

DA

R8

24

53

Pss

BR

IP3

48

23

Pss

BR

IP3

48

31

Pss

BR

IP3

48

99

67

Ta

ble

2.3

. C

har

acte

rist

ics

of

iso

late

s o

f P

. s.

syri

ng

ae

fro

m A

ust

rali

an v

iney

ard

s w

ith

sy

mp

tom

s o

f b

acte

rial

in

flo

resc

ence

ro

t. C

om

par

iso

n o

f P

. s.

syr

ing

ae

rpo

B

seq

uen

ces

wit

h t

hat

of

the

know

n T

um

bar

um

ba

bac

teri

al i

nfl

ore

scen

ce r

ot

iso

late

, D

AR

82

44

8.

Co

nti

nu

ed

Nu

mb

er o

f r

po

B n

t

dif

feren

t fr

om

DA

R8

24

48

(o

ut

of

42

6 n

t)

6

22

(P

mo

)

12

(P

sm)

23

(P

sm)

20

(P

sp)

20

(P

tab

)

19

(P

str)

37

(P

. fr

ag

i )

a Po

siti

ve

(+)

or

neg

ativ

e (-

) fo

r n

ecro

sis

on l

eaf

dis

c, G

rap

evin

e P

athog

enic

ity

Lea

f T

est

Ass

ay.

BIR

= B

acte

rial

in

flo

resc

ence

ro

t af

fect

ed v

iney

ard.

BL

S =

bac

teri

al l

eaf

spo

t af

fect

ed v

iney

ard

. N

D =

vin

eyar

d n

ot

affe

cted

by

lea

f sp

ot

or

bac

teri

al i

nfl

ore

scen

ce r

ot.

C =

can

ker

. P

ss:

P.

s. s

yrin

gae;

Pm

a: P

. s.

macu

lico

la;

Pst

r: P

. s.

str

iafa

cien

s; P

sm:

P.

s. m

ors

pru

no

rum

; P

mo:

P. s.

mo

ri;

Php

: P

. s.

pha

seo

lico

la;

Pst

ab:

P.

s.

tab

aci

. n

t =

nu

cleo

tide

GL

PT

Aa

+

- +

- - +

+

-

Ho

st o

rig

in

Mo

un

t T

ull

y,

C,

19

72

.

Sta

nth

orp

e, 1

980

.

Mar

coo

la B

each

, 19

78

.

Arm

idal

e, 1

97

5.

Ben

ora

Po

int,

197

7.

No

t sp

ecif

ied

(V

IC),

198

0

Wy

aga,

19

81

.

Han

gin

g R

ock

, 2

01

3.

Ho

st s

ym

pto

m

can

ker

no

t sp

ecif

ied

no

t sp

ecif

ied

leaf

bea

n s

po

t

no

t sp

ecif

ied

no

t sp

ecif

ied

can

e b

acte

rial

oo

ze

Ho

st

Pru

nu

s a

mer

ica

na

, (A

mer

ican

plu

m)

Mo

rus

alb

a L

. (w

hit

e m

ulb

erry

).

Bra

ssic

a o

lera

cea

(

cab

bag

e)

Pru

nu

s a

viu

m,

(wil

d c

her

ry)

Ph

ase

olu

s vu

lga

ris,

(co

mm

on

bea

n)

Gly

cin

e m

ax

(L.)

Mer

r. (

soyb

ean

)

Ave

na

sp

p. (o

ats)

V.

vin

ifer

a c

v S

auv

igno

n B

lanc

Iso

late

Pss

BR

IP3

86

70

Pm

o B

RIP

34

80

5

Psm

BR

IP3

88

17

Psm

DA

R3

34

19

Psp

BR

IP3

88

11

Pst

ab B

RIP

34

80

3

Pst

r B

RIP

34

83

2

P.

fra

gi

(ST

128

)

68

RNA polymerase β-subunit (rpoB) gene sequencing was used as the final step in

P. syringae pathovar identification. When purified rpoB PCR products from all

isolates were sequenced and BLAST searches conducted, grapevine P. syringae

isolates were identified as P. s. syringae, with 100% similarity to P. s. syringae

B728a (accession number CP000075) (Table 2.3). The rpoB sequence of the

representative grapevine P. s. syringae BIR isolate, DAR82448 from

Tumbarumba, was identical to the sequence for a leaf spot isolate (DAR73915)

originally collected from an Adelaide Hills vineyard in 2000. It was also identical

with P. s. syringae isolates from three Tumbarumba vineyards associated with

loss of crop yield in the field due to inflorescence necrosis: DAR82171 from a

diseased cane; DAR82169 from an infected rachis; and DAR8244 and

DAR82446 from sucker shoots. In contrast, some known BIR P. s. syringae

isolates differed by 3 to 7 nt (of 426 nt) from DAR82448, showing that this group

of pathogens include some genetically relatively dissimilar P. s. syringae isolates.

The rpoB sequences were used to produce a phylogenetic tree inferred using the

NJ method which showed P. s. syringae to be separated from the other

P. syringae pathovars, albeit with low bootstrap support (42%). The rpoB

sequences of seven of the Tumbarumba grapevine isolates clustered with the

Adelaide Hills isolate (DAR73915) with 87% bootstrap support (Fig. 2.3). The

NJ method was found to be superior to UPGMA for generating a useful

phylogenetic tree. The evolutionary distances in the UPGMA tree, were not well

expressed due to high levels of heterogeneity and weak bootstrap support (data

not shown).

69

MLST has been used to produce phylogenetic trees with higher discriminatory

power, resulting in clearer assumptions on evolutionary history. MLST was

performed on all isolates in this study to produce a phylogenetic tree from

concatenated sequence data. The MLST data were used to produce a NJ

phylogenetic tree with higher discrimination and stronger bootstrap support for

the separation of P. s. syringae from other pathovars of P. syringae. The analysis

indicates the existence of three clades with strong bootstrap support. Clade 1

(98% bootstrap support) contains mainly pathogenic grapevine P. s. syringae

isolates from New South Wales (Tumbarumba and Murrumbateman), South

Australia (Adelaide Hills and Coonawarra), Victoria (Macedon Ranges) and

Tasmania (Piper’s River). It also contains one cow pea isolate Queensland

(Toowoomba) that is pathogenic on grapevine (Table 2.3), and four non-

pathogenic grape isolates from Victoria. Clade 2 (100% bootstrap support)

contains isolates collected from apricot (one pathogenic and one non-pathogenic),

peach (pathogenic) and grapevine (one pathogenic and one non-pathogenic).

Clade 3 contains other pathovars tested in this study (including, tabai, mori,

morsprunorum, maculicola, striafaciens, and phaseolicola) (Fig. 2.4).

70

Fig. 2.3. Phylogenetic relationships between Pseudomonas spp based on rpoB sequence. The

evolutionary relatedness was inferred using the Neighbour-Joining method (Saitou & Nei, 1987).

Isolates that are pathogenic on grapevine leaves are shaded. Pss: P. syringae pv. syringae; Pma:

P. syringae pv. maculicola; Pstr: P. syringae pv. striafaciens; Psm: P. syringae pv.

morsprunorum; Pmo: P. syringae pv. mori; Php: P. syringae pv. phaseolicola; Pstab: P. syringae

pv. tabaci. *This sequence data was downloaded from GenBank and was not tested for

grapevine pathogenicity. Numbers on nodes are bootstrap values, the frequency (%) with which a

cluster appeared in a bootstrap test of 1000 runs.

71

Fig. 2.4. Phylogenetic relationships between Pseudomonas spp. based on gapA, gltA, gyrB and

rpoD concatenated MLST data. The evolutionary relatedness was inferred using the Neighbour-

Joining method (Saitou & Nei 1987). Isolates that are pathogenic on grapevine leaves are

shaded. Pss: P. syringae pv. syringae; Pma: P. syringae pv. maculicola; Pstr: P. syringae pv.

striafaciens; Psm: P. syringae pv. morsprunorum; Pmo: P. syringae pv. mori; Php: P. syringae

pv. phaseolicola; Pstab: P. syringae pv. tabaci. Numbers on nodes are bootstrap values, the

frequency (%) with which a cluster appeared in a bootstrap test of 1000 runs.

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2.4 Discussion

Distribution in Australian vineyards

This study has used phylogenetic and molecular techniques to investigate the

genetic diversity of P. s. syringae isolates from diseased vines of wine-grape

cultivars in Australian vineyards. Included were isolates from grapevines

displaying symptoms of BIR or BLS, alone or in combination, within eight

vineyards situated in six different viticultural regions across Australia.

P. s. syringae was isolated from diseased grapevines in the following cool climate

vineyards affected by BIR: three in Tumbarumba, New South Wales; one in

Murrumbateman, Southern Tablelands, NSW; one in Piper’s River, Tasmania,

and one in Coonawarra, South Australia. We also isolated P. s. syringae from

diseased grapevines in a cool climate vineyard with BLS symptoms only

(Hanging Rock, Macedon Ranges, Victoria) and a culture collection isolate from

an Adelaide Hills (South Australia) cool climate vineyard with BLS (Hall et al.,

2002). Finally, we isolated non-pathogenic P. s. syringae from apparently healthy

grapevines in cool climate Victorian vineyards not affected by BIR at Glenlofty,

Pyrenees and at Hallston, Gippsland (Fig. 2.5).

Hall et al. 2002 originally reported P. syringae as a weak pathogen of grapevines

in the Adelaide Hills region. In that investigation P. s. syringae caused no yield

loss although it did cause increasingly severe foliar symptoms in following

seasons. A table-grape vineyard in Mildura (Victoria), watered with overhead

water sprinklers, also recorded symptoms of P. syringae BIR in 1998, 2001, 2004

and 2014 on cultivars Sultana and Red Globe (C. Skyllas, Victorian Department

of Environment and Primary Industries, personal communication).

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Fig. 2.5. Pseudomonas syringae pv. syringae (P. s. syringae) isolated from eight cool climate

Australian grape vineyards, plus one warm climate table-grape region with overhead watering

system. : P. s. syringae from diseased grapevines within cool climate vineyards affected by

bacterial inflorescence rot: (1) Tumbarumba, New South Wales; (2) Murrumbateman in the

Southern Tablelands, New South Wales; (3) Piper’s River, Tasmania; (4) Coonawarra, South

Australia. : P. s. syringae from cool climate vineyards with grapevine bacterial leaf spot

symptoms only. (5) Adelaide Hills, South Australia; and (6) Hanging Rock in the Macedon

Ranges, Victoria. : P. s. syringae from apparently healthy grapevines in Victorian cool climate

vineyards not affected by bacterial inflorescence rot: (7) Glenlofty in the Pyrenees, Victoria; (8)

Hallston in Gippsland, Victoria. : P. s. syringae from Red Globe and Sultana table-grapes in

warm climate region Sunraysia, Victoria (9) (reported by Victoria Department of Environment

and Primary Industries Diagnostics Service, 16/11/1998, but not included in the current

phylogenetic studies).

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Bacterial isolates from the Mildura table-grape vineyard could not be obtained for

our study and therefore could not be compared with the cool climate wine-grape

Australian isolates of P. s. syringae. However the collection dates indicate that

symptoms of BIR caused by P. syringae may predate the report of Hall et al.

(2002).

Infection of grapevine

The observed symptoms produced by P. s. syringae on grapevine (necrotic

lesions on leaf tissue, shoots and inflorescences) are in agreement with those

previously reported for BIR by Whitelaw-Weckert et al. (2011) and Abkhoo

(2015). P. s. syringae, which has an extensive plant host range, can be widely

distributed on plant surfaces, water and soil. Following heavy spring rains the

bacterium spreads across wet plant surfaces on shoots, inflorescences, and

through the leaf stomata (Melotto et al., 2006). In grapevine, P. s. syringae

infection starts in the leaves (BLS), followed by systemic movement of bacteria

to the bunch rachis (BIR) (Whitelaw-Weckert et al., 2011). BIR of immature

grapevine inflorescences shows similarities to a disease of immature fruit

blossoms by P. s. syringae in other woody fruit trees: apple (Mansvelt & Hattingh

1989), lychee (Afrose et al. 2014b), mango (Cazorla et al., 1998; Golzar &

Cother, 2008; Young, 2008); pear (Mansvelt & Hattingh, 1987; Moragrega et al.,

2003), and stone fruit (Little et al., 1998).

As P. s. syringae is a relatively new pathogen to the wine industry, symptoms on

grapevine may be misidentified as other pathological or physiological conditions.

Some symptoms of BIR may have previously been attributed to Botrytis cinerea,

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which causes grey mould of grape bunches. B. cinerea can infect inflorescences

early in the season, causing necrosis (Keller et al., 2003). Similarly, P. s. syringae

infection also induces necrosis in inflorescences but with the additional symptom

of occasional visible ‘bacterial ooze’ emerging from the plant tissues. Under-

reporting may also have been caused by the misdiagnosis of P. s. syringae as a

physiological disorder. Before the Whitelaw-Weckert et al. (2011) study,

symptoms of BIR in Tumbarumba were attributed to physiological causes:

inflorescence necrosis and bunch-stem necrosis. Inflorescence necrosis and

bunch-stem necrosis are induced by ammonium toxicity (Keller & Koblet, 1995),

and their symptoms include rachis and/or pedicel lesions, and

inflorescence/bunch abscission (Capps & Wolf, 2000). The symptoms are similar

to those of BIR, except that the bacterial disease causes the additional symptom

of water-soaked appearance and bacterial ooze consisting of pure cultures of

P. s. syringae (Abkhoo, 2015; Whitelaw-Weckert et al., 2011). The similarity of

these symptoms may have led to P. s. syringae infections being under-reported

within the wine industry. Future investigations should include surveying

inflorescences early in the season and comparing bunch numbers at harvest

within vineyards containing known P. s. syringae infection.

Phenotypic identification

Phenotype-based methods for identifying P. syringae, such as LOPAT, can

produce variable results depending on pathovar, host origin or the nature of the

bacterium itself (Lelliott & Stead, 1987). The non-pathogenic grapevine

P. s. syringae isolates (DAR82449, DAR82450, DAR82451 and DAR82452)

were unable to cause tobacco leaf HR. As the LOPAT protocol was devised for

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pathogenic bacteria, the protocol appears to have successfully differentiated

between the pathogenic and non-pathogenic strains in our collection. This is

consistent with the findings of Diallo et al. (2012) who demonstrated that their

environmental P. syringae isolates were unable to cause HR in tobacco. The HR

is the result of a cell death program used by plants to combat infecting bacteria. It

is initiated by plant resistance proteins following recognition of effectors secreted

into the plant cell by the invading bacteria to suppress host defences. However,

HR does not occur unless the bacteria have a functional type III secretion system,

which is encoded by the hrp and hrc genes (He, 1996). The HR- negative isolates

identified by Diallo et al. (2012) lacked at least one gene in the canonical hrp/hrc

locus or the associated conserved effector locus which prevents them from

initiating HR on tobacco. Further studies are required to investigate whether the

non-pathogenic grapevine P. s. syringae isolates identified in the current study

also contain mutations in the hrp/hrc locus.

Molecular characterisation of isolates

The present study used rpoB sequence typing and MLST analysis to confirm the

identity of the DAR73915 isolate, originally collected by Hall et al. (2000) from

the Adelaide Hills, as P. s. syringae. Interestingly, five P. s. syringae isolates

collected from diseased grapevines in Tumbarumba, New South Wales were

found to have identical rpoB sequences to the DAR73915 Adelaide Hills isolate.

As the rpoB gene has been established as a reliable marker for bacterial strain

identification with high resolution for phylogenetic applications (Mollet et al.,

1997; Tayeb et al., 2005), these results indicate that the Adelaide Hills and

Tumbarumba P. s. syringae isolates may have originated from the same source.

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This study also used MLST data from four genes (gapA, gltA, gyrB, and rpoD) to

characterise the core genome of P. s. syringae isolates collected from grapevines

in Australian vineyards. The grape P. s. syringae isolates were grouped by MLST

into two separate clades with excellent bootstrap support. Each clade contained a

mixture of pathogenic (to grapevine) and non-pathogenic isolates. The clades also

contained non-grape P. s. syringae hosts, and a mixture of pathogenic (to

grapevine) and non-pathogenic stains, indicating that P. s. syringae in Australian

vineyards is genetically diverse.

Pathogenicity

Pseudomonas syringae pv. syringae isolates that were similar molecularly

differed in pathogenicity towards grapevine, and P. s. syringae isolates

pathogenic to grapevine had significantly different rpoB and MLST sequences. In

addition, some P. s. syringae isolates from non-grape hosts were positive for

GPLTA pathogenicity testing on grapevine leaf discs, indicating a lack of host

specificity and a potential source for cross-infection of grape from other

horticultural crops. However, further investigations are required to investigate

whether these non-grape isolates can cause grapevine BIR. These results are

consistent with the results of Najafi and Taghavi (2014) who reported that

P. s. syringae isolates obtained from diseased tissues of Prunus, beet, pear,

quince, oat, millet, wheat, barley and rice were all pathogenic to peach seedlings,

regardless of the original host or position within a phylogenetic tree. Sanz et al.

(2013) also showed that the core genome of P. syringae (by MLST) was only

weakly associated with the pathovar designation and the plant host from which

the bacteria were isolated. Pathogenicity and the preferred plant host are less

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likely to be directed by the core genome (e.g. as characterised by RAPD and

MLST) than by the ‘flexible genome’ which consists of genes encoding proteins

responsible for adaptation to specific niches, evolving through horizontal

exchange (Hwang et al., 2005). Further investigations are required to investigate

horizontal transfer of genes encoding pathogenicity within the ‘flexible genome’

of grapevine pathogenic P. s. syringae.

Source of inoculum

The rpoB and MLST sequences of P. s. syringae isolate DAR82446, which was

isolated from a necrotic rachis on a grapevine sucker shoot in a Tumbarumba

vineyard, were identical to those of the pathogenic P. s. syringae isolates causing

BIR within the same vineyard. As DAR82446 was also positive for the grapevine

pathogenicity leaf test assay, this implies that grapevine sucker shoots may allow

overwintering of pathogenic P. s. syringae. Similarly, Pseudomonas avellanae,

the causal agent of hazelnut bacterial canker, infects sucker shoots on hazelnut

trees and it has been suggested that latently infected sucker shoots used for

propagation may have been the main vehicle for wide-spread dispersal of the

pathogen in Italy (Scortichini, 2002). In many fruit crops, P. syringae can also

overwinter in buds, tissue around leaf scars and saprophytically at the margin of

necroses and cankers (Bultreys & Kaluzna, 2010). In addition, as P. s. syringae

has been isolated from weeds (Geranium sp. and Malva sp.) within Californian

stone fruit orchards affected by bacterial canker (Little et al., 1998), it is possible

that P. s. syringae may overwinter on weeds and ground cover within agricultural

fields. The role of other plant species within the vineyard as potential sources of

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P. s. syringae inoculum, and the progression of colonisation within the grapevine

host, needs to be investigated.

Dispersion

The results of the current study demonstrate the general spread of P. s. syringae

across cool climate Australian vineyards. Pseudomonas syringae may also be

dispersed by soil particles (Hollaway & Bretag, 1997), honey bees (Pattemore et

al., 2014), insects and mammals (Bashan, 1986), water sources (Morris et al.

2008) and precipitation from clouds (Clarke et al., 2010; Monteil et al., 2014;

Morris et al., 2008; Morris et al., 2013). Interestingly, P. s. syringae infections of

grapevines in cool climate regions of Australia are often associated with heavy

spring rains (Hall et al., 2002; Whitelaw-Weckert et al., 2011). This is also

supported by the phylogenetic data demonstrating similarity between isolates of

P. s. syringae from grapevine tissue originating from separate vineyards (e.g.

DAR82440 from Murrumbateman, New South Wales and DAR82442 from

Piper’s River, Tasmania). P. syringae may be dispersed by many mechanisms

including agricultural tools used in pruning and harvesting (Carroll et al., 2010).

Hall et al., 2002 previously showed that BLS symptoms and disease severity

increased in vineyards in subsequent seasons, and that transmission to other

nearby vineyards may result. This suggests that the shared use of contaminated

pruning equipment within and between vineyards may have resulted in disease

spread (Lamichhane et al., 2014). This mode of transmission has also been

observed with P. syringae from cherry (Carroll et al., 2010).

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Similarly, Hirano et al. (1996) demonstrated an association between bacterial

populations and rainfall on bean cultivars. A comprehensive review conducted by

Morris et al. (2013) indicated that the water cycle and rainfall are important for

P. s. syringae movement through the environment. However, as P. syringae from

water sources such as rainfall and snowmelt have been reported to lack essential

T3SS effectors for virulence (Mohr et al., 2008). Although environmental

reservoirs contain non-pathogenic strains of P. syringae, these only account for

approximately 20% of P. syringae strains isolated from the environment (Morris

et al., 2007; Morris et al., 2008). Humidity has been demonstrated to increase

bacterial motility in bean (Leben et al., 1970) and it is more likely that the

increased disease in vineyards with heavy spring rain and overhead water

sprinklers is caused by the increased humidity from these water sources. It will

be important to determine whether the combination of high humidity and unclean

pruning equipment play a major role in motility and dispersal of this pathogen.

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2.5 Conclusions

The results from this investigation provide the foundations for an improved

understanding of the genetic structure and diversity of grapevine pathogen,

P. s. syringae. We have demonstrated that infection of Australian grapevines with

pathogenic P. s. syringae occurs in at least six cool climate viticultural regions

plus one warmer region with over-head water spray irrigation. It is clear that

genetically and pathogenically distinct strain groups of P. s. syringae can be

isolated from grapevines, and that genetically distinct strain groups of

P. s. syringae from other plant hosts may infect grapevine.

On the basis of this study, we conclude that the presence of P. s. syringae in

Australian cool climate vineyards may pose a threat to the Australian wine

industry. Damage caused by P. s. syringae can lead to severe economic losses.

Furthermore, some isolates of P. s. syringae lack host specificity and therefore

may be transmitted from one crop to another. The isolation of pathogenic

P. s. syringae from grapevine sucker shoots also suggests that sucker shoots allow

overwintering of the pathogen. Protective measures may need to be introduced or

considered in vineyards susceptible to P. s. syringae, due to its easy dispersal

through pruning and other equipment. Appropriate protective measures and

sterilisation of pruning machinery are highly recommended in susceptible

vineyards.

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2.6 Acknowledgements

Dr. Thomas Hill, Colorado State University, USA; Dr Roger Shivas and Miss Yu

Pei Tan, Department of Agriculture, Fisheries and Forestry (DAFF) in

Queensland are thanked for their generous gifts of P. syringae isolates. We are

indebted to Mr. Nathan Scarlett (dec’d) who collected the Coonawarra vineyard

necrotic rachis samples.

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Chapter 3 Pseudomonas syringae pv. syringae From Cool

Climate Australian Grapevine Vineyards: Insight Into

Phenotypes and Virulence Associated With Bacterial

Inflorescence Rot

3.1 Introduction

Pseudomonas syringae pv. syringae (P. s. syringae) causes extensive yield losses

in winegrape production in some Australian cool climate viticultural regions.

Bacterial inflorescence rot (BIR) and bacterial leaf spot (BLS), relatively new

diseases to grapevine, are characterised by leaf spots (BLS only), necrotic lesions

on petioles and shoots, and necrosis of inflorescences. Here I report on the

phenotypic and genotypic differences between P. s. syringae isolated from

various winegrape (Vitis vinifera) hosts, using molecular multi-locus sequence

typing (MLST) and analysis of molecular variance (AMOVA). These results

show that all P. s. syringae isolates obtained from grapevines with BIR or BLS

symptoms were assessed as positive for the tobacco leaf hypersensitivity

response. Most pathogenic P. s. syringae (i.e. those positive for tobacco leaf

hypersensitivity response) were negative for tyrosinase activity. This absence of

tyrosinase activity may be associated with a lifestyle within the plant with little

need for protection from UV light, so that melanin is not required for survival.

Sensitivity to ampicillin was associated with pathogenicity, in line with a possible

programmed balance between antibiotic resistance and pathogenicity in some

bacterial plant pathogens. Syringopeptin production and the presence of the gene

for syringolin A (sylC) were also associated with BIR.

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3.2 Materials and methods

Bacterial Strains. Pseudomonas syringae pv. morsprunorum

(P. s. morsprunorum)(DAR33419) and P. s. syringae (DAR72042 and

DAR73915) were obtained from the NSW Industry and Investment Culture

Collection (Orange, Australia). Pseudomonas syringae isolates BRIP34823,

BRIP38670, BRIP34831, BRIP34899, BRIP38817, BRIP34832, BRIP34805,

BRIP38811 and BRIP34803 were obtained from the Department of Agriculture,

Fisheries and Forestry (DAFF), Queensland. Pseudomonas syringae isolates

DAR82449, DAR82450, DAR82451, DAR82432, and DAR82453 were obtained

from Dr. Thomas Hill, Colorado State University, USA.

Six vineyards affected by BIR, three in the Tumbarumba region, New South

Wales, one in the Canberra district (Murrumbateman, New South Wales), one in

the Coonawarra (South Australia) and one in Piper’s River (Tasmania), were

sampled between September and October in 2006 by M Weckert, and in 2011 and

2014. Samples were also collected from a vineyard with BLS, but no BIR, in the

Macedon Ranges region (Hanging Rock, Victoria), and from frost affected but

healthy grapevines in Glenlofty (Pyrenees, Victoria) and Hallston (Gippsland,

Victoria). Leaves, shoots, and rachii were collected with ethanol-sterilised

equipment, placed into Zip-Lock polyethylene bags and kept at 4°C before

bacterial isolation.

Bacteria were isolated from surface sterilised grapevine tissue, aseptically placed

on Pseudomonas Selective (PS) agar (Oxoid) and incubated in the dark for up to

85

3 days at 25°C. Pseudomonas syringae was identified by the production of

fluorescent pigments under UV light (354 nm) and the LOPAT identification

scheme and confirmed by the absence of 2-keto gluconate production and nitrate

reduction, and the production of acid from sucrose (Lelliott & Stead, 1987). All

isolates were maintained on King’s B (KB) agar at 25°C in darkness for 24 h and

suspended in sterile deionised water (SDW) to a concentration of approximately

108 CFU/mL determined by optical density. This suspension was then used for all

biochemical tests and incubated at 25°C unless otherwise stated. All tests were

carried out in triplicate.

Gelatin liquefaction. Gelatin liquefaction was determined by inoculating gelatin

medium in sterile bottles and incubated for up to 21 days (Latorre & Jones, 1979).

Liquefaction was determined by refrigerating the bottles for 30 min at 4°C and

tilting the medium on days 3, 7, 14, and 21. If the medium ran freely, that was

considered a positive reaction. Viscous samples were considered negative.

Aesculin hydrolysis. Bacteria were streaked over medium containing 10.0 g/L

peptone, 1.0 g/L aesculin, 0.5 g/L ammonium ferric citrate and 12.0 g/L agar

No.1 (Oxoid) and incubated for 3 days. Aesculin hydrolysis was determined by

the conversion of aesculin to aesculetin, as demonstrated by production of black

colouration of the medium (Fig. 3.1A and 3.1B) (Lelliott & Stead, 1987).

Tyrosinase Activity. Bacteria were streaked on tyrosinase-casein medium

containing 5 mL glycerol, 10.0 g/L casein hydrolysate (Oxoid), 0.5 g/L K2HPO4,

0.25 g/L MgSO4•7H2O, 1.0 g/L l-tyrosine, and 15.0 g/L Bacteriological agar No.3

86

(Oxoid) (pH7.2) and incubated for up to 10 days. Tyrosinase activity was

assessed as the production of a reddish brown pigment (Fig. 3.1C and 3.1D)

(Lelliott et al., 1966).

Carbohydrate source utilisation. Carbohydrate source tests were done on

mineral salts medium containing 1.0 g/L NH4H2PO4, 0.2 g/L KCl, 0.2 g/L

MgSO4•7H2O, 12.0 g/L agar, with 1.6% alcoholic bromothymol blue. Carbon

sources (tartaric acid, lactic acid or sorbitol) were sterilised and added aseptically

to molten medium for a final concentration of 0.1% (w/v). Bacteria that grew on

individual carbon sources were considered positive for that carbon source

utilisation (Barta & Willis 2005) after 3 to 5 days. Negative controls were used by

inoculating bacteria onto medium in the absence of a carbon source (Fig. 3.1E

and 3.1F) (Gašić et al., 2012; Scortichini et al., 2005).

Ice nucleation activity. Bacterial isolates were tested for ice nucleation activity

(INA) as described by others (Lindow et al., 1978). A 10 µL drop of 108 CFU/mL

bacteria suspension in SDW was kept at 5°C and applied to an aluminium foil

boat floating in a water/ethanol ice bath at -6°C. Bacterial suspensions that

rapidly froze were considered INA positive. Sterile deionised water and non-ice

nucleating Pseudomonas fluorescens were included as controls.

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Fig. 3.1. GATTa characterisation of P. syringae phenotypes. P. syringae present with aesculin

negative (A), or positive (B), tyrosinase negative (C), or positive (D), and negative for tartaric

acid, lactic acid, or sorbitol negative (E) or positive (F).

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Pectolytic Activity by Paton’s Pectate Medium Method. Pectolytic activity

was assessed by spot inoculating bacteria, using 10 µL drops, onto Paton’s

Pectate Medium (5.0 g/L peptone (Oxoid), 5.0 g/L lab-lemco (Oxoid), 5.0 g/L

calcium lactate (ChemSupply), 12.0 g/L bacteriological agar No. 3 (Oxoid), pH

7.0) with a pectate overlayer (10.0 g/L polygalacturonic acid sodium salt

(Sigma)), 0.1 g/L disodium ethylenediamine tetra acetate (Sigma), and 1.5 mL

0.54% bromothymol blue (Sigma)). Pectolytic Pseudomonads were determined to

by the production of shallow pits after 3-4 days (Lelliott & Stead, 1987; Liao et

al., 1994) (Fig. 3.2A and 3.2B).

Proteolytic Activity. Sterile molten nutrient agar was supplemented with 10g/L

skim milk powder. The production of a clearing of the milk around the bacterial

colonies is an indication of proetolytic activity (Fig. 3.2C and 3.2D) (Kitten et al.,

1998). Proteolytic Pseudomonas marginalis (isolated from a grapevine, Mildura,

Victoria) was used as a positive control.

Catalase. Bacterial colonies were applied to a sterile glass side with an applicator

stick and a drop of 3% (v/v) hydrogen peroxide was added. The production of gas

bubbles indicates a positive reaction for catalase and the conversion of hydrogen

peroxide into oxygen and water.

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Fig. 3.2. Pecto- and proteolytic activity of Pseudomonas spp. Pectolytic activity of negative (A)

and positive (B) isolates on Patons media with pectin overlayer. Proteolytic activity of negative

(C) and positive (D) isolates on milk agar.

Antibiotic Resistance. Bacteria were streaked onto KB agar containing either

chloramphenicol (25 µg/mL), ampicillin (100 µg/mL), tetracycline (15 µg/mL),

or streptomycin (100 µg/mL) (Sigma) and incubated. Bacterial growth was

checked every 24 hours for 3 days. The absence of growth, or failure to thrive,

indicated bacteria to be sensitive to the tested antibiotic (Hwang et al., 2005).

Pathogenicity Tests. Tobacco leaves were infiltrated with bacterial suspensions

at individual sites. This was achieved by making a small nick on the abaxial side

of the leaf lamina with a 26 gage hypodermic needle, and injecting 100 µL

bacterial suspensions into the mesophyll using a needle-less syringe. A positive

90

reaction for tobacco leaf HR is the presence of water-soaking within the first 24

hours post inoculation (hpi) and collapse of the mesophyll (Fig. 3.3). At 48 hpi a

light-brown necrosis should be produced along with collapsing of the mesophyll

at the site of infiltration (Lelliott & Stead, 1987). All individual leaves infiltrated

also included a negative control of SDW.

The lemon pathogenicity assay was carried out as described by Gašić et al. (2012)

with minor changes. Approximately 100 µL of bacterial suspension was injected

into individual lemons at six sites using a hypodermic needle, leaving a droplet at

the injection site. Lemons were then incubated at 25°C on wet filter paper to

promote high humidity in a sealed plastic container, and symptom development

was observed daily for seven days (Fig. 3.4).

The detached grapevine leaf assay was carried out as described by Arraiano et al.

(2001) with minor changes. Briefly, healthy leaves were detached from

V. vinifera cv. Chardonnay plants (grown under greenhouse conditions) and were

surface-sterilised in sodium hypochlorite (1% available chlorine) solution

containing 100 μL/L 190 TWEEN 80 (Sigma, Australia) for 3 min, followed by

four washes in SDW. Leaves were placed, abaxial side up, into Petri dishes

containing 1% agar, and allowed to dry. For inoculation, bacterial suspensions

(approximately 108 CFU/mL) were sprayed as a fine mist onto the grapevine

leaves until most of the leaf was covered. Plates were then incubated at 25°C in

light/dark conditions. Symptom development was observed daily for seven days

(Fig. 3.5).

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Fig. 3.3. Hypersensitivity reaction in tobacco leaves caused by potentially pathogenic isolates of

P. syringae. Collapse of the mesophyll at the site of infiltration 24 hpi indicates a positive reaction

(arrows). Water (control) (asterisks) and non-pathogenic P. syringae (§) did not produce HR-like

symptoms.

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Fig. 3.4. Pathogenicity test on mature lemon (cv. Yen Ben). Isolates of P. syringae that are

potentially pathogenic to lemon cause necrotic spots at the point of inoculation within 7 days

(‘pos’).

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Fig. 3.5. Pathogenicity test on detached grapevine (Chardonnay) leaves spray inoculated with

P. syringae. A) Detached grapevine leaf with no necrosis. Necrosis symptoms were observed over

seven days: (B) One day post inoculation (dpi), (C) 3 dpi, (D) 5 dpi, and (E) 7 dpi.

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Syringomycin and syringopeptin production. Syringomycin production was

determined by a method for general lipodepsipeptide detection (Kairu, 1997).

Bacterial cultures were spot inoculated onto potato dextrose agar (PDA) and

incubated for 4-6 days. Plates were then sprayed with a suspension

(approximately 106 CFU/mL) of Geotrichum candidum, Saccharomyces

cerevisiae or Bacillus megaterium in sterile 0.9% NaCl and incubated for 24 to 48

h. A zone of inhibition around the bacterial colony was considered a positive

result for lipodepsipeptide production (Gašić et al., 2012; Kairu, 1997).

Saccharomyces cerevisiae and G. candidum indicate the production of

syringomycin (Fig. 3.6A and 3.6B, and 3.6C and 3.6D, respectively) (Gašić et al.,

2012; Iacobellis et al., 1992; Lavermicocca et al., 1997; Vassilev et al., 1996)

whereas a zone of inhibition around colonies sprayed with B. megaterium

indicates syringopeptin production (Fig. 3.6E and 3.6F) (Lavermicocca et al.,

1997; Vassilev et al., 1996).

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Fig. 3.6. Determination of syringomycin and syringopeptin production in P. syringae. Isolates

positive for syringomycin produced zones of inhibition of S. cerevisiae around test colonies of

P. syringae (A negative, B positive), and/or G. candidum (C negative, D positive). Production of

syringopeptin was determined by inhibition of B. megaterium. No inhibition of B. megaterium

around test isolates indicates that syringopeptin is not produced (E) whereas inhibition around test

isolates indicates the production of syringopeptin (F).

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Detection of P. s. syringae toxin genotypes. To supplement the phenotypic data,

PCR was used to detect genes involved in production of four toxins:

syringomycin (syringomycin biosynthesis enzyme 1, syrB); syringolin A

(syringolin A biosynthesis enzyme, sylC); syringopeptin (syringopeptin

synthetase C, sypC); and coronatine (coronafactate ligase, cfl). Bacterial DNA

was extracted using Qiagen DNeasy Blood and Tissue Kit (Qiagen, Australia)

following the manufacturer’s protocol. PCR was carried out using GoTaq Green

Master Mix (Promega, Australia) with a final primer concentration of 0.3 µM and

100 ng sample DNA and performed with C1000 Thermal Cycler (BioRad,

Australia) under the following conditions: 94°C for 30 s, 60°C for 30 s, and 72°C

for 30 s for 35 cycles. PCR products were analysed on a 2% agarose gel and

visualised with ethidium bromide using a GelDock system (BioRad). In order to

validate gene detection gyrA (housekeeping) target was also amplified. Samples

containing detectable amounts of gyrA PCR product indicated that amplifiable

genomic DNA was present during PCR reactions. Isolate data were scored

according to the presence or absence of PCR products. All primers used are listed

in Table 3.1.

97

Ta

ble

3.1

. P

rim

ers

use

d f

or

the

det

ecti

on

of

DN

A g

yra

se s

ub

un

it A

(g

yrA

), s

yri

ng

om

yci

n b

iosy

nth

esis

e en

zym

e 1

(sy

rB),

sy

rin

go

lin

A

bio

syn

thes

is

enzy

me

(syl

C),

sy

rin

gop

epti

n s

yn

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ase

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syp

C),

an

d c

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naf

acta

te l

igas

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tho

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Th

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l et

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(2

00

5)

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m

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al. (1

99

5)

Th

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m

Am

rein

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(20

04)

Th

is s

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)

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19

94

)

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CP

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75

U2

51

30

AJ5

48

82

6

AF

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62

16

S7

39

73

Ta

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(bp

)

18

6

25

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22

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27

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Seq

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5’→

3’)

AC

AC

GC

TC

TT

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TC

GG

TG

AT

CC

AG

GA

AA

TC

CT

CA

AC

CA

GA

TA

TC

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GC

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TG

GA

GA

AG

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GA

AA

CC

GA

TG

GA

AA

CC

AC

GG

CA

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AA

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AG

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GG

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GA

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AG

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GT

AG

TA

CG

CA

AC

A

Pri

mer

gyr

A 4

F

gyr

A 4

R

syrB

3F

syrB

3R

sylC

2F

sylC

2R

syp

C 6

F

syp

C 6

R

cfl

8F

cfl

8R

98

Multilocus sequence typing. Multilocus sequence typing (MLST) of all

P. s. syringae isolates was carried out on glyceraldehyde-3-phosphate

dehydrogenase (gapA), citrate synthase (gltA), DNA gyrase B (gyrB), and sigma

factor 70 (rpoD). Target gene amplification, purification, sequencing and

production of a Neighbour-Joining tree are outlined in Chapter 2.

Statistical Analysis. Evolutionary relationships of P. s. syringae from BIR

affected grapevine vineyards were demonstrated using Arelquin software.

Analysis of molecular variance (AMOVA) was used to determine associations

between the relevant MLST sequence data previously presented in Chapter 2 and

selected functional data (tyrosinase activity, syringopectin and syringomycin,

lemon test, tobacco leaf HR, grapevine leaf pathogenicity, and resistance to

antibiotics) (Excoffier et al., 1992) as implemented by Arlequin, version 3.5.1.2

(Excoffier et al., 2005). Files were set up using the program DnaSP, version 5.10

(Librado & Rozas, 2009) from FASTA files created in MEGA5 (Tamura et al.,

2011). AMOVA determined the proportion of genetic variation between

populations, relative to the proportion of variation within populations.

Populations were defined as isolates that were either negative or positive for

specific genotypes/phenotypes (e.g. one population included all the isolates that

contain a positive genotype for the sylC gene). AMOVAs estimated ΦST fixation

indices of between-population genetic differentiation incorporating pair-wise

nucleotide distances scaled by a gamma correction of 0.18. Fixation indices were

assessed for significance against null distributions of the data generated by

permutation (10,000 replicates). In cases where n < 5 for populations,

significance testing was not done.

99

3.3 Results

Previously, in Chapter 2, I identified P. s. syringae from six vineyards affected by

BIR (three in Tumbarumba, one from Murrumbateman, one from Coonawarra,

and one from Piper’s River), from grapevines with BLS only (Hanging Rock),

and from apparently healthy grapevines (two from Hallston and two from

Glenlofty). In the current study, 32 isolates of P. syringae underwent a series of

biochemical and antibiotic resistance testing, with a focus on the characterisation

of P. s. syringae from grapevine hosts (n=26; Table 4.2). All isolates of

P. syringae in the current study were Gram-negative bacilli (Data not shown).

Most isolated P. s. syringae belonged to LOPAT group Ia, as determined by the

scheme of Lelliot and Stead (1986) (i.e. positive for levan and tobacco HR,

negative for oxidase, potato rot and arginine dihydrolase). Exceptions were non-

pathogenic isolates of P. s. syringae (DAR82449 to DAR82452, from “healthy”

but frost-affected Chardonnay grapevines) that were negative for tobacco leaf HR

(Table 3.2).

Production of fluorescent pigments, visualised under UV light at 354 nm on PS

agar, was also observed in all isolates of P. syringae (Table 3.2). Initially, isolates

appeared with a white/pale green to green fluorescence; however cultures older

than approximately five days began to produce blue fluorescent pigment (data not

shown).

100

Table 3.1. Biochemical and antibiotic reactions of P. s. syringae from grapevine hosts and from other pathovars of P. syringae. Isolates were collected in year indicated and number of isolates used in the current study indicated

within the brackets.

P. syringae pv. syringae

P. syringae pathovar

Tumbarumba1

2011 (4)

Tumbarumba2

2013 (6)

Tumbarumba3

2006 (4)

Tumbarumba4

2011 (3)

Adelaide

Hills5

2000 (1)

Murrumbateman6

2013 (1)

Hanging

Rock7

2013 (1)

Piper’s

River8

2014 (1)

Glenlofty9

2007 (2)

Hallston10

2007 (2)

Coonawarra11

2010 (1) Pstr12 Pma13 Pstab14 Pmo15 Php16 Psm17

Levan + + + + + + + + + + +

+ + + + + +

Oxidase - - - - - - - - - - - - - - - - -

Potato rot - - - - - - - - - - - - - - - - -

Arginine Dihydrolase - - - - - - - - - - - - - - - - -

Tobacco HR + + + + + + + + - - + + + + + + +

Fluorescence (254nm) + + + + + + + + + + + + + + + + -

Gelatin + + + + + + + + + + +

- - - - + -

Aesculin + + + + + + + + + + + + - - + - -

Tyrosinase Activity v - - v - + - - + - + + - - - - +

Tartaric Acid v - v - - - - - - - - - + - - - -

Lactic Acid + v + + + + + + + - +

- + - - + -

Sorbitol + + + + + + + + + + + + + - + + +

Nitrate Reduction - - - - - - - - - - -

- - - - - -

2-keto Gluconate - - - - - - - - - - - - - - - - -

Acid from Sucrose + + + + + + + + + + + + + + + + +

Ice Nucleation Activity + + + + + + - + + + +

+ - + - - -

Pectolytic + + + + + + + + + + + + + + + + -

Proteolytic - - - - - - - - - - - - - - - - -

Catalase + + + + + + + + + + + + + + + + +

Diffusible Brown Pigment - - - - - - - - - - - - - - - - +

Pathogenic to Grapevinea + + + v + + + + - - +

+ + + - - -

Pathogenic to Lemon v v + + + + + - - v + - - - + + +

Syringopeptin (B. megaterium) + + + + + + + + v + -

+ + - + + -

Syringomycin (S. cerevisiae) v v v v - + + + v - + - - - - - -

Syringomycin (G. candidum) v v + + - + + + v - - - - - - - -

Chloramphenicol Resistance v v v + + + - + + v +

+ + - - - +

Ampicillin Resistance - - - v - - - - + + + - + + + - -

Tetracycline Resistance v - - - - - - - - - - - - - - - -

Streptomycin Resistance - - - - - - - - - - - - - - - - -

1DAR82159 to DAR82162, 2DAR82443 to DAR82448. 3DAR77819, DAR77820, DAR82169 and DAR82170. 4DAR82165, DAR82166 and DAR82171. 5DAR73915. 6DAR82440. 7DAR82441. 8DAR82442. 9DAR82449 and DAR82450. 10DAR82451 and DAR82452. 11DAR82453. 12BRIP34832.

13BRIP38817. 14BRIP34803. 15BRIP34805. 65BRIP38811. 17DAR3341. a Pathogenic to grapevine determined by detached grapevine leaf assay. “v” indicates variable results between isolates. Pstr, P. syringae pv. striafaciens; Pma, P. syringae pv. maculicola; Pstab, P. syringae pv. tabaci; Pmo, P. syringae

pv. mori; Php, P. syringae pv. phaseolicola; Psm, P. syringae pv. morsprunorum.

101

Biochemical testing. All isolates were found to be positive for gelatin

liquefaction and aesculin hydrolase activity (Table 3.2). Tyrosinase activity in

isolates of P. s. syringae collected from grapevine was variable. With the

exception of P. s. syringae, each pathovar was able to produce its own unique set

of GATTa (Gelatin liquefaction, Aesculin hydrolase, Tyrosinase activity, and

Tartaric acid utilisation) test results. Pseudomonas syringae pv. syringae isolates

produced variable GATTa profiles. Two isolates of P. s. syringae were positive

for tartaric acid utilisation, albeit weakly, and variability was observed in isolates

collected from Tumbarumba vineyards. Most isolates of P. s. syringae were

positive for lactic acid utilisation and all isolates were able to utilise sorbitol as a

sole C-source (Table 3.2).

All P. s syringae isolates from grapevine were negative for nitrate reduction and

2-keto gluconate production, and produced acid from sucrose (Table 3.2). Most

P. s. syringae isolates were positive for ice nucleation activity (INA), with the

exception of one pathogenic isolate DAR82441. All P. s. syringae isolates were

positive for pectolytic activity (as determined using Paton’s Pectate Medium

Method), lacked proteolytic activity, were catalase positive, and did not produce a

diffusible brown pigment on KB agar (Table 3.2).

Pathovars other than P. s. syringae were included in the biochemical analyses to

determine the robustness of the identification methodology. The LOPAT results

for pathovars striafaciens, maculicola, tabaci, mori, phaseolicola and

morsprunorum, were consistent with the published data for these pathovars

(Gašić et al., 2012; Lelliott et al., 1966). They also produced consistent

102

phenotypes for secondary confirmatory tests (nitrate reduction, 2-keto gluconate

production, and production of acid from sucrose). All non-syringae pathovars,

with the exception of P. s. morsprunorum, fluoresced under UV light, were

positive for pectolytic activity (by Paton’s Pectate Method), negative for

proteolytic activity, were catalase positive, and did not produce a diffusible

brown pigment. P. s. morsprunorum did not fluoresce under UV light, was

negative for pectolytic activity (by Paton’s Pectate Medium Method) and negative

for proteolytic activity, was also catalase positive, and produced a diffusible

brown pigment on KB agar (Table 3.2).

Pathogenicity testing. In addition to infiltrating tobacco leaves as part of the

LOPAT identification assay (Table 3.2, Fig. 3.3) pathogenicity tests were also

carried out by inoculating mature lemons and detached grapevine leaves. Of the

24 P. s. syringae isolates found to be potentially pathogenic based on tobacco leaf

HR assay (Table 3.2), 22 also caused development of necrotic lesions on lemon

(Table 3.2, Fig. 3.4). Similarly, all P. s. syringae grape isolates, with the

exception of DAR82449, DAR82450, DAR82451 and DAR82452 from

“healthy” but frost-affected Chardonnay grapevines, were positive for grapevine

pathogenicity when tested on detached grapevine leaves (Table 3.2, Fig. 3.5).

Pathovars striafaciens, maculicola, and tabaci were potentially pathogenic to

detached grapevine leaves but not on mature lemon, whereas pathovars mori,

phaseolicola, and morsprunorum were potentially pathogenic on mature lemon

but not on detached grapevine leaves (Table 3.2).

103

Antibiotic resistance. All pathogenic P. s. syringae isolates, except one isolate

from a BIR affected Coonawarra grapevine (DAR82453), were sensitive to

ampicillin. In contrast, pathovars maculicola, tabaci, and mori, and all non-

pathogenic grapevine isolates (DAR82449 to DAR82452) were resistant to

ampicillin (Table 3.2). Most (73%) of the grapevine P. s. syringae (both

pathogenic and non-pathogenic) isolates were resistant to chloramphenicol.

Resistance to chloramphenicol was seen in pathovars striafaciens, maculicola,

and morsprunorum. All isolates of P. syringae tested were sensitive to

streptomycin. Only one isolate was resistant, albeit weakly, to tetracycline

(DAR82161) (Table 3.2).

Toxin production . As toxins produced by some P. syringae pathovars, such as

syringopeptin and syringomycins, exhibit antimicrobial activity, the detection of

such toxins can be determined using sensitive indicator microorganisms. This

study used B. megaterium inhibition to determine isolates that may produce

syringopeptin, and S. cerevisiae and G. candidum inhibition for the detection of

syringomycin in isolates for P. s. syringae from grapevine host (Fig. 3.6).

The presence of genes responsible for toxin production was also detected using

PCR techniques. The grapevine P. s. syringae isolates were tested for

syringomycin, syringolin, syringopeptin, and coronatine production by detection

of genes involved in their biosynthesis (syrB, sylC, sypC, and cfl respectively).

PCR amplification with primers syrB, sylC, sypC, and cfl gave rise to product

sizes of 256, 222, 274 and 507 bp, respectively (Fig. 3.7). Although the positive

control, P. syringae pv. tabaci (BRIP34803) from soybean, gave a positive result

104

for cfl, none of the P. s. syringae isolates were positive, indicating that they could

not produce coronatine. Detectable amounts of gyrB housekeeping gene were

produced by all P. s. syringae isolates (Data not shown).

Coronatine is a known molecular mimic of jasmonic acid and has been

thoroughly investigated for its role in plant-pathogen interactions and virulence.

Although it is commonly accepted that P. s syringae does not produce coronatine

it was included so it could be excluded as a possible mechanism for virulence in

grapevine P. s. syringae. None of the grapevine P. s. syringae isolates was able to

produce coronatine by detection of cfl gene required for coronatine synthesis

(Bultreys & Gheysen, 1999).

105

Fig. 3.7. PCR products amplified with syrB primers (syringomycin) (lanes 2 and 3), sylC primers

(syringolin) (lanes 4 and 5), sypC primers (syringopeptin) (lanes 6 and 7), and cfl primers (lanes 8

and 9) from isolates of P. s. syringae grown in culture from diseased grapevine (DAR77819,

DAR77820. DAR82162, DAR82170, DAR82440, and DAR82446), P. s. phaseolicola

(BRIP38811) from diseased bean, and P. s. tabaci (BRIP34803) from soybean. Negative controls

used are BRIP38811, DAR77819, DAR82446, and DAR82170 for syrB, sylC, sypC and cfl

reactions, respectively. Positive controls are DAR82162, DAR82440, DAR77820, and

BRIP34803 for syrB, sylC, sypC and cfl reactions, respectively. Products were separated on a

1.2% agarose gel stained with ethidium bromide.

106

Molecular multi-locus sequence typing (MLST). A linearised neighbour-

joining MLST phylogenetic tree was constructed from the combined rpoD, gyrB,

gltA, and gapA data set (data included in Chapter 2). The current analysis expands

on the functional data of each P. s. syringae strain isolated from grapevine hosts.

Previously I reported that P. s. syringae falls into two heterogenous clades,

separated from other P. syringae pathovars (Hall et al., 2016). Phylogenetic

analysis in the current study showed strong bootstrap support (93%), which

allowed for visual inspection of functional/phenotypic data (Fig. 3.8). Visual

inspection of Fig. 4.8 showed that all P. s. syringae isolates from grapevines with

BIR or BLS symptoms were positive for tobacco leaf hypersensitivity response.

Most pathogenic P. s. syringae isolates (i.e. those positive for tobacco leaf

hypersensitivity response) were negative for tyrosinase activity and sensitive to

ampicillin (Fig. 3.8).

Syringopeptin, syringomycin (by the S. cerevisiae test), and syringomycin (by the

G. candidum test) were produced in 95%, 60% and 70%, respectively, of

P. s. syringae isolated from grapevines affected by BIR. The same toxins were

produced in 73%, 46% and 54%, respectively, of total grapevine P. s. syringae.

Genes for syringolin (sylC), syringomycin (syrB), and syringopeptin (sypC) were

present in 95%, 75% and 85%, respectively, of P. s. syringae isolates from

grapevines affected by BIR. Genes for the same toxins were present in 73%, 58%

and 65%, respectively, of total grapevine P. s. syringae (Fig. 3.8).

107

Fig. 3.8. Phylogenetic tree of P. s. syringae isolates and distribution of pathogenicity, antibiotic resistance, and toxin phenotypes. A linearised neighbour-joining MLST tree from combined rpoD, gyrB, gltA, and gapA data set (from Hall et al.,

2016) is shown. The evolutionary history was inferred using the Neighbour-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 0.32928731 is shown. The percentage of replicate trees in which the associated

taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the Jukes-Cantor method (Jukes & Cantor, 1969) and are in the units of the number

of base substitutions per site. The analysis involved 28 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 2126 positions in the

final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Blacked out squares indicate that isolates were positive for that phenotype or genotype, or were positive for antibiotic resistance. BIR: P. s. syringae isolated

from grapevine with bacterial inflorescence rot; BLS: P. s. syringae isolated from grapevine with bacterial leaf spot. sypC: gene for syringopeptin; syrB: gene for syringomycin; sylC: gene for syringolin A.

108

AMOVA. Statistical comparisons between genetic data and phenotypic/genotypic

populations were then performed using AMOVA (Excoffier et al., 2005).

Analysis of molecular variance (AMOVA) was used to investigate any putative

associations between the MLST sequence and the functional data listed in Fig.

3.8. Analysis by AMOVA demonstrated that the following eight P. s. syringae

population groups positive for a factor differed genetically from populations

negative for that factor (e.g. isolation from BIR affected grapevines, tyrosinase

production, grapevine leaf pathogenicity, tobacco leaf HR, syringopeptin

production by B. megaterium, syringomycin production by G. candidum,

syringomycin production by S. cerevisiae, and ampicillin resistance). There was

also a non-significant (P = 0.054) trend for tartaric acid producers to differ

genetically from those not producing tartaric acid (Table 3.3). However, in most

cases, the genetic variations between the populations, compared with variation

within the populations, were very low. The two exceptions with large between

population variation were tartaric acid production (ΦST = 0.401, variation between

populations 40.1%, within populations 59.9%) and syringopeptin

(B. megaterium) (ΦST = 0.369, variation between populations 36.9%, within

populations 63.1%) (Table 3.3). Low sample replication in both of these cases did

not allow for meaningful permutation tests of significance of these high ΦST

values. However, in both cases the high ΦST resulted from fixation of alternate

haplotypes in compared populations. Interestingly, analysis by AMOVA showed

that the phenotype populations of P. s. syringae positive for syringomycin

production (using S. cerevisiae and G. candidum) were not associated with the

populations carrying syringomycin gene syrB using AMOVA (Table 3.3).

109

Analysis by AMOVA also demonstrated that the population of P. s. syringae

isolated from BIR affected grapevines was genetically distinct from the seven

following populations: tyrosinase positive, grape leaf pathogenicity negative,

tobacco leaf HR negative, lemon pathogenicity negative, syringopeptin (by

B. megaterium) negative, ampicillin resistant, and negative for the syringolin A

gene sylC. There was also a non-significant (P = 0.060) trend for tartaric acid

producers to differ genetically from those not producing tartaric acid (Table 3.4).

In most cases, the genetic variations between the populations, compared with

variation within the populations, were very low. The four exceptions with large

between population variation were tartaric acid production (ΦST = 0.454, variation

between populations 45.4%, within populations 54.6%), tobacco leaf HR negative

(ΦST = 0.250, variation between populations 25.0%, within populations 75.0%),

syringopeptin (B. megaterium) (ΦST = 0.504, variation between populations

50.4%, within populations 49.6%), and ampicillin resistance (ΦST = 0.201,

variation between populations 20.1%, within populations 78.9%). The between

population variations for tyrosinase positive, grapevine leaf pathogenicity

negative, and presence of syringolin A gene, sylC were lower (17.3%, 15.1% and

13.0% respectively) (Table 3.4). Although ΦST test demonstrated high level of

genetic fixation between sylC genotypes and syringopeptin production (by

B. megaterium inhibition), significance could not be associated as n < 5 in the

permutation test (Data not shown).

110

Table 3.3. Analysis of molecular variance (AMOVA) between sample populations using MLST

sequence data from grapevine P. s. syringae. Separate AMOVA tests (N=17) of paired

populations defined as positive or negative for various phenotypes. Genetic fixation index (ΦST)

estimates of differentiation between populations (incorporating pairwise nucleotide variation) as

reported. Probabilities (P) of ΦST estimates calculated by permutation (10,000 replicates), except

where not applicable (NA

) due to low sample size. Significance (*) set at P<0.05. The total sample

genetic variance attributed between and within populations also reported.

Phenotypes

aAll

P. s. syringae

tested

Variation (%)

between

populations,

variation (%)

within populations

BIR (N=20) vs non-BIR (N=6) P = 0.002 15.4

ΦST 0.154 * 84.6

Tryosinase neg (N=18) vs pos (N=8) P = 0.003 16.8

ΦST 0.168 * 83.2

Tartaric Acid neg (N=2) vs pos (N=24) NA 23.6

76.4

Lactic Acid neg (N=3) vs pos (N=23) NA 11.9

88.1

INA neg (N=1) vs pos (N=25) NA 0.6

99.4

Grapevine Leaf Pathogenicity neg (N=7) vs pos (N=19) P = 0.006 15.3

ΦST 0.153 * 84.7

Tobacco Leaf HR neg (N=4) vs pos (N=22) NA 17.4

82.6

Lemon Pathogenicity neg (N=7) vs pos (N=19) P = 0.063 6.7

ΦST 0.067 93.3

Syringopeptin (B. megaterium) neg (N=2) vs pos (N=24) NA 36.9

63.1

Syringomycin (G. candidum) neg (N=10) vs pos (N=16) P = 0.047 6.8

ΦST 0.068 93.2

Syringomycin (S. cerevisiae) neg (N=12) vs pos (N=14) P = 0.001 17.6

ΦST 0.176 * 82.4

Chloramphenicol resistance neg (N=7) vs pos (N=19) P = 0.840 1.5

ΦST 0.015 98.5

Ampicillin resistance neg (N=20) vs pos (N=6) P = 0.007 16.7

ΦST 0.167 * 83.3

Tetracycline resistance neg (N=25) vs pos (N=1) NA 3.8

96.2

Presence of syringolin A gene sylC neg (N=4) vs pos

(N=22) NA

7.2

92.8

Presence of syringopeptin gene sypC neg (N=5) vs pos

(N=21)

P = 0.290 1.6

ΦST 0.016 98.4

Presence of syringomycin gene syrB neg (N=7) vs pos

(N=19)

P = 0.210 2.7

ΦST 0.027 97.3

111

Table 3.4. Analysis of molecular variance (AMOVA) between sample populations using MLST

sequence data from BIR affected grapevine P. s. syringae isolates. Separate AMOVA tests

(N=17) of paired populations defined as positive or negative for various phenotypes. Genetic

fixation index (ΦST) estimates of differentiation between populations (incorporating pairwise

nucleotide variation) as reported. Probabilities (P) of ΦST estimates calculated by permutation

(10,000 replicates), except where not applicable (NA

) due to low sample size. Significance (*) set

at P<0.05. The total sample genetic variance attributed between and within populations also

reported.

Phenotypes/Genotypes

bP. s. syringae from

BIR positive

grapevines only

(n = 20)

Variation (%) between

populations, variation

(%) within

populations

Tryosinase pos (8) P = 0.008 17.3

ΦST 0.173 * 82.7

Tartaric Acid pos (24) P = 0.060 45.4

ΦST 0.454 54.6

Lactic Acid neg (3) P = 0.089 12.7

ΦST 0.127 87.3

INA neg (1) NA 10.2

89.8

Grapevine Leaf Pathogenicity neg (7) P = 0.009 15.1

ΦST 0.151 * 84.9

Tobacco Leaf HR neg (4) NA 25.0

75.0

Lemon Pathogenicity neg (7) P = 0.031 11.1

ΦST 0.111 * 88.9

Syringopeptin (B. megaterium) neg (2) NA 50.4

49.6

Syringomycin (G. candidum) pos (16) P = 0.284 1.5

ΦST 0.015 98.5

Syringomycin (S. cerevisiae) pos (14) P = 0.339 0.6

ΦST 0.006 99.4

Chloramphenicol resistance pos (19) P = 0.316 0.9

ΦST 0.009 99.1

Ampicillin resistance pos (6) P = 0.007 20.1

ΦST 0.201 * 78.9

Tetracycline resistance pos (1) NA 0.5

99.5

Presence of syringolin A gene sylC neg (4) NA 13.0

87.0

Presence of syringopeptin gene sypC neg (5) P = 0.578 2.8

ΦST 0.028 97.2

Presence of syringomycin gene syrB neg (7) P = 0.765 3.0

ΦST 0.030 97.0

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3.4 Discussion

This project aimed to undertake a comparative study of the biochemical

characteristics of P. s. syringae from grapevine tissues affected by BIR or

bacterial leaf spot (BLS). Additionally, PCR techniques were employed to make a

phylogenetic analysis and an evolutionary study of the presence of toxin

production genes and BIR symptoms. I used MLST to gain an insight into the

evolutionary history of P. s. syringae causing BIR in Australian vineyards (Hall

et al., 2016). The present study has further characterised these P. s. syringae

isolates using phenotypic data, with the aim of determining associations between

phenotypic data and reduction in crop yield seen in Chapter 2. By mapping

phenotypic traits into MLST phylogeny, the origins of these phenotypes may be

determined.

Numerous Pseudomonas spp. have been previously characterised according to

their phenotypic traits to establish an identification scheme for these plant

pathogens (Lelliott et al., 1966). These tests have been implemented by many

groups as a rapid, easy and convenient form of identification (Abkhoo, 2015;

Ferrante & Scortichini, 2009; Hall et al., 2002; Lelliott & Stead, 1987; Scortichini

et al., 2005; Whitelaw-Weckert et al., 2011). The subsequent development of

more sensitive methods such as gene sequencing and MLST have allowed for the

advancement and improvement of phylogenetic classification in the P. syringae

complex (Clarke et al., 2010; Hwang et al., 2005). The current study aimed to use

a combination of both biochemical and molecular techniques for the identification

and classification of isolates of P. s. syringae from grapevine.

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LOPAT

LOPAT distinguished pathogenic from non-pathogenic isolates in this study.

LOPAT is an established scheme for the identification of plant pathogenic

Pseudomonas spp. (Burkowicz & Rudolph, 1994; Lelliott et al., 1966; Lelliott &

Stead, 1987). In the previous study, most vineyard isolates recovered were

classified as P. syringae based on the LOPAT identification scheme. These

isolates were identified by production of levan type colonies on sucrose agar, and

HR in infiltrated tobacco leaves. Moreover, they were negative for oxidase

production, potato soft rot, and arginine dihydrolase activity. Four of these

P. s. syringae isolates which were collected from frost affected vineyards which

did not show symptoms of BIR or BLS, were unable to cause necrosis in lemon

or detached grape leaves, or produce HR in tobacco leaves, were identified as

P. s. syringae by rpoB sequence typing and MLST (Hall et al., 2016). As the

LOPAT protocol was devised for pathogenic bacteria, the protocol appears to

have successfully differentiated between the pathogenic and non-pathogenic

P. s. syringae isolates in my collection. This is consistent with the findings of

Diallo et al. (2012) who demonstrated that their environmental P. syringae

isolates were unable to cause HR in tobacco.

GATTa

GATTa showed some ability to disseminate between pathovars of P. syringae in

this study. Tests such as GATTa have long been used as reliable markers for

discrimination between pathovars of P. syringae (Gašić et al., 2012; Jones, 1971;

Lelliott et al., 1966). While some variability in GATTa results has previously

been observed with isolates of P. s. morsprunorum (Gilbert et al., 2009), GATTa

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discrimination is generally considered reliable for separation of P. syringae

pathovars (Latorre & Jones, 1979). Pseudomonas syringae pv. syringae has a

GATTa (+ + - -) profile, meaning it is capable of gelatin liquefaction and aesculin

hydrolase activity, but negative for tyrosinase activity and tartaric acid utilisation,

(Barta & Willis, 2005; Gašić et al., 2012; Gilbert et al., 2009; Natalini et al.,

2006; Vicente & Roberts, 2007). Although our results are in agreement with

previous reports regarding gelatin liquefaction and aesculin hydrolase activity

(Gašić et al., 2012), a small proportion of the P. s. syringae grapevine isolates

tested (7.7%) were weakly positive for tartaric acid utilisation. This is in

agreement with Monteil et al. (2014) who demonstrated that P. s. syringae from

rain sources metabolised D-tartrate. Additional tests for sorbitol and lactic acid

utilisation were also carried out, indicating that P. s. syringae can utilise these

sources for energy. The GATTa results for P. s. syringae isolates are in

agreement with other reports (Barta & Willis, 2005; Gašić et al., 2012), indicating

a level of discriminatory power between pathovars of P. syringae using this form

of identification.

Tyrosinase

In the current study 23% of the grapevine P. s. syringae isolates tested were

found to be positive for tyrosinase activity. This contrasts with reports that

P. s. syringae is reported to lack tyrosinase activity (Fahy & Hayward, 1983;

Gašić et al., 2012). The non-discriminative value for this particular GATTa test

has been previously reported (Burkowicz & Rudolph, 1994; Lelliott et al., 1966;

Young & Triggs, 1994). Tyrosinases are ubiquitous copper-containing enzymes

found in fungi, plants, mammals, bacteria and other organisms that are essential

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for the formation of melanin. The production of green pigmentation naturally

produced by Pseudomonas spp. is reported to hinder the visual production of the

reddish hue produced in tyrosinase-casein agar (Latorre & Jones, 1979). In the

current study, no green pigmentation was produced on tyrosinase-casein agar

leaving visual representation unambiguous. Due to the level of variability

between isolates, the next step was to determine whether there were associations

between pathogenicity/grapevine symptom production of BIR and the presence of

tyrosinase activity phenotypes.

Pseudomonas syringae pv. syringae isolates from BIR affected vineyards were

generally negative for tyrosinase activity, and AMOVA demonstrated that the

population of P. s. syringae isolated from BIR affected grapevines differed

genetically from the tyrosinase positive population. In BIR affected grapevine

tissue, P. s. syringae populates the xylem of leaf petioles and rachii (Whitelaw-

Weckert et al., 2011) so the absence of tyrosinase may be caused by a lifestyle

within the plant where there may be little need for protection from UV light and

melanin is not required for survival. Interestingly, the P. s. syringae isolates

collected in this study, that were found to be were positive for tyrosinase activity,

were isolated from vineyards with BLS symptoms only (no loss of

inflorescences), and from non-pathogenic P. s. syringae. These tyrosinase

positive P. s. syringae isolates may have originated from epiphytic populations

where tyrosinase activity and resultant melanin production play a role in UV light

protection and thus increase epiphytic fitness (Claus & Decker, 2006; Rozhavin,

1983).

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Ice nucleation activity

The grapevine P. s. syringae isolates collected in this study were generally INA

positive. This is in agreement with previous reports of P. s. syringae from other

host species (Bultreys & Kaluzna, 2010; Hwang et al., 2005; Natalini et al.,

2006). Positive INA has been reported to facilitate P. syringae to cause ice crystal

formation in plant tissue (Lindow et al., 1982). Extracellular freezing can occur

naturally at temperatures between -3 to -8°C but the presence of suitable ice

nuclei, such as P. s. syringae cells, can trigger nucleation at up to -1.5°C, or more

typically at -2 to -4˚C (Hill et al., 2014; Maki et al., 1974), causing damage to

plant tissue and enhanced pathogenicity (Feil et al., 2005, Hirano & Upper, 2000;

Lindow et al., 1982; Whitesides & Spotts, 1991). In the current investigation,

both non-pathogenic and pathogenic isolates were found to be INA positive, and

one pathogenic isolate (DAR82441) was INA negative, indicating that INA was

not correlated with pathogenicity for these plant derived P. s. syringae isolates. In

contrast, Morris et al. (2010) reported that plant pathogenicity/virulence of

P. syringae isolated from environmental water samples were positively correlated

with ice nucleation activity. These contrasting results may be explained by the

different sources from which the isolates were derived. Strains of P. s. syringae

that were INA negative may not be able to incite frost damage but may co-exist

on plants with other P. syringae strains to assure INA function (Hirano & Upper,

2000; Wilson & Lindow, 1994). Additionally, others have demonstrated that INA

positive P. s. syringae strains from Tumbarumba are able to cause significant

infection under humid conditions that are unaccompanied by freezing (Hall et al.,

2016; Whitelaw-Weckert et al., 2011). The results from the present investigation

indicate that INA activity may be valuable for pathovar identification of

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P. syringae, but appears to have no role in pathogenicity and virulence in

grapevine.

Pseudomonas syringae pv. tabaci and P. s. striafaciens were also INA positive

and P. s. morsprunorum, and P. s. phaseolicola were INA negative, in agreement

with Gross et al. (1984), Hwang et al. (2005) and Lindow et al. (1982).

Pseudomonas syringae pv. mori and P. s. maculicola were INA negative,

although positive INA has been reported by Hwang et al. (2005).

Pectolytic activity

In the present study, all P. s. syringae isolates tested negative for pectolytic

activity by the potato soft rot method, but positive by the Paton’s Pectate Medium

Method. Microbial pectolytic activity is important for tissue maceration and

pathogenicity (Liao et al., 1988; Marín‐Rodríguez et al., 2002). The most

common method for determination of microbial pectolytic activity is the LOPAT

potato soft rot test which involves the degradation of pectic substances in potato

tuber cell walls. Alternatively, the Paton’s Pectate Medium Method involves the

liquefaction of an over-layer consisting of polygalacturonic acid (sodium

polypectate) and EDTA, resulting in cavities within the bacterial colonies. A

possible reason for this disparity might be that the polygalacturonic acid pectic

substances in Paton’s Pectate Medium may be easier to degrade than the complex

mixture, rich in galactan (oligomer of β-1, 4-linked galactosyl residues) in potato

tubers (Sørensen et al., 2000). Alternatively, the Paton’s medium contains EDTA,

a calcium chelator. As calcium plays an important role in cross-linking plant wall

acidic pectin residues, chelation of calcium may aid in the degradation of pectates

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by pectolytic enzymes (Hepler, 2005). Another possible reason for the disparity

might be the different nutrients and pH of the media used in the different tests. It

is known that the many different microbial pectolytic enzymes differ widely in

the conditions under which they are active (Hildebrand, 1971).

Feil et al. (2005) reported that fully sequenced P. s. syringae B728a did contain a

pectate lyase gene (Psyr0852). However, it is not known if this gene is expressed

as no pectolytic testing was undertaken (Feil et al., 2005). In BIR affected tissue,

soft rot is seen in necrotic rachii, resulting in a soft and water-soaked appearance.

As non-pathogenic P. s. syringae were also positive for pectolytic activity in the

current study and AMOVA showed that pectolytic activity was not associated

with BIR. It is possible that co-existence with other pectolytic organisms could

help the invasion of P. s. syringae by causing the initial plant wound (Danhorn &

Fuqua, 2007). The role of pectolytic P. s. syringae in pathogenicity and virulence

in grapevine requires further investigation.

Pathogenicity

Every isolate of P. s. syringae from BIR affected grapevines induced HR-like

spots in leaves of tobacco plants. Although the tobacco leaf test is generally

reported to be a reliable marker for pathogenicity of Pseudomonas spp. it has

been suggested that it is wise to include a number of host pathogenicity tests

when determining the virulence of P. s. syringae isolates (Gašić et al., 2012).

Thus, bacterial isolates in this study were assessed not only on their ability to

cause tobacco HR, but also to cause necrotic lesions in detached grapevine leaves

and lemon fruit.

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Indeed, AMOVA showed that P. s. syringae from BIR affected vineyards were

genetically distinct from grapevine isolates that did not induce tobacco leaf HR.

Potentially pathogenic P. s. syringae isolates recovered from grapevine hosts

were also able to cause necrosis on grapevine leaves, in agreement with previous

studies (Abkhoo, 2015; Whitelaw-Weckert et al., 2011). However, not all isolates

collected from grapevines produced necrotic spots on detached grapevine leaves.

This discrepancy may be caused by an atypical host-pathogen interaction in

detached leaves as compared with leaves attached to living plants. In the absence

of the living plant, bacterial inoculation of detached leaves may need to be

performed by infiltration rather by surface application (Randhawa & Civerlo,

1985).

Most potentially pathogenic P. s. syringae BIR isolates were also found to cause

necrosis on mature lemon fruit. Although P. s. syringae is a known pathogen of

lemon (Little et al., 1998; Scortichini et al., 2003), the present study found that

development of necrotic lesions on lemon could not predict pathogenicity of

P. s. syringae on tobacco. This is in contrast to the findings of Scortichini et al.

(2003) who demonstrated that lemon fruit inoculation is an effective means for

P. s. syringae pathogenicity assessment. The finding that most P. s. syringae from

BIR affected grapevine lacked strict host specificity is in agreement with the

results seen in Chapter 2. This is not surprising as P. s. syringae has the

propensity to have a wide host range and cause disease on numerous host plant

species (Najafi & Taghavi, 2014). In future directions of this project, we would

like to repeat these tests on an increased number of hosts and enumerate the

population sizes of the bacteria over time

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Production of toxins by grapevine P. s. syringae isolates

Two classes of lipodepsipeptides are produced by P. s. syringae:

lipodesinonapeptides such as syringomycin, syringostatin, or pseudomycin, and

syringopeptins (Bender et al., 1999; Bultreys et al., 1999). Features of toxin

production can be associated with the identification of P. syringae pathovars

either by gene detection or direct detection of these secondary metabolites.

Syringomycin

Analysis by AMOVA showed that neither the syringomycin phenotypes, as

assayed by the S. cerevisiae or G. candidum plate test methods, nor the presence

of syringomycin gene (syrB) were associated with BIR. Syringomycin production

in this study was assessed through the detection of the syrB gene by PCR and by

inhibition of S. cerevisiae and/or G. candidum. Syringomycins are a class of

cyclic lipodepsipeptide phytotoxins capable of inducing necrosis. Symptoms

produced by syringomycin are caused by the formation of pores in the plasma

membrane leading to an influx of H+ and Ca

2+ ions and cytoplasm acidification,

resulting in cellular death. Sacchromyces cerevisiae and G. candidum are

inhibited by syringomycin producing isolates of P. syringae (Bultreys &

Gheysen, 1999; Gašić et al., 2012; Quigley, 1994; Wang et al., 2006).

Seventy-five percent of the vineyard isolates were positive for syrB. This is in

agreement with the report that most isolates of P. s. syringae share the common

characteristic of having a syrB gene (Scortichini et al., 2003). Analysis by

AMOVA also demonstrated that there was no association between the phenotypic

plate tests for syringomycin and the presence of the syringomycin gene syrB. The

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negative results of the plate tests may be a consequence of a lack of certain plant

metabolites in the media that are required for syringomycin production. It has

been shown that P. s. syringae may sense specific plant metabolites, such as

arbutin and/or D-fructose, to modulate syringomycin production (Mo & Gross,

1991; Quigley, 1994). Whether the media used should contain these metabolites

was not assessed and needs further investigation. The current study indicates that

syringomycin production determination as tested by S. cerevisiae and

G. candidum inhibition should be approached with caution.

Syringolin

Currently there is no biochemical or microbiological assay available for the

detection of syringolin. Therefore PCR detection of the syringolin gene, sylC, was

employed. This gene has been shown to be involved in the biosynthesis of

syringolin A by catalysing ureido bond formation (Imker et al., 2009).

Syringolins act on the plant proteasome (Misas-Villamil et al., 2013). In the

current study, AMOVA demonstrated that the population of P. s. syringae

without syringolin A gene sylC was genetically distinct from P. s. syringae

isolated from BIR affected grapevines. Syringolin A has been reported to greatly

reduce plant abscisic acid (ABA)-induced stomatal closure (Schellenberg et al.,

2010). This is consistent with the description of grapevine pathogenic

P. s. syringae producing stomata with a star-like locked-open appearance

(Whitelaw-Weckert et al., 2011). Others have demonstrated that syringolin A

facilitated the colonisation of tissue by increasing mobility and suppression of

phytohormone signalling in adjacent tissues (Misas-Villamil et al., 2013). The

current investigation is the first to report the link of the presence of sylC in

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P. s. syringae from grapevine BIR symptoms. This indicates that syringolin A

may be an important virulence factor for colonisation and suppression of host

defences in the grapevine host.

Syringopeptin

Analysis by AMOVA showed that isolates positive for syringopeptin production

by plate test were strongly associated with BIR. Syringopeptin is composed of a

peptide chain of 22 amino acids attached to a 3-hydroxyl fatty acid tail (Ballio et

al., 1995). This fatty acid tail allows for insertion of the toxin into the plant

plasma membrane, forming transmembrane pores and leading to ion leakage

(Hutchison & Gross, 1997). Syringopeptin production was assessed both through

the detection of the sypC gene by PCR and inhibition of B. megaterium by plate

test. Indicator bacterium B. megaterium is highly sensitive to syringopeptin

(Bensaci et al., 2011; Grgurina et al., 1996). This is in agreement with the results

of Lavermicocca et al. (1997) who found syringopeptin production a common

trait of P. s. syringae. However, there was negligible association between BIR

and the presence of syringopeptin gene, sypC. Moreover, there was little

association between results of the syringopeptin plate test and presence of sypC,

indicating that the plate test and the presence of the sypC gene were not

equivalent. As different P. s. syringae strains produce different syringopeptins

with activity against B. megaterium (Grgurina et al., 2002; Scholz-Schroeder et

al., 2003), the possibility that a different primer pair may be needed for PCR of

grapevine sypC requires further investigation.

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Previous studies have shown that mutant sypA P. s. syringae isolates exhibit

attenuated virulence in Prunus avium (wild cherry). It has also been demonstrated

that larger pores are produced by syringopeptin than syringomycin (Bensaci et al.,

2011), highlighting the importance of syringopeptin production in plant-pathogen

interactions (Scholz-Schroeder et al., 2001). This is the first report to demonstrate

syringopeptin production by P. s. syringae in grapevine isolates from grapevine.

Resistance to antibiotics

Resistance to either streptomycin or tetracycline (with one exception) was not

detected in the isolates investigated in this study. Resistance to four antibiotics

were investigated in this study: tetracycline, a broad-spectrum antibiotic that

inhibits protein synthesis via blocking aminoacyl tRNA; streptomycin, which

inactivates the 30S ribosome involved in protein synthesis; chloramphenicol,

which inhibits peptidyl transferases during translation by binding to the 70S

ribosome; and ampicillin which inhibits cell wall biosynthesis. Analysis by

AMOVA demonstrated that chloramphenicol resistance was not related with

P. s. syringae populations from BIR affected grapevines. Antibiotic resistance for

these compounds may not be expressed by core genome phylogeny but may be

transferred horizontally (Hwang et al., 2005). Interestingly, ampicillin sensitivity

was observed in P. s. syringae originating from BIR affected vineyards, whereas

resistance was observed in the non-BIR P. s. syringae isolates. Analysis by

AMOVA found that the population of P. s. syringae with ampicillin resistance

was genetically distinct from P. s. syringae isolated from grapevines affected by

BIR. These results are in accord with those of Bartoli et al. (2015) who found that

antibiotic resistance was inversely correlated with pathogenicity in

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Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae

complex. Since many P. syringae strains are typically resistant to ampicillin, this

finding suggests that strains of P. s. syringae capable of infecting grape are

distinct from those having a more environmental reservoir where they would

interact with other bacteria capable of producing ampicillin, necessitating them to

be resistant to this antibiotic (S. Lindow, personal communication).

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3.5 Conclusions

The results from this investigation provide the foundations for an improved

understanding of the biochemical characteristics of P. s. syringae from grapevine

affected by BIR. This study has also shown that identification schemes, indicator

strains and pathogenicity tests may need to be carefully considered and

interpreted during the identification process to ensure proper classification.

Unfortunately, a clear connection between most phenotypes and virulence could

not be observed. A clearer picture may be observed with an increase of isolates

and to extend the survey of grapevines to a greater geographical region, and are

being considered for any future research. The results of this investigation

demonstrate that a positive tobacco HR may aid in predicting the ability of

P. s. syringae grapevine isolates to cause BIR (or BLS). In addition, lack of

tyrosinase activity appears to be associated with P. s. syringae population

pathogenic to grapevine, indicating that tyrosinase activity may not be needed for

a pathogenic lifestyle within the plant. Sensitivity to ampicillin was also

associated with pathogenicity, in line with a possible programmed balance

between antibiotic resistance and pathogenicity in some bacterial plant pathogens.

Syringopeptin production and the presence of the gene for syringolin A (sylC)

also appear to be associated with BIR in grapevine P. s. syringae. As these two

toxins are known to be major virulence determinants in P. s. syringae virulence,

they may have a future role as indicators of pathogenicity in viticulture and

should be considered as virulence determinants to grape and other plants. Finally,

this study indicates that to understand the evolution of P. s. syringae in plant

hosts, future approaches should include genetic based analyses.

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3.6 Acknowledgments

Dr. Thomas Hill, Colorado State University, USA; Dr Roger Shivas and Miss Yu

Pei Tan, Department of Agriculture, Fisheries and Forestry (DAFF) in

Queensland are thanked for their generous gifts of P. syringae isolates. I am

indebted to Mr. Nathan Scarlett (dec’d) who collected the Coonawarra vineyard

necrotic rachis samples. Dr. David Gopurenko (NSW Department of Primary

Industries) is thanked for his valuable advice regarding AMOVA. Professor

Steven Lindow (Berkeley, University of California, USA) for his review of the

manuscript and further interpretation.

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Chapter 4 Vitis vinifera Defence Responses to

Pseudomonas syringae pv. syringae

4.1 Introduction

The pathogen-induced modulation of plant defence responses has been reported

to contribute to virulence. These induced hormonal changes have outcomes that

are dependent on pathogen lifestyle and host and can indicate the pathogenic

lifestyles of organisms. It is generally considered that the salicylic acid (SA)

pathway is induced by pathogens with a biotrophic lifestyle, whereas the

jasmonic acid/ethylene (JA/ET) pathways are induced by necrotrophic pathogens.

To date there have been no studies on the plant defence gene expression in

grapevine in response to Pseudomonas syringae pv. syringae (P. s. syringae).

Plant defence responses to pathogenic and non-pathogenic P. s. syringae were

performed on potted Chardonnay grapevines. Callose deposition was observed by

aniline blue staining under epifluorescence and defence gene expression for SA,

JA, ET, and stilbene synthase (STS) were monitored by qPCR.

Pathogenic P. s. syringae caused increases in the activity of STS and the SA and

JA/ET mediated pathways in potted Chardonnay. The failure to significantly

increase any of the plant defence gene targets at 24 hpi may suggest the transient

breakdown of plant defences that accompany the onset of disease and successful

colonisation of host plants. Concomitant expression of both pathways was

observed in later stages (96 and 120 hpi). No significant increase in any defence

gene targets was observed in plants inoculated with non-pathogenic

P. s. syringae. The results of the current study provide insight into the grapevine

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defence responses to pathogenic P. s. syringae. This may open up knowledge for

effective targeted treatment and effective disease management in affected regions.

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4.2 Materials and methods

Plant Material. Lignified cuttings of V. vinifera cv. Chardonnay were hot water

treated for 30 mins at 50°C in potable water, allowing 1L/cutting, and then

maintained at room temperature in potable water for another 30 mins (Waite et

al., 2014). Cuttings were then treated with 3g/L indole-3-butyric acid (hormone

rooting gel; Yates, Australia), planted in fine vermiculite and watered twice daily.

Upon root formation, cuttings were placed in individual pots containing double

autoclaved premium potting mix (Hortico, Australia) and grown in a glasshouse

maintained at ~15–25˚C. Plants were supplemented with 25 g trace element

granules per plant once (Osmocote, Australia), and watered daily.

Pseudomonas syringae pv. syringae inoculation. One pathogenic and one non-

pathogenic P. s. syringae isolate (DAR82161 and DAR82450 respectively) was

used in this experiment. DAR82161 was selected based on its isolation source

(necrotic rachis of BIR infected vineyard), hypersensitivity reaction (HR) in

tobacco, necrosis on lemon fruit and detached V. vinifera grapevine leaf (see

Chapter 2) and ability to produce toxins unique to P. s. syringae (determined by

biochemical and molecular analysis outlined in Chapter 3). Similarly, DAR82450

was determined as non-pathogenic by its inability to produce HR-like symptoms

in tobacco leaf, inability to produce necrosis on lemon fruit and on V. vinifera leaf

(Chapter 3).

Bacterial isolates were grown on King’s B agar at 25°C for 2 days and then

suspended in sterile deionised water (SDW). The bacterial suspension was

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adjusted to an optical density of 0.6 at 600nm (~1x108 CFU/mL). For grapevine

inoculation, individual V. vinifera cv. Chardonnay plants (3 plants per treatment)

were sprayed with a fine mist of bacterial suspension (sterile distilled water for

control plants), bagged to promote high humidity (~100% RH) and placed in

growth chambers (Thermoline Scientific, Australia) with a 16/8 hour light/dark

photoperiod on a 28/18°C day/night cycle. Leaves were collected immediately

after inoculation (time zero), 12 h post inoculation (hpi), then every 24 h up to

120 hpi and snap frozen in liquid nitrogen.

Quantification of disease progression. Photos of leaves were taken at collection

and modified using Adobe Photoshop CS4 (version 11.0.2) to remove

background features and replaced with a solid colour. The extent of lesion

development was calculated using Assess 2.0 Image Analysis Software for Plant

Disease Quantification (Lamari, 2008) on modified images.

Callose Deposition. Estimation of callose deposition was carried out on leaf discs

excised from spray-inoculated leaves on live potted V. vinifera cv. Chardonnay

grapevines, as previously described (Adam & Somerville, 1996; Misas-Villamil

et al., 2013). Briefly, leaf discs were cleared in 95% (v/v) ethanol at 37°C in

darkness. Discs were rinsed in 50% (v/v) ethanol, then in SDW for 30 mins each

before staining in 0.01% (w/v) aniline blue dissolved in 150 mM K2HPO4 (pH

9.3) for 30 mins. Samples were mounted on glass slides in 50% (v/v) glycerol and

callose deposition was viewed under epifluorescence using Olympus Provis

AX70 light microscope with a 365nm excitation filter. Callose deposits were

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quantified using ImageJ software v1.48 (Abràmoff et al., 2004) from digital

photographs as described by Luna et al., (2011). Briefly, pixels of high intensity

(callose deposits) were quantified relative to the total number of pixels covering

the plant material.

Primer Design. Primers for actin, pathogenesis-related 1 (PR1) and stilbene

synthase (STS) qPCR were derived from GenBank accessions of previously

reported gene sequences from the V. vinifera genome. Primer design was

achieved using the online tool OligoPerfect™ Designer (Life Technologies) with

the limitations of 50-60% GC content, primer size between 20-22 nucleotides,

and a melting temperature (Tm) of 60°C using the Tm°C = 4(G+C) + 2(A+T).

Primers sequences were checked for homology against the V. vinifera genome by

nucleotide alignment

(EMBL:www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html) (Table 4.1).

Other primer pairs used in this study were previously published: VvTL1 (I. Dry,

personal communication), PR10 (Kortekamp et al., 2006), VvJAZ5 (Zhang et al.,

2012) and ETR2 (Böttcher et al., 2013)(Table 4.1).

132

Ta

ble

4.1

. S

equ

ence

of

V. vi

nif

era

pri

mer

s u

sed

for

qP

CR

Pri

mer

Au

tho

r

Th

is s

tud

y

Th

is s

tud

y

I D

ry,

per

son

al

com

mu

nic

atio

n

Ko

rtek

amp

et

al. (2

00

6)

Zh

ang

et

al. (2

01

2)

Bö

ttch

er e

t al

. (2

01

3)

Th

is s

tud

y

Eff

icie

ncy

(%)

97

98

99

10

7

10

0

93

95

Ta

rget

Len

gth

(bp

)

26

3

11

4

18

8

74

4

22

0

18

0

11

3

Rev

erse

Pri

mer

(5

' →

3')

AC

GG

AA

TC

TC

TC

AG

CT

CC

AA

AC

CA

TA

AG

GC

CC

AC

CT

GA

GT

CA

TT

GA

TG

TC

AA

CG

GA

GC

AC

GC

AA

TA

GA

AC

AT

CA

CA

AA

TA

CT

CC

GG

AG

GT

TG

CT

TC

CA

TC

CT

AT

T

CT

AT

GC

CG

CA

AG

CT

GG

AT

GT

TG

CT

GC

TT

CC

TT

AC

CG

AG

TT

Fo

rwa

rd P

rim

er (

5'

→ 3

')

AT

GC

AC

TT

CC

CC

AT

GC

TA

TC

CA

AG

TT

GG

CG

TT

GG

GT

CT

AT

CG

AA

AC

TG

GT

GA

CT

GC

AA

TG

CT

TA

CG

AG

AG

TG

AG

GT

CA

CT

TC

TA

AC

GG

AA

GG

AT

CT

GC

GT

TT

TC

TG

CT

GG

AT

GG

AA

TT

GC

TG

AG

CC

GA

AG

AA

AT

GC

TT

GA

GG

AG

Acc

essi

on

Nu

mb

er

AF

36

95

24

AJ5

36

32

6

AF

00

30

07

AJ2

91

70

5

XM

_0

02

27

776

9

CB

97

57

99

JN8

58

96

4

Gen

e

Ta

rget

AC

TIN

-8

PR

1-8

VvT

L1

PR

10

VvJ

AZ

5

ET

R2

ST

S

133

RNA Extraction & cDNA synthesis. Whole frozen leaves were ground to a fine

powder using a mortar and pestle in the presence of liquid nitrogen. Total RNA

was extracted from 50 mg ground leaf tissue using the Qiagen RNeasy Plant Mini

Kit (Qiagen, Australia) with some modifications (MacKenzie et al., 1997). RNA

purity and quantity was determined using a Nanodrop 2000 (Thermo Scientific,

Australia). Equal amounts of total RNA from individual plant samples were

reverse transcribed to cDNA (between 0.2µg - 0.5µg) with a High Capacity

cDNA Reverse Transcription Kit (Applied Biosystems, Australia) according to

manufacturer’s instructions using a C1000 ThermalCycler (BioRad, Australia).

Expression of Defence related genes. Relative gene expression was analysed

using a Rotor-Gene 6000 (Corbett Research) running Rotor-Gene 6000 v1.7

software. Reactions (25 μL) were carried out with a QuantiTect SYBR Green

PCR Kit (Qiagen) using 2 µL target cDNA and a final primer concentration of

0.3µM. Cycle conditions were 94°C for 15 s, 60°C for 30 s and 72°C for 45 s.

Relative expression of each target was carried out in biological and technical

triplicates.

The amplification efficiency of each primer set was calculated using the formula:

E = 10(-1/slope)

-1.

This was to ensure that amplification efficiencies for primer sets were within 10%

of each other for comparative 2-ΔΔCT

analysis (Livak & Schmittgen, 2001;

Schmittgen & Livak, 2008). Melt curve analysis was also carried out to ensure

target specificity and that no artefacts, primer dimerisation, or non-specific

amplification occurred. These analyses were carried out using Rotor-Gene 6000

134

software v1.7. PCR products were also analysed on a 1.7% agarose gel stained

with ethidium bromide and viewed using a BioRad GelDock system.

Before determining relative gene expression of defence targets, all cDNA

samples were tested for consistency using V. vinifera actin housekeeping gene

(AF369524). Only cDNA samples that produced CT values with less than a 6-fold

difference in actin expression against time zero treatments were used for relative

gene expression analysis. All gene of interest targets were normalised against the

expression of actin with relative expression conveyed in arbitrary units using time

zero as equivalent to one.

The expression of selected defence-related genes was compared between

pathogenic and non-pathogenic P. s. syringae and was normalised to control

group (Schmittgen & Livak, 2008).

Fold change due to treatment = 2-ΔΔCT

ΔΔCT = [(CT, target – CT, actin)treatment] – [(CT, target – CT, actin)control]

Statistics. All data values are expressed as the mean ± standard error of the mean

(SEM). Direct comparisons of control versus inoculated plants were carried out

using single factor ANOVA in GraphPad Prism v5 using the natural log.

ANOVA was also performed on callose deposition with significance set at 0.05.

135

4.3 Results

Lesion development. Chardonnay leaves inoculated with the pathogenic

P. s. syringae isolate DAR82161 developed lesions within 24 hpi and the size of

lesions increased over the course of the experiment (Fig. 4.1). Lesion

development (Fig. 4.2) continued until whole leaf senescence. Control plants,

treated with SDW, and plants treated with non-pathogenic P. s. syringae failed to

produce necrotic lesions. Plants treated with non-pathogenic P. s. syringae

showed no signs of HR over the 120 h period.

Fig. 4.1. Lesion development in V. vinifera cv. Chardonnay leaves treated with water (control),

non-pathogenic and pathogenic isolates of P. s. syringae. Healthy Chardonnay plants were spray-

inoculated and lesion development was observed on attached leaves over 120 h

136

0 24 48 72 96 120

0

20

40

60Control

P. s. syringae

Hours Post Inoculation

Pe

rce

nt

(%)

Le

af

Are

a

Co

nta

inin

g L

esi

on

s

Fig. 4.2. Lesion development in grapevine V. vinifera cv. Chardonnay leaves infected with

pathogenic P. s. syringae. Healthy Chardonnay plants were sprayed with 108 cfu/ml P. s. syringae

(n=3) and lesion development on attached leaves was assessed over time using Assess 2.0

software. Data is presented as mean ± SEM.

137

Callose deposition. Callose deposition was observed using aniline blue staining

with epifluorescence microscopy, and quantified by signal intensity from digital

photographs (Fig. 4.3). Callose deposition appeared to increase in V. vinifera cv.

Chardonnay leaves treated with the non-pathogenic P. s. syringae isolate

(DAR82450) within 12 hpi and was significantly increased over callose levels in

control leaves by 24 hpi. This increase in callose deposition continued until the

end of the experiment at 120 hpi (Fig. 4.4). Control leaves did not show any

increase in callose deposition over the course of the experiment. Inoculation of

the grapevine leaves with a pathogenic P. s. syringae isolate also led to callose

deposition but at lower levels than observed in leaves inoculated with the non-

pathogenic P. s. syringae isolate (Fig. 4.4). No bright callose deposits were found

to be associated with stomata guard cells. This suggests that the pathogenic

P. s. syringae isolate was able to suppress PTI plant defence responses more

effectively than the non-pathogenic isolate.

138

Fig. 4.3. Histochemical analysis of Chardonnay leaves infected with P. s. syringae. Callose

deposition was induced in grapevine leaves infected with pathogenic and non-pathogenic

P. s. syringae. Leaves were stained with aniline blue and examined by fluorescent microscopy.

Callose spots were quantified by pixel intensity using ImageJ software and measured with SEM

(below image). No callose deposits were associated with stomata. Bar = 200µm

139

0 12 24 48 72 96 120

0

10

20

30

40

50

Control

Pathogenic P. s. syringae

Non-pathogenic P. s. syringae

** * *

**

*

**

*

**

Hours Post Inoculation

Pe

rce

nt

(%)

Ca

llo

se D

ep

osi

ts

Fig. 4.4. Effect of inoculation with pathogenic and non-pathogenic P. s. syringae isolates on

cellular defence responses in grapevine leaves. V. vinifera cv. Chardonnay plants were spray

inoculated with either pathogenic or non-pathogenic P. s. syringae (108 cfu/ml) or water (control).

Callose deposition was observed in cleared leaves stained with 0.01% (w/v) aniline blue and

quantified by pixel intensity of digital photographs. * indicates significant difference in callose

deposition relative to control leaves (P <0.05). ** indicates significant differences in callose

deposition relative to control and pathogenic P. s. syringae treated leaves (P <0.05). Data are

presented as mean ± SEM.

140

Amplification Efficiencies. Vitis vinifera cv. Chardonnay cDNA was obtained

from grapevine leaves by reverse transcription of total RNA. Amplification

efficiencies of all primer sets listed in Table 4.1 was achieved using a dilution

series of cDNA to produce five data points. Standard curves were generated using

the Rotor-Gene 6000 software v1.7 under the conditions described above, and by

plotting the log of cDNA against CT values. For application of the comparative

2-ΔΔCT

method amplification efficiencies were determined as previously described

(Livak & Schmittgen, 2001; Schmittgen & Livak, 2008). For this study the

housekeeping gene (actin) was determined to have an E value of 0.97 (97%

efficiency). Defence gene targets Pathogenesis-related 1 (PR1), V. vinifera

thaumatin-like 1 (VvTL1), Pathogenesis-related 10 (PR10), V. vinifera

jasmonate-ZIM-domain 5 (VvJAZ5), ethylene receptor 2 (ETR2) and stilbene

synthase (STS) produced amplification efficiencies of 98, 99, 107, 100, 93, and

95%, respectively.

Melt curve analysis was also carried out to determine primer specificity. All

primer pairs resulted in one single peak in the melt curve, indicating high

specificity for their targets to produce precise amplification. Additionally, these

PCR products were also visualised on an agarose gel stained with ethidium

bromide. In all cases, a single product corresponding to each products target

length was produced (data not shown).

Expression analysis. The quality of the cDNA was assessed by analysing the

expression of the actin housekeeping gene. Diluted (1:10) samples of cDNA were

prepared for real time (quantitative) polymerase chain reaction (qPCR) using the

141

sequence specific primers listed in Table 4.1. No significant fold changes in actin

expression were observed in response to the treatments applied over the time

course of the experiment (Fig. 4.5), indicating that actin is suitable housekeeping

gene to use as an internal control for normalisation of defence gene transcription.

Expression of V. vinifera defence-related genes was analysed in the grapevine

leaves at 0, 12, 24, 48, 72, 96, and 120 hpi after inoculation with either

pathogenic P. s. syringae (DAR82161), or non-pathogenic P. s. syringae

(DAR82450). Inoculations were carried out with 48 hpi cultures grown on KB

agar at concentrations of ~1x108

CFU/ml. This allowed for the analysis of gene

expression to be compared between pathogenic and non-pathogenic bacterial

pathogens to V. vinifera grapevine.

142

0 12 24 48 72 96 120

0

2

4

6

8

10Control

Pathogenic P. s. syringae

Non-pathogenic P. s. syringae

LSD

Hours Post Inoculation

Fo

ld c

ha

ng

es

in a

ctin

ge

ne

ex

pre

ssio

n

Fig. 4.5. Housekeeping gene expression. Transcript accumulation of internal control, actin, gene

in untreated (control) and pathogenic and non-pathogenic P. s. syringae treated. Analysis was

performed by qPCR. Relative transcript levels were calculated using the 2-ΔΔCT

(Livak &

Schmittengen, 2001). Results represent the mean fold increase of cDNA levels plotted against 1x

expression (0 h). Results are the mean ± SEM of three experiments.

143

Expression of defence-related Vitis vinifera grapevine genes in response to

P. s. syringae infection. Chardonnay leaves were challenged with either

pathogenic or non-pathogenic P. s. syringae at a concentration of ~1x108

CFU/ml, and the expression of defence-related gene markers was quantified using

real time qPCR. Figures 4.6 to 4.11 show the relative accumulation of transcripts

corresponding to genes encoding PR-proteins and other defence-related genes in

the grapevine leaves treated with pathogenic and non-pathogenic P. s. syringae

against uninoculated control plants, calculated using the comparative CT method

(Schmittgen & Livak, 2008). The defence-related genes chosen for analysis are

representative of SA- (PR1 and PR10, Fig. 4.6 and Fig. 4.7, respectively), JA-

(VvTL1 and VvJAZ5, Fig 4.8 and Fig. 4.9, respectively), and ET-mediated (ETR2,

Fig. 4.10) defence pathways in V. vinifera. Stilbene synthase (STS, Fig. 4.11) was

also analysed because it has been shown previously to be induced by in response

to a number of other grapevine pathogens including Erysiphe necator (powdery

mildew), P. viticola (downy mildew) and Botrytis cinerea (Chong et al., 2008; Le

Henanff et al., 2009).

Treatment of V. vinifera plants with non-pathogenic P. s. syringae caused no

significant fold changes in leaf defence gene transcript, relative to control leaves,

during the 120 h incubation period. In contrast, treatment of V. vinifera plants

with pathogenic P. s. syringae caused rapid and transient up-regulation at 12 hpi

in some defence genes. Salicylic acid-mediated PR1 was increased 6.4-fold (Fig.

4.6); SA-mediated PR10 increased 996.4-fold (Fig. 4.7); JA-mediated VvTL1

increased 6.9-fold (Fig. 4.8); ET-mediated ETR2 increased 7.3-fold (Fig. 4.10);

and STS increased 30.4-fold (Fig. 4.11). Surprisingly, at 24 hpi there was no

144

significant defence-related gene expression in response to pathogenic

P. s. syringae. Later, however, pathogenic P. s. syringae caused elevated

expression of PR10, VvTL1, VvJAZ5 and STS from 48hpi (Figs. 4.7, 4.8, 4.9 and

4.11 respectively) and PR1 and ETR2 from 96hpi (Figs. 4.6 and 4.10

respectively).

145

0 12 24 48 72 96 1200

5

10

15

20

* *

*

Non-pathogenic P. s. syringae

Pathogenic P. s. syringae

Hours Post Inoculation

Re

lati

ve

PR

1G

en

e E

xp

ress

ion

Fig. 4.6. Transcript accumulation of PR1 (indicator of SA-mediated defence), in pathogenic

P. s. syringae and non-pathogenic P. s. syringae treated V. vinifera cv. Chardonnay grapevine

leaves. Analysis was performed by qPCR. Transcript accumulation was calculated by the 2-ΔΔCT

method from triplicate data with grapevine actin gene as the internal control and non-treated

leaves (control) as the reference sample. Results represent the average fold changes in transcript

level relative to control leaves with error bars for the SEM. * indicates significant difference fold

change of transcript accumulation between pathogenic and non-pathogenic P. s. syringae isolates,

P <0.05.

146

0 12 24 48 72 96 1200

500

1000

1500

2000

*

** * *

Non-pathogenic P. s. syringae

Pathogenic P. s. syringae

Hours Post Inoculation

Re

lati

ve

PR

10

Gen

e E

xp

ress

ion

Fig. 4.7. Transcript accumulation of PR10 (indicator of SA-mediated defence) in pathogenic

P. s. syringae and non-pathogenic P. s. syringae treated V. vinifera cv. Chardonnay grapevine

leaves. Analysis was performed by qPCR. Transcript accumulation was calculated by the 2-ΔΔCT

method from triplicate data with grapevine actin gene as the internal control and non-treated

leaves (control) as the reference sample. Results represent the average fold changes in transcript

level relative to control leaves with error bars for the SEM. * indicates significant difference fold

change of transcript accumulation between pathogenic and non-pathogenic P. s. syringae isolates,

P <0.05.

147

0 12 24 48 72 96 1200

20

40

60

80

*

*

*

*

*

Pathogenic P. s. syringae

Non-pathogenic P. s. syringae

Hours Post Inoculation

Re

lati

ve

VvT

L1

Gen

e E

xp

ress

ion

Fig. 4.8. Transcript accumulation of VvTL1 (indicator of JA-mediated defence), in pathogenic

P. s. syringae and non-pathogenic P. s. syringae treated V. vinifera cv. Chardonnay grapevine

leaves. Analysis was performed by qPCR. Transcript accumulation was calculated by the 2-ΔΔCT

method from triplicate data with grapevine actin gene as the internal control and non-treated

leaves (control) as the reference sample. Results represent the average fold changes in transcript

level relative to control leaves with error bars for the SEM. * indicates significant difference fold

change of transcript accumulation between pathogenic and non-pathogenic P. s. syringae isolates,

P <0.05.

148

0 12 24 48 72 96 1200

10

20

30

40

50

*

*

*

*Non-pathogenic P. s. syringae

Pathogenic P. s. syringae

Hours Post Inoculation

Re

lati

ve

VvJ

AZ

5 G

en

e E

xp

ress

ion

Fig. 4.9. Transcript accumulation of VvJAZ5 (indicator of JA-mediated defence, in pathogenic

P. s. syringae and non-pathogenic P. s. syringae treated V. vinifera cv. Chardonnay grapevine

leaves. Analysis was performed by qPCR. Transcript accumulation was calculated by the 2-ΔΔCT

method from triplicate data with grapevine actin gene as the internal control and non-treated

leaves (control) as the reference sample. Results represent the average fold changes in transcript

level relative to control leaves with error bars for the SEM. * indicates significant difference fold

change of transcript accumulation between pathogenic and non-pathogenic P. s. syringae isolates,

P <0.05.

149

0 12 24 48 72 96 1200

2

4

6

8

10

*

*

Non-pathogenic P. s. syringae

Pathogenic P. s. syringae

Hours Post Inoculation

Re

lati

ve

ET

R2

Gen

e E

xp

res

sio

n

Fig. 4.10. Transcript accumulation of ETR2 (indicator of ET-mediated defence), in pathogenic

P. s. syringae and non-pathogenic P. s. syringae treated V. vinifera cv. Chardonnay grapevine

leaves. Analysis was performed by qPCR. Transcript accumulation was calculated by the 2-ΔΔCT

method from triplicate data with grapevine actin gene as the internal control and non-treated

leaves (control) as the reference sample. Results represent the average fold changes in transcript

level relative to control leaves with error bars for the SEM. * indicates significant difference fold

change of transcript accumulation between pathogenic and non-pathogenic P. s. syringae isolates,

P <0.05.

150

0 12 24 48 72 96 1200

20

40

60

*

*

*

* *

Non-pathogenic P. s. syringae

Pathogenic P. s. syringae

Hours Post Inoculation

Re

lati

ve

ST

SG

en

e E

xp

ress

ion

Fig. 4.11. Transcript accumulation of STS (Stilbene Synthase, indicator of pathogenesis) in

pathogenic P. s. syringae and non-pathogenic P. s. syringae treated V. vinifera cv. Chardonnay

grapevine leaves. Analysis was performed by qPCR. Transcript accumulation was calculated by

the 2-ΔΔCT

method from triplicate data with grapevine actin gene as the internal control and non-

treated leaves (control) as the reference sample. Results represent the average fold changes in

transcript level relative to control leaves with error bars for the SEM. * indicates significant

difference fold change of transcript accumulation between pathogenic and non-pathogenic

P. s. syringae isolates, P <0.05.

151

4.4 Discussion

As a hemibiotrophic species, P. syringae exhibits two distinct life cycle phases:

an early biotrophic stage and a late necrotrophic stage (Alfano and Collmer,

1996; Xin and He, 2013). Although the role of P. s. syringae in plant-pathogen

interactions has been widely researched (Brooks et al., 2005; Engl et al., 2014;

Mammarella et al., 2014; Melotto et al., 2006; Misas-Villamil et al., 2013) little is

known regarding its role in grapevine-pathogen interactions. As P. s. syringae can

cause extensive V. vinifera grapevine crop losses (Abkhoo, 2015; Hall et al.,

2016; Whitelaw-Weckert et al., 2011), it is important to understand the infection

mechanisms for this pathogen. Moreover, it is important to understand the

involvement of the different grapevine defence pathways in the response to

P. s. syringae infection. In this experiment we studied the effect of P. s. syringae

infection on the V. vinifera defence system using selected markers for

phytohormone-mediated defences.

Callose deposition

Callose deposition, recognised as one of the first plant defences during PTI, forms

a physical barrier to prevent and hinder pathogen invasion (Voigt, 2014). In the

current investigation there was a strong, early (24 hpi) leaf callose deposition in

leaf cells of Chardonnay plants inoculated with non-pathogenic P. s. syringae.

This finding is in accordance with previous reports that callose deposition became

visible in V. vinifera leaves as early as 7-24 hpi following P. viticola infection

(Gindro et al., 2003; Hamiduzzaman et al., 2005).

152

The current study showed that inoculation of V. vinifera leaves with a pathogenic

P. s. syringae isolate also led to callose deposition but at lower levels than

observed in leaves inoculated with the non-pathogenic P. s. syringae isolate.

Similarly, studies with Arabidopsis also demonstrated greater callose deposits

following inoculation with avirulent than virulent bacteria (DebRoy et al., 2004;

Hauck et al., 2003), suggesting basal defence suppression by virulent bacteria

(Nomura et al., 2005). This suggests that the pathogenic P. s. syringae isolate

was able to suppress the callose plant defence response more effectively than the

non-pathogenic isolate.

Salicylic acid-mediated defence gene expression

In the current study, both pathogenic and non-pathogenic strains of P. s. syringae

were applied to grapevine leaves and the level of defence-related gene expression,

measured relative to actin, was assessed against non-treated control plants. Little

or no increase in gene expression was observed when non-pathogenic

P. s. syringae was applied to Chardonnay plants, and there were no signs of

disease or HR. Pathogenic P. s. syringae, however, resulted in disease and

increased expression of all genes tested, indicating compatible interactions

between grapevine and pathogenic P. s. syringae.

Expression of PR1 (Pathogenesis-related 1) was used as an indicator for SA-

mediated defence responses. Although PR1 transcription has been reported to be

a general response to unspecific stimuli (Wielgoss & Kortekamp, 2006), this

study showed that a pathogenic isolate of P. s. syringae caused a moderate

increase in PR1 transcription, whereas a non-pathogenic isolate did not (Fig. 4.6).

153

There was a transient up-regulation of PR1 in response to the pathogenic

P. s. syringae isolate DAR82161 within 12 hpi (< 10-fold), returning to basal

levels before increasing again at 96 and 120 hpi. This finding is in accordance

with a previous report that PR1 relative expression showed a brief moderate

(<10-fold) peak at 24 hpi in Arabidopsis infected with P. s. tomato, although

there was no evidence of a further peak as that study did not continue after 48 hpi

(Langlois-Meurinne et al., 2005). One of the most well studied defence-related

proteins in plants, PR1 is known to be enhanced by SA-mediated responses

(Chong et al., 2008; Li et al., 2011; Wielgoss & Kortekamp, 2006). Although

transgenic expression of grapevine PR1 in tobacco revealed that basic PR1 was

capable of conferring resistance to P. s. tabaci (Li et al., 2011), in the current

study moderate PR1 accumulation did not appear to have prevented infection of

grapevine leaves by P. s. syringae.

Expression of PR10 (Pathogenesis-related 10) was used as a second indicator for

SA-mediated defence responses. A large (~1000-fold) increase in transcript levels

of PR10 was first observed in V. vinifera leaves 12 h post inoculation with

pathogenic P. s. syringae (Fig. 4.7). At 24 hpi, transcript levels returned to basal

levels before increasing again at 48 hpi and remaining high however to a lesser

extent than at 12 hpi. Similarly, in a V. vinifera cv. Riesling study, Kortekamp et

al. (2006) reported that PR10 was strongly up-regulated in at 12 hpi following

infection by the grapevine pathogen P. viticola, although in that study PR10

remained up-regulated at 24 hpi.

154

PR10 is known to be strongly expressed in tissue adjacent to necrotic zones and

in distant tissues (Breda et al., 1996). It has been demonstrated that PR10 may be

involved in induced cell death of host tissue (Xu et al., 2014). Others have also

shown that PR10 in V. vinifera is rapidly up-regulated in response to infiltrated

P. syringae pv. pisi (Robert, et al., 2001) and UV-C exposure (Bonomelli et al.,

2004). In the current study, high transcript levels were observed in grapevine

leaves infected with pathogenic P. s. syringae. Furthermore, these leaves also

demonstrated increasing levels of necrosis. These results together suggest that

PR10 is highly expressed in tissues exhibiting and preceding lesion development

in grapevine.

Jasmonic acid-mediated defence gene expression

Expression of VvTL1 (thaumatin-like 1) was used as an indicator for JA-mediated

defence responses. Although VvTL1 has been mostly reported to be involved in

berry ripening, it is induced upon methyl-JA treatment in V. vinifera (I. Dry,

personal communication). Pathogenic P. s. syringae caused moderate increases

in transcript levels of VvTL1 in V. vinifera leaves 12 h after inoculation, before

large increases at 48 hpi and thereafter. These findings are consistent with those

of Jacobs et al. (1999) who, in a V. vinifera cv. Sultana study, reported up-

regulation in leaf VvTL1 by grapevine powdery mildew pathogen E. necator.

Grapevine thaumatin-like protein was also up-regulated in V. vinifera leaves by

two grapevine pathogens, powdery mildew pathogen E. necator and Phomopsis

viticola (Monteiro et al., 2003).

155

Anti-fungal activity of VvTL1 has been demonstrated in several studies. The

VvTL1 protein significantly inhibited spore germination and hyphal growth of

grapevine anthracnose pathogen Elsinoe ampelina on V. vinifera cv. Chardonnay

(Jayasankar et al., 2003), and thaumatin extracted from grape also showed

antifungal activity towards E. necator, Phomopsis viticola and B. cinerea in vitro

(Monteiro et al., 2003).

Expression of Jasmonate-ZIM-domain (VvJAZ5) was used as another indicator

for JA-mediated defence responses. The current study used a V. vinifera JAZ

target VvJAZ5, as reported in Zhang et al. (2012). This gene was significantly

upregulated upon JA and MeJA treatment in V. vinifera (Crimson Seedless) cell

suspensions (Zhang et al., 2012). Increased transcript levels of VvJAZ5 were first

observed in grapevine leaves 48 h after inoculation with pathogenic

P. s. syringae. Expression of VvJAZ5 transcripts then remained elevated for the

remainder of the study. This is in agreement with previous reports showing that

P. syringae was capable of increasing JAZ expression. Pseudomonas syringae

pv. tomato DC3000 induced a subset of JAZ proteins in the early stages of

infection in Arabidopsis (24 hpi), including JAZ5, which has extensive homology

with VvJAZ5 (Demianski et al., 2012).

The current study also indicates that when V. vinifera was challenged with

pathogenic P. s. syringae the JAZ proteins may act as negative regulators for JA-

signalling, as expression of the SA marker PR10 was elevated before the increase

in JAZ transcript accumulation at 48 h, after which it began to decline. This is in

agreement with Demianski et al. (2012) and Thines et al. (2007) who report that

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JAZ proteins are negative regulators for JA-signalling in Arabidopsis. Others

have also shown that SA-mediated gene expression enhances JAZ to represses

JA-mediated responses in Arabidopsis (Van der Does et al., 2013).

Ethylene-mediated defence gene expression

Increased transcript levels of ETR2, commonly used as a marker for ET-mediated

responses (Böttcher et al., 2013), were first observed in V. vinifera leaves 12 hpi

after inoculation with pathogenic P. s. syringae. Transcript expression then

returned to control levels, before increasing again at 96 and 120 h. These results

are in accordance with a previous report that infection with P. s. maculicola

induced the ET signalling pathway in Arabidopsis (Groen et al., 2013). Ethylene

(ET) is known to regulate a number of growth and developmental processes in

higher plants (Wang et al., 2002). It is also generally accepted that ET cooperates

in conjunction with JA-mediated pathways, although interactions with SA have

also been reported during pathogen attack (Adie et al., 2007).

Stilbene synthase induction

Expression of STS was used as a measure of pathogenesis. A large spike in

transcript levels of STS were first observed in V. vinifera leaves 12 hpi with

pathogenic P. s. syringae. STS then decreased at 24 hpi, before increasing again

from 48 to 120 hpi. These results are in agreement with the findings of

Kortekamp (2006) who, in a V. vinifera cv. Riesling study, reported that STS was

strongly up-regulated at 12 hpi with the grapevine pathogen P. viticola, although

in that study STS remained up-regulated at 24 hpi. Stilbene synthases have also

been shown to be induced in V. vinifera by a number of grapevine pathogens with

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various lifestyles including hemi-biotrophic P. s. syringae (Robert et al., 2001)

and the necrotroph B. cinerea (Chong et al., 2008). Transient up-regulation of

STS genes in grapevine leaves has also been demonstrated in response to

ethephon treatment (Belhadj et al., 2008).

The STS gene family encode synthetases that are responsible for the synthesis of

antimicrobial compounds (phytoalexins) in V .vinifera (Adrian et al., 1997;

Jeandet et al., 2010; Pezet et al., 2003; Timperio et al., 2012), and STS has

become a well-known indicator for the common response in V. vinifera to

pathogen infection, and an indication of resistance response activation (Chong et

al., 2008; Kortekamp, 2006).

Several important observations can be made regarding the response of defence-

related genes in grapevine leaves infected with a pathogenic isolate of

P. s. syringae. 1) There was a predominant increase in the expression of the SA-

meditated genes PR1 and PR10 at 12 hpi with a small but significant increase in

the JA-mediated gene VvTL1. 2) The expression of the JA pathway genes VvTL1

and VvJAZ5 occurred predominately at 48 hpi. 3) The decrease in JA-mediated

VvTL1 expression at 72 hpi was preceded by a peak in VvJAZ5 expression. 4)

P. s. syringae infection resulted in a peak in the expression of ET-mediated gene

ETR2 12 hpi prior to the peak in VvTL1 expression and co-incident with the peak

in PR1 and PR10 expression.

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Antagonism between the JA- and SA-mediated defence pathways

Results of the current study indicate antagonism between the JA/SA defence

pathways. This study showed that pathogenic P. s. syringae first dramatically

increased the SA pathways genes, PR1 and PR10, at 12 hpi, but these were then

reduced by 24 hpi. This decrease could have been caused by the activation of JA

signalling pathway, as indicated by the peaks in JA-mediated VvJAZ5 and VvTL1

at 48 hpi. Generally, the activation of JA signalling is known to inhibit SA-

mediated defence signalling, which is required for resistance in plants against

P. syringae (Anderson et al., 2004; Brooks et al., 2005; Petersen et al., 2000;

Spoel et al., 2003). The stability of the JAZ protein functions independently of

SA/JA antagonism (Van der Does et al., 2013) but the JAZ-mediated modulation

of JA signalling does affect SA-dependent defences (Pieterse et al., 2014). One

postulated reason for this is that DELLA stabilisation by flagella perception

results in positive JA signalling (Pieterse et al., 2014). One result of this increase

in JA signalling might be that hemi-biotrophic pathogens such as P. s. syringae

are able to suppress SA-dependent defences that would otherwise provide

resistance or limit pathogen growth (Navarro et al., 2008; Pieterse et al., 2012).

Alternatively, P. s. syringae produces syringolin A (Chapter 3, Amrein et al.,

2004; Ramel et al., 2009) which reduces SA signalling by inhibition of the

proteasome required for NPR1 (SA transcription factor) turnover (Groll et al.,

2008; Misas-Villamil et al., 2013; Vierstra, 2009). Schellenberg et al. (2010) have

also demonstrated SA antagonism by syringolin A producing P. s. syringae

greatly reducing PR1 expression in Arabidopsis.

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An interesting finding from the current data was the repression, or failure to show

significant increase, in transcript accumulation at 24 hpi for all defence related

targets. Milli et al. (2011) also demonstrated similar expression patterns

suggesting that transient breakdown of the plant defence responses in response to

pathogen attack may accompany the onset of disease. This may also indicate

successful colonisation by pathogens after suppression of plant host defences,

which has been demonstrated by others (Kelley et al., 2010; Milli et al., 2012).

Because both SA and JA/ET pathways were enhanced in response to pathogenic

P. s. syringae, these results could indicate a unique result from the grapevine in

response to this pathogen. This may be explained by P. s. syringae having an

early hemi-biotrophic and late necrotrophic stages during plant infection (Alfano

and Collmer, 1996; Xin and He, 2013). This understanding of grapevine defence

responses to P. s. syringae may open up novel procedures to enhance resistance

against this pathogen.

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4.5 Conclusions

The results of this study show that V. vinifera up-regulates both the JA- and SA-

mediated defence pathways in response to pathogenic P. s. syringae. The results

indicate that antagonistic interactions may take place between these two pathways

during the early stages of infection. Several plant defence genes exhibited

temporal oscillation after inoculation with pathogenic P. s. syringae. While these

genes were induced strongly after inoculation, the expression of the defence

genes decreased transiently before increasing again later in the infection process.

These patterns are seldom seen and may provide an interesting example of the

interplay between SA and JA signal transduction in grape (S. Lindow, personal

communication). It was also found that callose deposition in Chardonnay leaves

was greater in response to non-pathogenic than to pathogenic P. s. syringae

strains suggesting that the pathogenic strain is more effective at suppressing PTI.

Further work from these preliminary findings may lead to a new understanding of

how P. s. syringae manipulates, and possibly modulates, the grapevine defences

to promote disease.

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4.6 Acknowledgments

Professor Steven Lindow (Berkeley, University of California, USA) for his kind

review and assistance with data interpretation.

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Chapter 5 General Discussion

Pseudomonas syringae pv. syringae (P. s. syringae) is known to be a widespread

plant pathogen but its effects on grapevine hosts have only recently been

recognised. Grapevine disease symptoms observed in this study were in

agreement with those previously reported (Hall et al., 2002; Whitelaw-Weckert et

al., 2011). In the current study, two types of symptoms were observed: grapevines

with bacterial leaf spot (BLS; leaf spots and necrotic stem lesions only), and

grapevines with bacterial inflorescence rot (BIR; leaf spots, necrotic stem lesions

and necrotic rachii, resulting in loss of yield). In vineyards affected by BLS,

symptom severity has been observed to increase in following seasons (Hall et al.,

2002). Similarly, in vineyards affected by BIR, loss of inflorescences also

increased with time. Anecdotally, a vineyard owner observed minor yield losses

soon after P. s. syringae infection, whereas in later seasons there was total crop

loss (J. Cullen personal communication). The yield losses reported to be caused

by P. s. syringae in Chapter 2 showed that this pathogen can have devastating

consequences for the Australian wine industry.

Genetic diversity

The advancement of molecular techniques, such as gene sequencing, has allowed

for the refinement of the characterisation of P. syringae among plant host species.

These techniques can include multi-locus sequence typing (MLST), DNA-DNA

hybridisation (Gardan et al., 1999), and DNA fingerprinting such as random

amplified polymorphic DNA (RAPD) (Afrose et al., 2014). The results presented

in Chapter 2 demonstrated using MLST that, in cool climate vineyards,

genetically diverse groups of P. s. syringae were isolated from grapevine hosts

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affected by BLS or BIR (Hall et al., 2016). Had the BIR-causing P. s. syringae

population in Australia been clonal, descended from the same recent ancestor, the

phylogenetic trees produced in this study would have shown an initial line of

P. s. syringae giving rise to subsequent lines diverging from the initial line

(McCann et al., 2013; Feil et al., 2000). However, the data in Chapter 2 and

Chapter 3 show that there are multiple genetic lines with different parental lines

spread across phylogenetic trees (Figs. 2.3, 2.4, and 3.8). The host specificity

studies presented in both Chapter 2 and 3 indicated that P. s. syringae isolated

from grapevine lacked host specificity, which is consistent with the findings of

others (Najafi & Taghavi, 2014). A lack of host specificity may have been

indicated by MLST and rpoB phylogenetic analysis showing that P. s. syringae

from cowpea and apple hosts were contained within BIR clades with grapevine

P. s. syringae, and these were capable of infecting grapevine leaves (Chapter 2).

Host range studies conducted by inoculating detached grapevine leaves, intact

tobacco leaves, and mature lemons, demonstrated that P. s. syringae isolated from

grapevines affected by BIR was potentially pathogenic to all three test host plants

(Chapter 3).

Mechanism of transmission

The mechanism of P. s. syringae transmission from vineyard to vineyard is

something that remains unclear. The RNA polymerase β-subunit (rpoB)

sequences of several Tumbarumba (New South Wales) isolates were identical to

Adelaide Hills (South Australia) P. s. syringae isolates. This was also the case

with grapevine P. s. syringae isolates from Murrumbateman (New South Wales)

and Piper’s River (Tasmania) that were identical to each other. These results were

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also supported by MLST phylogentic analysis (Chapter 2). As rpoB is a reliable

marker for bacterial strain identification and provides high resolution for

phylogenetics (Mollet et al., 1997; Tayeb et al., 2005) the phylogenetic results

indicate that the spread of P. s. syringae to these vineyards may have originated

from similar sources, possibly due to unsound nursery practices (Waite et al.,

2014), contamination of water sources (Morris et al., 2008) or through

contaminated pruning equipment (Carroll et al., 2010; Lamichhane et al., 2014).

The overwintering of P. s. syringae, allowing for survival of the pathogen over

time, may indicate systemic infection occurs in grapevine. P. s. syringae isolates

recovered from sucker shoots of grapevines in Tumbarumba vineyards were

identical to other P. s. syringae causing BIR within the same vineyards. In

hazelnut, Pseudomonas avellanae infects sucker shoots, and their propagation can

result in wide-spread dispersal (Scortichini, 2002). A similar mechanism for

spread may be possible with P. s. syringae. Weeds within vineyards have been

implicated as a vehicle for the spread of P. s. syringae by acting as an

overwintering host (Little et al., 1998). Future research is required to investigate

the potential sources of P. s. syringae and its progression throughout the

grapevine.

Pseudomonas syringae pv. syringae phenotypes

Testing using the gelatin, aesculin, tyrosinase, tartartic acid assay (GATTa) in

Chapter 3 showed that P. s. syringae from grapevine hosts had a variable

tyrosinase expression phenotype. A positive phenotype was observed by the

production of a reddish pigment on tyrosinase-casein agar (Latorre & Jones,

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1979) and could be readily distinguished in the current study. Positive tyrosinase

phenotypes were found to be associated with P. s. syringae populations from

healthy and BLS affected grapevines only, whereas negative phenotypes were

associated with P. s. syringae from BIR affected vineyards. This negative

phenotype may be an indication of environmentally driven lifestyle changes from

epiphytic to pathogenic as P. s. syringae isolates causing BIR have been reported

to populate within the xylem (Whitelaw-Weckert et al., 2011). In healthy and

BLS affected grapevine, it is likely that tyrosinase activity in P. s. syringae is

involved in epiphytic fitness (Claus & Decker, 2006) and may act as an indicator

of bacteria from sources that encounter UV damage. The role of tyrosinases in

virulence requires future investigation.

Virulence

In Chapter 3, the production of syringomycin and syringopeptins by

P. s. syringae isolates was determined using both biochemical and genotypic

methods. Production of syringomycin, determined by inhibition of

Saccharomyces cerevisiae and/or G. candidum, was observed in isolates of

P. s. syringae from grapevine. Similarly, syringopeptin production was observed

in P. s. syringae from grapevine using the indicator strain Bacillus megaterium.

Genes involved in the biosynthesis of these toxins were also shown to be present

in the P. s. syringae isolates. Both biochemical and genotypic methods suggested

that syringomycin and syringopeptin were produced by many strains of

P. s. syringae tested. This is consistent with the findings of Feil et al. (2005) and

Thomidis et al. (2005) who suggest these phytotoxins are expressed by strains of

P. s. syringae with wide host ranges. Analysis by MLST showed that

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syringopeptin was associated with BIR. To the best of my knowledge, this is the

first study to demonstrate syringopeptin production by P. s. syringae in grapevine

isolates.

As there is currently no biochemical assay for the detection of syringolins in

P. syringae, syringolin A production was assumed by detecting the presence of

the sylC gene which is involved in its biosynthesis. Syringolins are proteasome

inhibitors that can antagonise salicylic acid (SA)-mediated defence responses

(Misas-Villamil et al., 2013) and control stomatal aperture size changes

(Schellenberg et al., 2010). Others have demonstrated that P. s. syringae from

BIR affected grapevine causes stomata to be ‘locked open’ (Whitelaw-Weckert et

al., 2011). Although stomatal aperture was not studied in this project, future

research should investigate changes in grapevine stomata over the course of

infection with syringolin A-producing P. s. syringae. The presence of the

syringolin A gene (sylC) in P. s. syringae isolated from BIR affected grapevines

indicates that syringolins may be an important virulence effector involved in the

production of BIR symptoms in grapevine hosts. To the best of my knowledge,

the current investigation is the first to demonstrate the presence of the sylC gene

in grapevine P. s. syringae, and to link it with BIR symptoms.

Defence pathways

During plant-pathogen interactions, one of the first PAMP-triggered immunity

(PTI) plant defences is callose deposition. In Chapter 4, callose deposition was

assessed by aniline blue staining followed by epifluorescence microscopy. It was

found that both pathogenic and non-pathogenic P. s. syringae isolates were able

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to induce callose deposition in attached leaves of spray-inoculated potted

Chardonnay plants. Interestingly, larger amounts of callose deposits were

observed in leaves inoculated with a non-pathogenic P. s. syringae isolate, than in

plants inoculated with a pathogenic P. s. syringae isolate. This indicates that a

stronger PTI response is mounted during non-pathogenic P. s. syringae/resistant

grapevine interactions, and the defence response has been suppressed by the

pathogenic P. s. syringae. Mechanisms by which pathogenic P. s. syringae

isolates can overcome or evade these basal immune responses in grapevine

requires future research.

When the relative gene expression in the attached inoculated grapevine leaves

was monitored, both the SA and JA defence pathways were found to be up

regulated (Chapter 4). In the early stages of infection, P. s. syringae infection led

to elevated expression of PR1 and PR10 (SA-mediated defence markers) and

VvTL1 (JA-mediated defence markers. In the later stages of infection (i.e. from 24

hpi), SA-mediated defence markers PR1 and PR10 were decreased while the JA

mediated defence markers VvTL1 and VvJAZ5 were increased. Similarly, others

have reported that, in the later stages of infection, the JA-mediated defence

responses eventually overcome the SA responses, prioritising JA-mediated

pathways (Navarro et al., 2008; Pieterse et al., 2014). Indeed, Arabidopsis

infection with hemi-biotrophic P. s. tomato DC3000 caused DELLA repression of

SA signalling after 24 hours post infection (Navarro et al., 2008). DELLA

proteins are also known to enhance JA-defendant defences. It has been postulated

that flagellin-mediated DELLA stabilisation can result in biotrophic pathogen

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suppression of SA-dependent defences that would otherwise limit pathogen

growth (Pieterse et al., 2014).

The relatively low level of PR1 transcripts compared with PR10 may be

attributed to syringolin A production. Schellenberg et al. (2010) demonstrated

reduced PR1 expression in Arabidopsis being attributed to syrigolin A production

in P. s. syringae. In the current study, pathogenic P. s. syringae was positive for

the presence of the syringolin A gene (sylC). The failure to increase PR1

transcript accumulation between 24 and 72 hours post inoculation could be a

result of syringolin A production in the pathogenic P. s. syringae. Future

investigations are required to elucidate the role of syringolin A in grapevine

defence.

The most interesting finding relating to plant defences was the suppression, or

lack of significant increase in plant defences at 24 hours post inoculation, as

indicated by the lack of expression of all gene targets investigated and absence of

callose deposition in response to pathogenic P. s. syringae. This has also been

demonstrated in grapevine during Plasmopara viticola infection (Milli et al.,

2012), and may indicate that successful colonisation of the pathogen occurs by

suppressing host defences early during the early infection process (Kelley et al.,

2010). Suppression of early defences in combination with late necrosis may be

indicative of pathogen lifestyle from hemi-biotrophic to later necrotrophic

(Alfano & Collmer, 1996; Kelley et al., 2010; Xin & He, 2013). The findings in

the current study may be the first account of a grapevine-specific defence

response to P. s. syringae infection.

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Future Directions

This project has demonstrated that P. s. syringae causes infection of grapevine

cultivars across a range of Australian cool climate vineyards. Furthermore, this

pathogen is genetically diverse and lacks host-specificity, which could result in

transmission from other growing regions and crops. Future research should

consider surveying symptomatic grapevine samples from a wider geographical

area, and include other host plant species in the survey. A comprehensive survey

would confirm how wide-spread this pathogen is, and enhance our understanding

of P. s. syringae host-range in Australia.

The P. s. syringae isolates from BIR-affected vineyards were shown to contain

the sylC gene, suggesting that they may produce syringolin A. Surprisingly, these

isolates lacked tyrosinase activity as has been previously reported (Fahy &

Hayward, 1983; Gašić et al., 2012) although this may be due to the bacterium’s

response to different environmental conditions. The role of syringolin A in

grapevine should be further investigated as this is the first study to suggest a role

in grapevine pathogenicity. The role of tyrosinases in P. s. syringae from various

environments may also provide an understanding of lifestyle changes of

P. s. syringae from epiphytic to pathogenic.

Finally, P. s. syringae infection in grapevine was found to produce moderate PTI

responses (measured by callose deposition) and to up-regulate both SA and JA-

mediated pathways. The exact mechanisms underlying P. s. syringae

pathogenicity in the grapevine and the plant defence responses are still not fully

understood. Future experiments involving more detailed analysis of grapevine

170

defence and phytohormone responses could increase our current understanding of

the progression of P. s. syringae infection in grapevine, and may lead to effective

treatments within vineyards.

171

Chapter 6 Literature Cited

Abbasi, V., Rahimian, H., & Tajick-Ghanbari, M. (2013). Genetic variability of

Iranian strains of Pseudomonas syringae pv. syringae causing bacterial

canker disease of stone fruits. European Journal of Plant Pathology,

135(2), 225-235.

Abel, S., Ngu`yen, M. D., Chow, W., & Theologis, A. (1995). ASC4, a primary

indoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-

carboxylate synthase in Arabidopsis thaliana - Structural characterization,

expression in Escherichia coli, and expression characteristics in response

to auxin. Journal of Biological Chemistry, 270(32), 19093-19099.

Abkhoo, J. (2015). Bacterial inflorescence rot of grapevine caused by

Pseudomonas syringae pv. syringae in Iran. Australian Journal of Applied

Mathematics, 5(3), 29-32.

Abràmoff, M. D., Magalhães, P. J., & Ram, S. J. (2004). Image processing with

ImageJ. Biophotonics international, 11(7), 36-42.

Achard, P., Renou, J.-P., Berthomé, R., Harberd, N. P., & Genschik, P. (2008).

Plant DELLAS restrain growth and promote survival of adversity by

reducing the levels of reactive oxygen species. Current Biology, 18(9),

656-660.

Adam, L., & Somerville, S. C. (1996). Genetic characterization of five powdery

mildew disease resistance loci in Arabidopsis thaliana. The Plant Journal,

9(3), 341-356.

Adie, B. A. T., Pérez-Pérez, J., Pérez-Pérez, M. M., Godoy, M., Sánchez-Serrano,

J. J., Schmelz, E. A., & Solano, R. (2007). ABA is an essential signal for

plant resistance to pathogens affecting JA biosynthesis and the activation

of defenses in Arabidopsis. The Plant Cell Online, 19(5), 1665-1681.

Adie, B., Chico, J., Rubio-Somoza, I., & Solano, R. (2007). Modulation of plant

defenses by ethylene. Journal of Plant Growth Regulation, 26(2), 160-

177.

Adrian, M., Jeandet, P., Duillet-Breuil, A., Tesson, L., & Bessis, R. (1997).

Biological activity of resveratrol, a stilbenic compound from grapevines,

against Botrytis cinerea, the causal agent for gray mold. J Chem Ecol, 23,

1689 - 1702.

Afrose, S., Hossain, I., Hossain, M., & Khan, M. (2014b). Genetic diversity of

Pseudomonas syringae pv. syringae causing leaf blight of litchi in

Bangladesh. SAARC Journal of Agriculture, 12(1), 150-161.

Afrose, S., Hossain, I., Islam, M. R., Hossain, M. D., & Khan, M. A. H. (2014a).

RAPD analyses reveals the genetic diversity of the litchi leaf blight

pathogen, Pseudomonas syringae pv. syringae in Bangladesh. Current

Research in Microbiology and Biotechnology, 2(1), 301-309.

172

Alfano J,R., Collmer A. (1996). Bacterial pathogens in plants: Against the wall.

The Plant Cell, 8, 1683-1698.

Alonso, J. M., & Stepanova, A. N. (2004). The ethylene signaling pathway.

Science, 306(5701), 1513-1515.

Amrein, H., Makart, S., Granado, J., Shakya, R., Schneider-Pokorny, J., &

Dudler, R. (2004). Functional analysis of genes involved in the synthesis

of syringolin A by Pseudomonas syringae pv. syringae B301D-R.

Molecular Plant-Microbe Interactions, 17(1), 90-97.

Anderson, J. P., Badruzsaufari, E., Schenk, P. M., Manners, J. M., Desmond, O.

J., Ehlert, C., Maclean, D. J., Ebert, P. R., & Kazan, K. (2004).

Antagonistic interaction between abscisic acid and jasmonate-ethylene

signaling pathways modulates defense gene expression and disease

resistance in Arabidopsis. The Plant Cell Online, 16(12), 3460-3479.

Apel, K., & Hirt, H. (2004). Reactive oxygen species: Metabolism, oxidative

stress, and signal transduction. Annu. Rev. Plant Biol., 55, 373-399.

Argueso, C. T., Ferreira, F. J., Epple, P., To, J. P. C., Hutchison, C. E., Schaller,

G. E., Dangl, J. L., & Kieber, J. J. (2012). Two-component elements

mediate interactions between cytokinin and salicylic acid in plant

immunity. PLoS Genetics, 8(1), e1002448.

Arnold, D. L., Lovell, H. C., Jackson, R. W., & Mansfield, J. W. (2011).

Pseudomonas syringae pv. phaseolicola: from ‘has bean’ to supermodel.

Molecular Plant Pathology, 12(7), 617-627.

Arraiano, L. S., Brading, P. A., & Brown, J. K. M. (2001). A detached seedling

leaf technique to study resistance to Mycosphaerella graminicola

(anamorph Septoria tritici) in wheat. Plant Pathology, 50(3), 339-346.

Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu, W.-L., Gomez-Gomez,

L., Boller, T., Ausubel, F. M., & Sheen, J. (2002). MAP kinase signalling

cascade in Arabidopsis innate immunity. Nature, 415(6875), 977-983.

Asselbergh, B., Achuo, A. E., Höfte, M., & Van Gijsegem, F. (2008). Abscisic

acid deficiency leads to rapid activation of tomato defence responses upon

infection with Erwinia chrysanthemi. Molecular plant pathology, 9(1),

11-24.

Audenaert, K., De Meyer, G. B., & Höfte, M. M. (2002). Abscisic acid

determines basal susceptibility of tomato to Botrytis cinerea and

suppresses salicylic acid-dependent signaling mechanisms. Plant

Physiology, 128(2), 491-501.

Australia, P. H. (2013). Notifiable plant pests from Australian state and territory

legislation. Retrieved 17/06/2015, 2013, from

http://www.planthealthaustralia.com.au/biosecurity/notifiable-plant-pests/

173

Badel, J. L., Shimizu, R., Oh, H.-S., & Collmer, A. (2006). A Pseudomonas

syringae pv. tomato avrE1/hopM1 mutant is severely reduced in growth

and lesion formation in tomato. Molecular Plant-Microbe Interactions,

19(2), 99-111.

Balaž, J., Ilicic, R., Masirevic, S., Kojic, S., & Josic, D. (2014). First report of

Pseudomonas syringae pv. syringae causing bacterial leaf spots of oil

pumpkin (Cucurbita pepo L.) in Serbia. Plant Disease, 98(5), 684.

Ballio, A., Bossa, F., Di Giorgio, D., Di Nola, A., Manetti, C., Paci, M., Scaloni,

A., & Segre, A. L. (1995). Solution conformation of the Pseudomonas

syringae pv. syringae phytotoxic lipodepsipeptide syringopeptin 25-A.

European Journal of Biochemistry, 234(3), 747-758.

Ballio, A., Bossa, F., Di Giorgio, D., Ferranti, P., Paci, M., Pucci, P., Scaloni, A.,

Segre, A., & Strobel, G. A. (1994a). Novel bioactive lipodepsipeptides

from Pseudomonas syringae: The pseudomycins. FEBS letters, 355(1),

96-100.

Ballio, A., Collina, A., Di Nola, A., Manetti, C., Pad, M., & Segre, A. (1994b).

Determination of structure and conformation in solution of syringotoxin, a

lipodepsipeptide from Pseudomonas syringae pv. syringae by 2D NMR

and molecular dynamics. Structural Chemistry, 5(1), 43-50.

Baltrus, D. A., Nishimura, M. T., Romanchuk, A., Chang, J. H., Mukhtar, M. S.,

Cherkis, K., Roach, J., Grant, S. R., Jones, C. D., & Dangl, J. L. (2011).

Dynamic evolution of pathogenicity revealed by sequencing and

comparative genomics of 19 Pseudomonas syringae isolates. PLoS

Pathogens, 7(7), e1002132.

Barta, T. M., & Willis, D. K. (2005). Biological and molecular evidence that

Pseudomonas syringae pathovars coronafaciens, striafaciens and garcae

are likely the same pathovar. Journal of Phytopathology, 153(7-8), 492-

499.

Bartoli, C., Lamichhane, J. R., Berge, O., Varvaro, L., & Morris, C. E. (2015).

Mutability in Pseudomonas viridiflava as a programmed balance between

antibiotic resistance and pathogenicity. Molecular Plant Pathology, 16(8),

860-869.

Bashan, Y. (1986). Field dispersal of Pseudomonas syringae pv. tomato,

Xanthomonas campestris pv. vesicatoria, and Alternaria macrospora by

animals, people, birds, insects, mites, agricultural tools, aircraft, soil

particles, and water sources. Canadian Journal of Botany, 64(2), 276-281.

Behringer, M., Miller, W. G., & Oyarzabal, O. A. (2011). Typing of

Campylobacter jejuni and Campylobacter coli isolated from live broilers

and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR.

Journal of Microbiological Methods, 84(2), 194-201.

174

Belhadj, A., Telef, N., Cluzet, S., Bouscaut, J., Corio-Costet, M., & M rillon, J.

M. (2008). Ethephon elicits protection against Erysiphe necator in

grapevine. Journal of Agricultural and Food Chemistry, 56(14), 5781-

5787.

Bender, C. L., Alarcón-Chaidez, F., & Gross, D. C. (1999). Pseudomonas

syringae phytotoxins: Mode of action, regulation, and biosynthesis by

peptide and polyketide synthetases. Microbiology and Molecular Biology

Reviews, 63(2), 266-292.

Bensaci, M. F., Gurnev, P. A., Bezrukov, S. M., & Takemoto, J. Y. (2011).

Fungicidal activities and mechanisms of action of Pseudomonas syringae

pv. syringae lipodepsipeptide syringopeptins 22A and 25A. Frontiers in

Microbiology, 2, 216.

Benschop, J. J., Mohammed, S., O'Flaherty, M., Heck, A. J., Slijper, M., &

Menke, F. L. (2007). Quantitative phosphoproteomics of early elicitor

signaling in Arabidopsis. Molecular & Cellular Proteomics, 6(7), 1198-

1214.

Berge, O., Monteil, C.L., Bartoli, C., Chandeysson, C., Guibaud, C., Sands, D.C.,

& Morris, C.E. (2014). A user’s guide to a data base of the diversity of

Psuedomonas syringae and its application to classifying strains on the

phylogenetic complex. PLoS ONE, 9(9), e105547,

Bethke, G., Unthan, T., Uhrig, J. F., Pöschl, Y., Gust, A. A., Scheel, D., & Lee, J.

(2009). Flg22 regulates the release of an ethylene response factor

substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene

signaling. Proceedings of the National Academy of Sciences, 106(19),

8067-8072.

Bézier, A., Lambert, B., & Baillieul, F. (2002). Cloning of a grapevine Botrytis

responsive gene that has homology to the tobacco hypersensitivity related

hsr203J. Journal of Experimental Botany, 53(378), 2279-2280.

Bigeard, J., Colcombet, J., & Hirt, H. (2015). Signaling mechanisms in pattern-

triggered immunity (PTI). Molecular Plant, 8(4), 521-539.

Biosecurity Australia. (2002). Import Risk Analysis for the Importation of Unshu

Mandarin Fruit from Japan. Retrieved from

http://www.daff.gov.au/SiteCollectionDocuments/ba/plant/ungroupeddocs

/Unshu_Mandarins_Japan_Provisional_Final_IRA_20090406.pdf.

Blume, B., Nürnberger, T., Nass, N., & Scheel, D. (2000). Receptor-mediated

increase in cytoplasmic free calcium required for activation of pathogen

defense in parsley. The Plant Cell, 12(8), 1425-1440.

Bonomelli, A., Mercier, L., Franchel, J., Baillieul, F., Benizri, E., & Mauro, M.-

C. (2004). Response of grapevine defenses to UV-C exposure. American

Journal of Enology and Viticulture, 55(1), 51-59.

175

Bordiec, S., Paquis, S., Lacroix, H., Dhondt, S., Ait Barka, E., Kauffmann, S.,

Jeandet, P., Mazeyrat-Gourbeyre, F., Clément, C., Baillieul, F., & Dorey,

S. (2011). Comparative analysis of defence responses induced by the

endophytic plant growth-promoting rhizobacterium Burkholderia

phytofirmans strain PsJN and the non-host bacterium Pseudomonas

syringae pv. pisi in grapevine cell suspensions. Journal of experimental

botany, 62(2), 595-603.

Böttcher, C., Harvey, K. E., Boss, P. K., & Davies, C. (2013). Ripening of grape

berries can be advanced or delayed by reagents that either reduce or

increase ethylene levels. Functional Plant Biology, 40(6), 566-581.

Breda, C., Sallaud, C., El-Turk, J., Buffard, D., De Kozak, I., Esnault, R., et al.

(1996). Defense reaction in Medicago sativa: A gene encoding a class 10

PR protein is expressed in vascular bundles. MPMI-Molecular Plant

Microbe Interactions, 9(8), 713-719.

Brooks, D. M., Bender, C. L., & Kunkel, B. N. (2005). The Pseudomonas

syringae phytotoxin coronatine promotes virulence by overcoming

salicylic acid-dependent defences in Arabidopsis thaliana. Molecular

plant pathology, 6(6), 629-639.

Buell, C. R., Joardar, V., Lindeberg, M., Selengut, J., Paulsen, I. T., Gwinn, M.

L., Dodson, R. J., Deboy, R. T., Durkin, A. S., Kolonay, J. F., Madupu,

R., Daugherty, S., Brinkac, L., Beanan, M. J., Haft, D. H., Nelson, W. C.,

Davidsen, T., Zafar, N., Zhou, L., Liu, J., Yuan, Q., Khouri, H., Fedorova,

N., Tran, B., Russell, D., Berry, K., Utterback, T., Van Aken, S. E.,

Feldblyum, T. V., D'Ascenzo, M., Deng, W. L., Ramos, A. R., Alfano, J.

R., Cartinhour, S., Chatterjee, A. K., Delaney, T. P., Lazarowitz, S. G.,

Martin, G. B., Schneider, D. J., Tang, X., Bender, C. L., White, O., Fraser,

C. M., & Collmer, A. (2003). The complete genome sequence of the

Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato

DC3000. Proceedings of the National Academy of Sciences of the United

States of America, 100(18), 10181-10186.

Bultreys, A., & Gheysen, I. (1999). Biological and molecular detection of toxic

lipodepsipeptide-producing Pseudomonas syringae strains and PCR

identification in plants. Applied and Environmental Microbiology, 65(5),

1904-1909.

Bultreys, A., & Kaluzna, M. (2010). Bacterial cankers caused by Pseudomonas

syringae on stone fruit species with special emphasis on the pathovars

syringae and morsprunorum race 1 and race 2. Journal of Plant

Pathology, 92(1), S21-S33.

Bureau of Meteorology (2012). Annual and monthly potential frost days.

Retrieved August 26, 2014, from

http://www.bom.gov.au/jsp/ncc/climate_averages/frost/index.jsp

176

Burkowicz, A., & Rudolph, K. (1994). Evaluation of pathogenicity and of

cultural and biochemical tests for identification of Pseudomonas syringae

pathovars syringae, morsprunorum and persicae from fruit trees. Journal

of Phytopathology, 141(1), 59-76.

Cao, H., Li, X., & Dong, X. (1998). Generation of broad-spectrum disease

resistance by overexpression of an essential regulatory gene in systemic

acquired resistance. Proceedings of the National Academy of Sciences,

95(11), 6531-6536.

Capps, E. R., & Wolf, T. K. (2000). Reduction of bunch stem necrosis of

Cabernet Sauvignon by increased tissue nitrogen concentration. American

Journal of Enology and Viticulture, 51(4), 319-328.

Carroll, J., Robinson, T., Burr, T., Hoying, S., & Cox, K. (2010). Evaluation of

pruning techniques and bactericides to manage bacterial canker of sweet

cherry. New York Fruit Quarterly, 18, 9-15.

Cazorla, F. M., Tores, J. A., Olalla, L., Perez-Garcia, A., Farre, J. M., & de

Vicente, A. (1998). Bacterial apical necrosis of mango in Southern Spain:

A disease caused by Pseudomonas syringae pv. syringae. Phytopathology,

88(7), 614-620.

Chang, C., & Shockey, J. A. (1999). The ethylene-response pathway: signal

perception to gene regulation. Current opinion in plant biology, 2(5), 352-

358.

Chang, J. H., Urbach, J. M., Law, T. F., Arnold, L. W., Hu, A., Gombar, S.,

Grant, S. R., Ausubel, F. M., & Dangl, J. L. (2005). A high-throughput,

near-saturating screen for type III effector genes from Pseudomonas

syringae. Proceedings of the National Academy of Sciences of the United

States of America, 102(7), 2549-2554.

Chang, X., & Nick, P. (2012). Defence signalling triggered by flg22 and harpin is

integrated into a different stilbene output in Vitis cells. PloS one, 7(7),

e40446.

Chatterjee, A., Cui, Y., Yang, H., Collmer, A., Alfano, J. R., & Chatterjee, A. K.

(2003). GacA, the response regulator of a two-component system, acts as

a master regulator in Pseudomonas syringae pv. tomato DC3000 by

controlling regulatory RNA, transcriptional activators, and alternate sigma

factors. Molecular Plant-Microbe Interactions, 16(12), 1106-1117.

Chen, J., Zhang, Y., Wang, C., Lü, W., Jin, J., & Hua, X. (2011). Proline induces

calcium-mediated oxidative burst and salicylic acid signaling. Amino

Acids, 40(5), 1473-1484.

Chen, Z., Agnew, J. L., Cohen, J. D., He, P., Shan, L., Sheen, J., & Kunkel, B. N.

(2007). Pseudomonas syringae type III effector AvrRpt2 alters

Arabidopsis thaliana auxin physiology. Proceedings of the National

Academy of Sciences, 104(50), 20131-20136.

177

Chen, Z., Kloek, A. P., Cuzick, A., Moeder, W., Tang, D., Innes, R. W., Klessig,

D. F., McDowell, J. M., & Kunkel, B. N. (2004). The Pseudomonas

syringae type III effector AvrRpt2 functions downstream or independently

of SA to promote virulence on Arabidopsis thaliana. The Plant Journal,

37(4), 494-504.

Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J.

D. G., Felix, G., & Boller, T. (2007). A flagellin-induced complex of the

receptor FLS2 and BAK1 initiates plant defence. Nature, 448(7152), 497-

500.

Chisholm, S. T., Coaker, G., Day, B., & Staskawicz, B. J. (2006). Host-microbe

interactions: Shaping the evolution of the plant immune response. Cell,

124(4), 803-814.

Cho, H., & Kang, H. (2012). The PseEF efflux system is a virulence factor of

Pseudomonas syringae pv. syringae. The Journal of Microbiology, 50(1),

79-90.

Choi, J., Huh, S. U., Kojima, M., Sakakibara, H., Paek, K.-H., & Hwang, I.

(2010). The cytokinin-activated transcription factor ARR2 promotes plant

immunity via TGA3/NPR1-dependent salicylic acid signaling in

Arabidopsis. Developmental Cell, 19(2), 284-295.

Chong, J., Le Henanff, G., Bertsch, C., & Walter, B. (2008). Identification,

expression analysis and characterization of defense and signaling genes in

Vitis vinifera. Plant Physiology and Biochemistry, 46(4), 469-481.

Clarke, C. R., Cai, R., Studholme, D. J., Guttman, D. S., & Vinatzer, B. A.

(2010). Pseudomonas syringae strains naturally lacking the classical P.

syringae hrp/hrc locus are common leaf colonizers equipped with an

atypical type III secretion system. Molecular Plant-Microbe Interactions,

23(2), 198-210.

Claus, H., & Decker, H. (2006). Bacterial tyrosinases. Systematic and Applied

Microbiology, 29(1), 3-14.

Clerc, J., Groll, M., Illich, D. J., Bachmann, A. S., Huber, R., Schellenberg, B.,

Dudler, R., & Kaiser, M. (2009). Synthetic and structural studies on

syringolin A and B reveal critical determinants of selectivity and potency

of proteasome inhibition. Proceedings of the National Academy of

Sciences, 106(16), 6507-6512.

Cohen, Y., Reuveni, M., & Baider, A. (1999). Local and systemic activity of

BABA (DL-3-aminobutyric acid) against Plasmopara viticola in

grapevines. European Journal of Plant Pathology, 105(4), 351-361.

Collier, S. M., & Moffett, P. (2009). NB-LRRs work a “bait and switch” on

pathogens. Trends in Plant Science, 14(10), 521-529.

Cooksey, D. A. (1994). Molecular mechanisms of copper resistance and

accumulation in bacteria. FEMS Microbiology Reviews, 14(4), 381-386.

178

Cowan, A. K., Cairns, A. L. P., & Bartels-Rahm, B. (1999). Regulation of

abscisic acid metabolism: Towards a metabolic basis for abscisic acid-

cytokinin antagonism. Journal of Experimental Botany, 50(334), 595-603.

Crosse, J. (1956). Bacterial canker of stone fruits II. Leaf scar infection of cherry.

Journal of Horticultural Science, 31(3), 212-224.

Cugusi, M., Garau, R., Prota, U., & Dore, M. (1986). A bark necrosis of

grapevine caused by Pseudomonas syringae v. Hall, in Sardinia. Journal

of Phytopathology, 116(2), 176-185.

Cui, H., Wang, Y., Xue, L., Chu, J., Yan, C., Fu, J., Chen, M., Innes, R. W., &

Zhou, J.-M. (2010). Pseudomonas syringae effector protein AvrB perturbs

Arabidopsis hormone signaling by activating MAP kinase 4. Cell Host &

Microbe, 7(2), 164-175.

Cunnac, S., Lindeberg, M., & Collmer, A. (2009). Pseudomonas syringae type III

secretion system effectors: repertoires in search of functions. Current

Opinion in Microbiology, 12(1), 53-60.

Curnev, P. A., Kaulin, Y. A., Tikhomirova, A. V., Wangspa, R., Takemoto, D.,

Malev, V. V., & Schagina, L. V. (2002). Activity of toxins produced by

Pseudomonas syringae pv. syringae in model and cell membranes.

Tsitologiya, 44(3), 302-304.

Danhorn, T., & Fuqua, C. (2007). Biofilm formation by plant-associated bacteria.

Annual Review of Microbiology, 61, 401-422.

De Lorenzo, G., D'Ovidio, R., & Cervone, F. (2001). The role of

polygalacturonase-inhibiting proteins (PGIPs) in defense against

pathogenic fungi. Annual Review of Phytopathology, 39(1), 313.

De Soete, G. (1986). Optimal variable weighting for ultrametric and additive tree

clustering. Quality and Quantity, 20(2-3), 169-180.

de Torres-Zabala, M., Truman, W., Bennett, M. H., Lafforgue, G., Mansfield, J.

W., Rodriguez Egea, P., Bogre, L., & Grant, M. (2007). Pseudomonas

syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling

pathway to cause disease. EMBO J, 26(5), 1434-1443.

De Vos, M., Van Oosten, V. R., Van Poecke, R. M. P., Van Pelt, J. A., Pozo, M.

J., Mueller, M. J., Buchala, A. J., Métraux, J.-P., Van Loon, L. C., Dicke,

M., & Pieterse, C. M. J. (2005). Signal signature and transcriptome

changes of Arabidopsis during pathogen and insect attack. Molecular

Plant-Microbe Interactions, 18(9), 923-937.

DebRoy, S., Thilmony, R., Kwack, Y.-B., Nomura, K., & He, S. Y. (2004). A

family of conserved bacterial effectors inhibits salicylic acid-mediated

basal immunity and promotes disease necrosis in plants. Proceedings of

the National Academy of Sciences of the United States of America,

101(26), 9927-9932.

179

Demianski, A. J., Chung, K. M., & Kunkel, B. N. (2012). Analysis of

Arabidopsis JAZ gene expression during Pseudomonas syringae

pathogenesis. Molecular Plant Pathology, 13(1), 46-57.

Devadas, S. K., Enyedi, A., & Raina, R. (2002). The Arabidopsis hrl1 mutation

reveals novel overlapping roles for salicylic acid, jasmonic acid and

ethylene signalling in cell death and defence against pathogens. The Plant

Journal, 30(4), 467-480.

Di Giorgio, D., Camoni, L., Mott, K. A., Takemoto, J. Y., & Ballio, A. (1996).

Syringopeptins, Pseudomonas syringae pv. syringae phytotoxins,

resemble syringomycin in closing stomata. Plant Pathology, 45(3), 564-

571.

Diallo, M. D., Monteil, C. L., Vinatzer, B. A., Clarke, C. R., Glaux, C., Guilbaud,

C., Desbiez, C., & Morris, C. E. (2012). Pseudomonas syringae naturally

lacking the canonical type III secretion system are ubiquitous in

nonagricultural habitats, are phylogenetically diverse and can be

pathogenic. The ISME Journal, 6(7), 1325-1335.

Donadio, S., Monciardini, P., & Sosio, M. (2007). Polyketide synthases and

nonribosomal peptide synthetases: The emerging view from bacterial

genomics. Natural Product Reports, 24(5), 1073-1109.

Dudler, R. (2013). The role of bacterial phytotoxins in inhibiting the eukaryotic

proteasome. Trends in Microbiology, 22(1), 28-35.

Dudnik, A., & Dudler, R. (2014). Genomics-based exploration of virulence

determinants and host-specific adaptions of Pseudomonas syringae strains

Isolated from grasses. Pathogens, 3(1), 131-148.

Dufour, M. C., Lambert, C., Bouscaut, J., Mérillon, J. M., & Corio-Costet, M. F.

(2012). Benzothiadiazole-primed defence responses and enhanced

differential expression of defence genes in Vitis vinifera infected with

biotrophic pathogens Erysiphe necator and Plasmopara viticola. Plant

Pathology, 62(2), 370-382..

Duke, S. O., & Dayan, F. E. (2011). Modes of action of microbially-produced

phytotoxins. Toxins, 3(8), 1038-1064.

Dye, D. W. (1953). Control of Pseudomonas syringae with streptomycin. Nature,

172(4380), 683-684.

Ellinger, D., Naumann, M., Falter, C., Zwikowics, C., Jamrow, T., Manisseri, C.,

Somerville, S. C., & Voigt, C. A. (2013). Elevated early callose

deposition results in complete penetration resistance to powdery mildew

in Arabidopsis. Plant Physiology, 161(3), 1433-1444.

Ellis, J. G., Dodds, P. N., & Lawrence, G. J. (2007). Flax rust resistance gene

specificity is based on direct resistance-avirulence protein interactions.

Annual Review of Phytopathology, 45(1), 289-306.

180

Engl, C., Waite, C. J., McKenna, J. F., Bennett, M. H., Hamann, T., & Buck, M.

(2014). Chp8, a diguanylate cyclase from Pseudomonas syringae pv.

tomato DC3000, suppresses the pathogen-associated molecular pattern

flagellin, increases extracellular polysaccharides, and promotes plant

immune evasion. mBio, 5(3), e01168-01114.

Everett, K. R., Taylor, R. K., Romberg, M. K., Rees-George, J., Fullerton, R. A.,

Vanneste, J. L., & Manning, M. A. (2011). First report of Pseudomonas

syringae pv. actinidiae causing kiwifruit bacterial canker in New Zealand.

Aust. Plant Dis. Notes, 6(67-71).

Excoffier, L., Laval, G., & Schneider, S. (2005). Arlequin (version 3.0): An

integrated software package for population genetics data analysis.

Evolutionary Bioinformatics Online, 1, 47-50.

Excoffier, L., Smouse, P. E., & Quattro, J. M. (1992). Analysis of molecular

variance inferred from metric distances among DNA haplotypes:

Application to human mitochondrial DNA restriction data. Genetics,

131(2), 479-491.

Fahy, P. C., & Hayward, A. C. (1983). Media and methods for isolation and

diagnositic tests. Sydney: Academic Press.

Feil, E.J., Smith, J.M., Enright, M.C., & Spratt, B.G. (2000). Estimating

recombinational parameters in Streptococcus pneumoniae from multilocus

sequence typing data. Genetics, 154(4), 1439-1450.

Feil, H., Feil, W. S., Chain, P., Larimer, F., DiBartolo, G., Copeland, A., Lykidis,

A., Trong, S., Nolan, M., Goltsman, E., Thiel, J., Malfatti, S., Loper, J. E.,

Lapidus, A., Detter, J. C., Land, M., Richardson, P. M., Kyrpides, N. C.,

Ivanova, N., & Lindow, S. E. (2005). Comparison of the complete

genome sequences of Pseudomonas syringae pv. syringae B728a and pv.

tomato DC3000. Proceedings of the National Academy of Sciences of the

United States of America, 102(31), 11064-11069.

Felix, G., Duran, J. D., Volko, S., & Boller, T. (1999). Plants have a sensitive

perception system for the most conserved domain of bacterial flagellin.

The Plant Journal, 18(3), 265-276.

Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the

bootstrap. Evolution, 39(4), 783-791.

Ferrante, P., & Scortichini, M. (2009). Identification of Pseudomonas syringae

pv. actinidiae as causal agent of bacterial canker of yellow kiwifruit

(Actinidia chinensis Planchon) in central Italy. Journal of Phytopathology,

157(11‐12), 768-770.

Feys, B., Benedetti, C. E., Penfold, C. N., & Turner, J. G. (1994). Arabidopsis

mutants selected for resistance to the phytotoxin coronatine are male

sterile, insensitive to methyl jasmonate, and resistant to a bacterial

pathogen. The Plant Cell, 6(5), 751-759.

181

Finkel, T. (1998). Oxygen radicals and signaling. Current Opinion in Cell

Biology, 10(2), 248-253.

Frigerio, M., Alabadí, D., Pérez-Gómez, J., García-Cárcel, L., Phillips, A. L.,

Hedden, P., & Blázquez, M. A. (2006). Transcriptional regulation of

gibberellin metabolism genes by auxin signaling in Arabidopsis. Plant

Physiology, 142(2), 553-563.

Fu, X., & Harberd, N. P. (2003). Auxin promotes Arabidopsis root growth by

modulating gibberellin response. Nature, 421(6924), 740-743.

Fukuchi, N., Isogai, A., Nakayama, J., Takayama, S., Yamashita, S., Suyama, K.,

& Suzuki, A. (1992). Isolation and structural elucidation of syringostatins,

phytotoxins produced by Pseudomonas syringae pv. syringae lilac isolate.

Journal of the Chemical Society, Perkin Transactions 1(7), 875-880.

Gardan, L., Shafik, H., Belouin, S., Broch, R., Grimont, F., & Grimont, P. A. D.

(1999). DNA relatedness among the pathovars of Pseudomonas syringae

and description of Pseudomonas tremae sp. nov. and Pseudomonas

cannabina sp. nov. (ex Sutic and Dowson 1959). International Journal of

Systematic Bacteriology, 49(2), 469-478.

Gašić, K., Prokić, A., Ivanović, M., Kuzmanović, N., & Obradović, A. (2012).

Differentiation of Pseudomonas syringae pathovars originating from stone

fruits. Pesticidi i fitomedicina, 27(3), 219-229.

Gehring, C. A., Irving, H. R., McConchie, R., & Parish, R. W. (1997).

Jasmonates induce intracellular alkalinization and closure of

Paphiopedilum guard cells. Annals of Botany, 80(4), 485-489.

Gfeller, A., Dubugnon, L., Liechti, R., & Farmer, E. E. (2010). Jasmonate

biochemical pathway. Science Signaling, 3(109), cm3.

Gilbert, V., Legros, F., Maraite, H., & Bultreys, A. (2009). Genetic analyses of

Pseudomonas syringae isolates from Belgian fruit orchards reveal genetic

variability and isolate-host relationships within the pathovar syringae, and

help identify both races of the pathovar morsprunorum. European Journal

of Plant Pathology, 124(2), 199-218.

Gilbert, V., Planchon, V., Legros, F., Maraite, H., & Bultreys, A. (2010).

Pathogenicity and aggressiveness in populations of Pseudomonas

syringae from Belgian fruit orchards. European Journal of Plant

Pathology, 126(2), 263-277.

Gimenez-Ibanez, S., Boter, M., Fernandez-Barbero, G., Chini, A., Rathjen, J. P.,

& Solano, R. (2014). The bacterial effector HopX1 targets JAZ

transcriptional repressors to activate jasmonate signaling and promote

infection in Arabidopsis. PLoS Biol, 12(2), e1001792.

Gindro, K., Pezet, R., & Viret, O. (2003). Histological study of the responses of

two Vitis vinifera cultivars (resistant and susceptible) to Plasmopara

viticola infections. Plant Physiology and Biochemistry, 41(9), 846-853.

182

Glauser, G., Grata, E., Dubugnon, L., Rudaz, S., Farmer, E. E., & Wolfender, J.-

L. (2008). Spatial and temporal dynamics of jasmonate synthesis and

accumulation in Arabidopsis in response to wounding. Journal of

Biological Chemistry, 283(24), 16400-16407.

Glazebrook, J. (2005). Contrasting mechanisms of defense against biotrophic and

necrotrophic pathogens. Annual Review of Phytopathology, 43(1), 205-

227.

Glickmann, E., Gardan, L., Jacquet, S., Hussain, S., Elasri, M., Petit, A., &

Dessaux, Y. (1998). Auxin production is a common feature of most

pathovars of Pseudomonas syringae. Molecular Plant-Microbe

Interactions, 11(2), 156-162.

Goel, A. K., Lundberg, D., Torres, M. A., Matthews, R., Akimoto-Tomiyama, C.,

Farmer, L., Dangl, J. L., & Grant, S. R. (2008). The Pseudomonas

syringae type III effector HopAM1 enhances virulence on water-stressed

plants. Molecular Plant-Microbe Interactions, 21(3), 361-370.

Golzar, H., & Cother, E. J. (2008). First report of bacterial necrosis of mango

caused by Pseudomonas syringae pv. syringae in Australia. Australasian

Plant Disease Notes, 3(1), 107-109.

Govrin, E. M., Rachmilevitch, S., Tiwari, B. S., Solomon, M., & Levine, A.

(2006). An elicitor from Botrytis cinerea induces the hypersensitive

response in Arabidopsis thaliana and other plants and promotes the gray

mold disease. Phytopathology, 96(3), 299-307.

Greenberg, J. T., & Yao, N. (2004). The role and regulation of programmed cell

death in plant–pathogen interactions. Cellular Microbiology, 6(3), 201-

211.

Grgurina, I., Barca, A., Cervigni, S., Gallo, M., Scaloni, A., & Pucci, P. (1994).

Relevance of chlorine-substituent for the antifungal activity of

syringomycin and syringotoxin, metabolites of the phytopathogenic

bacterium Pseudomonas syringae pv. syringae. Experientia, 50(2), 130-

133.

Grgurina, I., Gross, D. C., Iacobellis, N. S., Lavermicocca, P., Takemoto, J. Y., &

Benincasa, M. (1996). Phytotoxin production by Pseudomonas syringae

pv. syringae: Syringopeptin production by syr mutants defective in

biosynthesis or secretion of syringomycin. FEMS Microbiology Letters,

138(1), 35-39.

Grgurina, I., Mariotti, F., Fogliano, V., Gallo, M., Scaloni, A., Iacobellis, N. S.,

Cantore, P. L., Mannina, L., van Axel Castelli, V., & Greco, M. L. (2002).

A new syringopeptin produced by bean strains of Pseudomonas syringae

pv. syringae. Biochimica et Biophysica Acta (BBA)-Protein Structure and

Molecular Enzymology, 1597(1), 81-89.

183

Groen, S. C., Whiteman, N. K., Bahrami, A. K., Wilczek, A. M., Cui, J., Russell,

J. A., Cibrian-Jaramillo, A., Butler, I. A., Rana, J. D., Huang, G.-H., Bush,

J., Ausubel, F. M., & Pierce, N. E. (2013). Pathogen-triggered ethylene

signaling mediates systemic-induced susceptibility to herbivory in

Arabidopsis. The Plant Cell, 25(11), 4755-4766.

Groll, M., Schellenberg, B., Bachmann, A. S., Archer, C. R., Huber, R., Powell,

T. K., Lindow, S., Kaiser, M., & Dudler, R. (2008). A plant pathogen

virulence factor inhibits the eukaryotic proteasome by a novel mechanism.

Nature, 452(7188), 755-758.

Gross, D. C., Cody, Y. S., Proebsting Jr, E. L., Radamaker, G. K., & Spotts, R. A.

(1984). Ecotypes and pathogenicity of ice-nucleation-active Pseudomonas

syringae isolated from deciduous fruit tree orchards Phytopathology,

74(2), 241-248.

Großkinsky, D. K., van der Graaff, E., & Roitsch, T. (2014). Abscisic acid–

cytokinin antagonism modulates resistance against Pseudomonas syringae

in tobacco. Phytopathology, 104(12), 1283-1288.

Guenzi, E., Galli, G., Grgurina, I., Gross, D. C., & Grandi, G. (1998).

Characterization of the syringomycin synthetase gene cluster. Journal of

Biological Chemistry, 273(49), 32857-32863.

Hacker, J., & Carniel, E. (2001). Ecological fitness, genomic islands and bacterial

pathogenicity. EMBO Reports, 2(5), 376-381.

Hall, B. G. (2004). Phylogenetic trees made easy: A how-to manual (3rd ed.).

Sunderland, USA: Sinauer Associates, Inc.

Hall, B. H., Cother, E. J., Noble, D., McMahon, R., & Wicks, T. J. (2003). First

report of Pseudomonas syringae on olives (Olea europaea) in South

Australia. Australasian Plant Pathology, 32(1), 119-120.

Hall, B., McMahon, R., Noble, D., Cother, E., & McLintock, D. (2002). First

report of Pseudomonas syringae on grapevines (Vitis vinifera) in South

Australia. Australasian Plant Pathology, 31(4), 421-422.

Hall, S. J., Dry, I. B., Blanchard, C. L., & Whitelaw-Weckert, M. A. (2016).

Plylogenetic relationships of Pseudomonas syringae pv. syringae isolates

associated with bacterial inflorescence rot in grapevine. [Accepted

manuscript: doi: 10.1094/pdis-07-15-0806-re]. Plant Disease, 100(3),

607-616.

Hamiduzzaman, M. M., Jakab, G., Barnavon, L., Neuhaus, J. M., & Mauch-Mani,

B. (2005). β-aminobutyric acid-induced resistance against downy mildew

in grapevine acts through the potentiation of callose formation and

jasmonic acid signaling. Molecular Plant-Microbe Interactions, 18(8),

819-829.

184

Hann, D. R., Domínguez-Ferreras, A., Motyka, V., Dobrev, P. I., Schornack, S.,

Jehle, A., Felix, G., Chinchilla, D., Rathjen, J. P., & Boller, T. (2014). The

Pseudomonas type III effector HopQ1 activates cytokinin signaling and

interferes with plant innate immunity. New Phytologist, 201(2), 585-598.

Hauck, P., Thilmony, R., & He, S. Y. (2003). A Pseudomonas syringae type III

effector suppresses cell wall-based extracellular defense in susceptible

Arabidopsis plants. Proceedings of the National Academy of Sciences,

100(14), 8577-8582.

He, P., Chintamanani, S., Chen, Z., Zhu, L., Kunkel, B. N., Alfano, J. R., Tang,

X., & Zhou, J. M. (2004). Activation of a COI1-dependent pathway in

Arabidopsis by Pseudomonas syringae type III effectors and coronatine.

The Plant Journal, 37(4), 589-602.

He, S. Y. (1996). Elicitation of plant hypersensitive response by bacteria. Plant

Physiology, 112(3), 865-869.

Heeb, S., & Haas, D. (2001). Regulatory roles of the GacS/GacA two-component

system in plant-associated and other Gram-negative bacteria. Molecular

Plant-Microbe Interactions, 14(12), 1351-1363.

Heese, A., Hann, D. R., Gimenez-Ibanez, S., Jones, A. M., He, K., Li, J.,

Schroeder, J. I., Peck, S. C., & Rathjen, J. P. (2007). The receptor-like

kinase SERK3/BAK1 is a central regulator of innate immunity in plants.

Proceedings of the National Academy of Sciences, 104(29), 12217-12222.

Hepler, P. K. (2005). Calcium: A central regulator of plant growth and

development. The Plant Cell, 17, 2142-2155.

Hildebrand, D. (1971). Pectolytic enzymes of Pseudomonas. Paper presented at

the Plant pathogenic bacteria, Proceedings of the 3rd International

Conference on Plant Pathogenic Bacteria. Centre for Agricultural

Publishing and Documentation, Wageningen, The Netherlands.

Hill, T. C. J., Moffett, B. F., DeMott, P. J., Georgakopoulos, D. G., Stump, W. L.,

& Franc, G. D. (2014). Measurement of ice nucleation-active bacteria on

plants and in precipitation by quantitative PCR. Applied and

Environmental Microbiology, 80(4), 1256-1267.

Hirano, S. S., & Upper, C. D. (2000). Bacteria in the leaf ecosystem with

emphasis on Pseudomonas syringae - a pathogen, ice nucleus, and

epiphyte. Microbiology and Molecular Biology Reviews, 64(3), 624-653.

Hirano, S. S., Baker, L. S., & Upper, C. D. (1996). Raindrop momentum triggers

growth of leaf-associated populations of Pseudomonas syringae on field-

grown snap bean plants. Applied and Environmental Microbiology, 62(7),

2560-2566.

185

Hollaway, G. J., & Bretag, T. W. (1997). Survival of Pseudomonas syringae pv.

pisi in soil and on pea trash and their importance as a source of inoculum

for a following field pea crop. Australian Journal of Experimental

Agriculture, 37(3), 369-375.

Howden, A. J. M., Rico, A., Mentlak, T., Miguet, L., & Preston, G. M. (2009).

Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-

acetonitrile to the plant hormone indole-3-acetic acid. Molecular Plant

Pathology, 10(6), 857-865.

Hutchison, M. L., & Gross, D. C. (1997). Lipopeptide phytotoxins produced by

Pseudomonas syringae pv. syringae: Comparison of the biosurfactant and

ion channel-forming activities of syringopeptin and syringomycin.

Molecular Plant-Microbe Interactions, 10(3), 347-354.

.Hwang, M. S. H., Morgan, R. L., Sarkar, S. F., Wang, P. W., & Guttman, D. S.

(2005). Phylogenetic characterization of virulence and resistance

phenotypes of Pseudomonas syringae. Applied and Environmental

Microbiology, 71(9), 5182-5191.

Iacobellis, N. S., Lavermicocca, P., Grgurina, I., Simmaco, M., & Ballio, A.

(1992). Phytotoxic properties of Pseudomonas syringae pv. syringae

toxins. Physiological and Molecular Plant Pathology, 40(2), 107-116.

Ichinose, Y., Shimizu, R., Ikeda, Y., Taguchi, F., Marutani, M., Mukaihara, T.,

Inagaki, Y., Toyoda, K., & Shiraishi, T. (2003). Need for flagella for

complete virulence of Pseudomonas syringae pv. tabaci: Genetic analysis

with flagella-defective mutants ΔfliC and ΔfliD in host tobacco plants.

Journal of General Plant Pathology, 69(4), 244-249.

Igari, K., Endo, S., Hibara, K.-i., Aida, M., Sakakibara, H., Kawasaki, T., &

Tasaka, M. (2008). Constitutive activation of a CC-NB-LRR protein alters

morphogenesis through the cytokinin pathway in Arabidopsis. The Plant

Journal, 55(1), 14-27.

Imker, H. J., Walsh, C. T., & Wuest, W. M. (2009). SylC catalyzes ureido-bond

formation during biosynthesis of the proteasome inhibitor syringolin A.

Journal of the American Chemical Society, 131(51), 18263-18265.

Innes, R. W., Bent, A. F., Kunkel, B. N., Bisgrove, S. R., & Staskawicz, B. J.

(1993). Molecular analysis of avirulence gene avrRpt2 and identification

of a putative regulatory sequence common to all known Pseudomonas

syringae avirulence genes. Journal of Bacteriology, 175(15), 4859-4869.

Irving, H. R., Gehring, C. A., & Parish, R. W. (1992). Changes in cytosolic pH

and calcium of guard cells precede stomatal movements. Proceedings of

the National Academy of Sciences, 89(5), 1790-1794.

186

Irwin, J., Singh, S., Kochman, J., & Murray, G. (1999). Pathogen risks associated

with bulk maize imports to Australia from the United States of America.

Retrieved from

http://www.daff.gov.au/__data/assets/pdf_file/0013/21901/TWGP_3.pdf.

Jabs, T., Tschöpe, M., Colling, C., Hahlbrock, K., & Scheel, D. (1997). Elicitor-

stimulated ion fluxes and O2− from the oxidative burst are essential

components in triggering defense gene activation and phytoalexin

synthesis in parsley. Proceedings of the National Academy of Sciences,

94(9), 4800-4805.

Jacobs, A. K., Dry, I. B., & Robinson, S. P. (1999). Induction of different

pathogenesis-related cDNAs in grapevine infected with powdery mildew

and treated with ethephon. Plant Pathology, 48(3), 325-336.

Jakobek, J. L., & Lindgren, P. B. (1993). Generalized induction of defense

responses in bean is not correlated with the induction of the hypersensitive

reaction. The Plant Cell, 5(1), 49-56.

Jasinski, S., Piazza, P., Craft, J., Hay, A., Woolley, L., Rieu, I., Phillips, A.,

Hedden, P., & Tsiantis, M. (2005). KNOX action in Arabidopsis Is

mediated by coordinate regulation of cytokinin and gibberellin activities.

Current Biology, 15(17), 1560-1565.

Jayasankar, S., Li, Z., & Gray, D. J. (2003). Constitutive expression of Vitis

vinifera thaumatin-like protein after in vitro selection and its role in

anthracnose resistance. Functional Plant Biology, 30(11), 1105-1115.

Jeandet, P., Delaunois, B., Conreux, A., Donnez, D., Nuzzo, V., Cordelier, S.,

Clement, C., & Courot, E. (2010). Biosynthesis, metabolism, molecular

engineering, and biological functions of stilbene phytoalexins in plants.

BioFactors (Oxford, England), 36, 331 - 341.

Jeandet, P., Douillet-Breuil, A.-C., Bessis, R., Debord, S., Sbaghi, M., & Adrian,

M. (2002). Phytoalexins from the Vitaceae: biosynthesis, phytoalexin

gene expression in transgenic plants, antifungal activity, and metabolism.

Journal of Agricultural and Food Chemistry, 50(10), 2731-2741.

Joardar, V., Lindeberg, M., Jackson, R. W., Selengut, J., Dodson, R., Brinkac, L.

M., Daugherty, S. C., DeBoy, R., Durkin, A. S., Giglio, M. G., Madupu,

R., Nelson, W. C., Rosovitz, M. J., Sullivan, S., Crabtree, J., Creasy, T.,

Davidsen, T., Haft, D. H., Zafar, N., Zhou, L., Halpin, R., Holley, T.,

Khouri, H., Feldblyum, T., White, O., Fraser, C. M., Chatterjee, A. K.,

Cartinhour, S., Schneider, D. J., Mansfield, J., Collmer, A., & Buell, C. R.

(2005). Whole-genome sequence analysis of Pseudomonas syringae pv.

phaseolicola 1448A reveals divergence among pathovars in genes

involved in virulence and transposition. Journal of Bacteriology, 187(18),

6488-6498.

Jones, A. (1971). Bacterial canker of sweet cherry in Michigan. Plant Disease

Reporter, 55(11), 961-965.

187

Jones, J. D. G., & Dangl, J. L. (2006). The plant immune system. Nature,

444(7117), 323-329.

Jukes, T. H., & Cantor, C. R. (1969). Evolution of protein molecules. In H. N.

Munro (Ed.), Mammalian Protein Metabolism (pp. 21-132). New York:

Academic Press.

Kairu, G. M. (1997). Biochemical and pathogenic differences between Kenyan

and Brazilian isolates of Pseudomonas syringae pv. garcae. Plant

Pathology, 46(2), 239-246.

Kaluzna, M., Ferrante, P., Sobiczewski, P., & Scortichini, M. (2010).

Characterisation and genetic diversity of Pseudomonas syringae from

stone fruites and hazelnut using repetitive-PCR and MLST. Journal of

Plant Pathology, 92(3), 781-787.

Katsir, L., Chung, H. S., Koo, A. J., & Howe, G. A. (2008a). Jasmonate

signaling: a conserved mechanism of hormone sensing. Current opinion

in plant biology, 11(4), 428-435

Katsir, L., Schilmiller, A. L., Staswick, P. E., He, S. Y., & Howe, G. A. (2008b).

COI1 is a critical component of a receptor for jasmonate and the bacterial

virulence factor coronatine. Proceedings of the National Academy of

Sciences, 105(19), 7100-7105.

Keen, N. T. (1990). Gene-for-gene complementarity in plant-pathogen

interactions. Annual Review of Genetics, 24(1), 447-463.

Keller, M., & Koblet, W. (1995). Stress-induced development of inflorescence

necrosis and bunch-stem necrosis in Vitis vinifera L. in response to

environmental and nutritional effects. Vitis, 34(3), 145-150.

Keller, M., Viret, O., & Cole, F. M. (2003). Botrytis cinerea infection in grape

flowers: Defense reaction, latency, and disease expression.

Phytopathology, 93(3), 316-322.

Kelley, B. S., Lee, S. J., Damasceno, C. M. B., Chakravarthy, S., Kim, B. D.,

Martin, G. B., & Rose, J. K. C. (2010). A secreted effector protein (SNE1)

from Phytophthora infestans is a broadly acting suppressor of

programmed cell death. The Plant Journal, 62(3), 357-366.

Kennelly, M. M., Cazorla, F. M., de Vicente, A., Ramos, C., & Sundin, G. W.

(2007). Pseudomonas syringae diseases of fruit trees: Progress toward

understanding and control. Plant Disease, 91(1), 4-17.

Kitten, T., Kinscherf, T. G., McEvoy, J. L., & Willis, D. K. (1998). A newly

identified regulator is required for virulence and toxin production in

Pseudomonas syringae. Molecular Microbiology, 28(5), 917-929.

Klement, Z. (1963). Rapid detection of the pathogenicity of phytopathogenic

Pseudomonads. Nature 199, 299-300.

188

Klingner, A. E., Palleroni, N. J., & Pontis, R. E. (1976). Isolation of

Pseudomonas syringae from lesions on Vitis vinifera. Journal of

Phytopathology, 86(2), 107-116.

Kolodziejek, I., Misas-Villamil, J. C., Kaschani, F., Clerc, J., Gu, C., Krahn, D.,

Niessen, S., Verdoes, M., Willems, L. I., Overkleeft, H. S., Kaiser, M., &

van der Hoorn, R. A. L. (2011). Proteasome activity imaging and profiling

characterizes bacterial effector syringolin A. Plant Physiology, 155(1),

477-489.

Koornneef, A., & Pieterse, C. M. (2008). Cross talk in defense signaling. Plant

Physiology, 146(3), 839-844.

Kortekamp, A. (2006). Expression analysis of defence-related genes in grapevine

leaves after inoculation with a host and a non-host pathogen. Plant

Physiology and Biochemistry, 44(1), 58-67.

Kortekamp, A., Wind, R., & Zvprian, E. (1997). The role of callose deposits

during infection of two downy mildew-tolerant and two-susceptible Vitis

cultivars. Vitis, 36, 103-104.

Kotan, R., & Şahin, F. (2002). First record of bacterial canker caused by

Pseudomonas syringae pv. syringae, on apricot trees in Turkey. Plant

Pathology, 51(6), 798-798.

Kumar, S., Tamura, K., & Nei, M. (2004). MEGA3: Integrated software for

molecular evolutionary genetics analysis and sequence alignment.

Briefings in bioinformatics, 5(2), 150-163.

Kusajima, M., Yasuda, M., Kawashima, A., Nojiri, H., Yamane, H., Nakajima,

M., Akutsu, K., & Nakashita, H. (2010). Suppressive effect of abscisic

acid on systemic acquired resistance in tobacco plants. Journal of General

Plant Pathology, 76(2), 161-167.

Kwaaitaal, M., Huisman, R., Maintz, J., Reinstädler, A., & Panstruga, R. (2011).

Ionotropic glutamate receptor (iGluR)-like channels mediate MAMP-

induced calcium influx in Arabidopsis thaliana. Biochem. J., 440(355-

365).

Lamari, L. (2008). Assess 2.0: Image analysis software for disease quantification:

APS Press.

Lamichhane, J. R., Varvaro, L., Parisi, L., Audergon, J.-M., & Morris, C. E.

(2014). Disease and frost damage of woody plants caused by

Pseudomonas syringae: Seeing the forest for the trees. Adv Agron, 126,

235-295.

Langlois-Meurinne, M., Gachon, C. M. M., & Saindrenan, P. (2005). Pathogen-

responsive expression of glycosyltransferase genes UGT73B3 and

UGT73B5 is necessary for resistance to Pseudomonas syringae pv. tomato

in Arabidopsis. Plant Physiology, 139(4), 1890-1901.

189

Latorre, B. A., & Jones, A. L. (1979). Pseudomonas morsprunorum, the cause of

bacterial canker of sour cherry in Michigan, and its epiphytic association

with P. syringae. Phytopathology, 69(8), 335-339.

Lavermicocca, P., Sante Iacobellis, N., Simmaco, M., & Graniti, A. (1997).

Biological properties and spectrum of activity of Pseudomonas syringae

pv. syringae toxins. Physiological and Molecular Plant Pathology, 50(2),

129-140.

Le Henanff, G., Heitz, T., Mestre, P., Mutterer, J., Walter, B., & Chong, J.

(2009). Characterization of Vitis vinifera NPR1 homologs involved in the

regulation of pathogenesis-related gene expression. BMC Plant Biology,

9, 54.

Leben, C., Schroth, M., & Hildebrand, D. (1970). Colonization and movement of

Pseudomonas syringae on healthy bean seedlings. Phytopathology, 60(4),

677-680.

Lelliott, R. A., & Stead, D. E. (1987). Methods for the diagnosis of bacterial

diseases of plants (Vol. 2). Oxford: Blackwell Publishing Ltd/ British

Society for Plant Plathology.

Lelliott, R. A., Billing, E., & Hayward, A. C. (1966). A determinative scheme for

the fluorescent plant pathogenic Pseudomonads. Journal of Applied

Microbiology, 29(3), 470-489.

Leon-Reyes, A., Du, Y., Koornneef, A., Proietti, S., Korbes, A. P., Memelink, J.,

Pieterse, C. M., & Ritsema, T. (2010). Ethylene signaling renders the

jasmonate response of Arabidopsis insensitive to future suppression by

salicylic acid. Molecular Plant-Microbe Interactions, 23(2), 187-197.

Leon-Reyes, A., Van der Does, D., De Lange, E. S., Delker, C., Wasternack, C.,

Van Wees, S. C., Ritsema, T., & Pieterse, C. M. (2010b). Salicylate-

mediated suppression of jasmonate-responsive gene expression in

Arabidopsis is targeted downstream of the jasmonate biosynthesis

pathway. Planta, 232(6), 1423-1432.

Li, X., Lin, H., Zhang, W., Zou, Y., Zhang, J., Tang, X., & Zhou, J. M. (2005).

Flagellin induces innate immunity in nonhost interactions that is

suppressed by Pseudomonas syringae effectors. Proceedings of the

National Academy of Sciences of the United States of America, 102(36),

12990-12995.

Li, Z. T., Dhekney, S. A., & Gray, D. J. (2011). PR-1 gene family of grapevine: A

uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers

high level resistance to bacterial disease in transgenic tobacco. Plant Cell

Reports, 30(1), 1-11.

Liao, C. H., Hung, H. Y., & Chatterjee, A. K. (1988). An extracellular pectate

lyase is the pathogenicity factor of the soft-rotting bacterium

Pseudomonas viridiflava. Mol Plant-Microbe Interact, 1, 199-206.

190

Liao, C. H., McCallus, D. E., & Fett, W. F. (1994). Molecular characterization of

two gene loci required for production of the key pathogenicity factor

pectate lyase in Pseudomonas viridiflava. Molecular Plant Microbe

Interactions, 7(3), 391-400.

Librado, P., & Rozas, J. (2009). DnaSP v5: A software for comprehensive

analysis of DNA polymorphism data. Bioinformatics, 25(11), 1451-1452.

Lindeberg, M., Cartinhour, S., Myers, C. R., Schechter, L. M., Schneider, D. J., &

Collmer, A. (2006). Closing the circle on the discovery of genes encoding

Hrp regulon members and type III secretion system effectors in the

genomes of three model Pseudomonas syringae strains. Molecular Plant-

Microbe Interactions, 19(11), 1151-1158.

Lindeberg, M., Cunnac, S., & Collmer, A. (2009). The evolution of Pseudomonas

syringae host specificity and type III effector repertoires. Molecular Plant

Pathology, 10(6), 767-775.

Lindeberg, M., Cunnac, S., & Collmer, A. (2012). Pseudomonas syringae type III

effector repertoires: Last words in endless arguments. Trends in

Microbiology, 20(4), 199-208.

Lindow, S. E., Arny, D. C., & Upper, C. D. (1978). Distribution of ice

nucleation-active bacteria on plants in nature. Applied and environmental

microbiology, 36(6), 831-838.

Lindow, S. E., Arny, D. C., & Upper, C. D. (1982). Bacterial ice nucleation: a

factor in frost injury to plants. Plant Physiology, 70(4), 1084-1089.

Little, E. L., Bostock, R. M., & Kirkpatrick, B. C. (1998). Genetic

characterization of Pseudomonas syringae pv. syringae strains from stone

fruits in California. Applied and Environmental Microbiology, 64(10),

3818-3823.

Liu, R., Wang, L., Zhu, J., Chen, T., Wang, Y. H., & Xu, Y. (2015). Histological

responses to downy mildew in resistant and susceptible grapevines.

Protoplasma, 252(1), 259-270.

Liu, Y., & Zhang, S. (2004). Phosphorylation of 1-aminocyclopropane-1-

carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated

protein kinase, induces ethylene biosynthesis in Arabidopsis. The Plant

Cell Online, 16(12), 3386-3399.

Liu, Y., Wang, L., Cai, G., Jiang, S., Sun, L., & Li, D. (2013). Response of

tobacco to the Pseudomonas syringae pv. tomato DC3000 is mainly

dependent on salicylic acid signaling pathway. FEMS Microbiology

Letters, 344(1), 77-85.

Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression

data using real-time quantitative PCR and the 2−ΔΔCT

method. Methods,

25(4), 402-408.

191

Lorenzo, O., Chico, J. M., Sánchez-Serrano, J. J., & Solano, R. (2004).

Jasmonate-insensitive 1 encodes a MYC transcription factor essential to

discriminate between different jasmonate-regulated defense responses in

Arabidopsis. The Plant Cell Online, 16(7), 1938-1950.

Lu, D., Lin, W., Gao, X., Wu, S., Cheng, C., Avila, J., Heese, A., Devarenne, T.

P., He, P., & Shan, L. (2011). Direct ubiquitination of pattern recognition

receptor FLS2 attenuates plant innate immunity. Science, 332(6036),

1439-1442.

Lu, S. E., Scholz-Schroeder, B. K., & Gross, D. C. (2002). Characterization of the

salA, syrF, and syrG regulatory genes located at the right border of the

syringomycin gene cluster of Pseudomonas syringae pv. syringae.

Molecular Plant-Microbe Interactions, 15(1), 43-53.

Lu, S.-E., Soule, J. D., & Gross, D. C. (2003). Characterization of the argA gene

required for arginine biosynthesis and syringomycin production by

Pseudomonas syringae pv. syringae. Applied and Environmental

Microbiology, 69(12), 7273-7280.

Ludwig, A. A., Saitoh, H., Felix, G., Freymark, G., Miersch, O., Wasternack, C.,

Boller, T., Jones, J. D., & Romeis, T. (2005). Ethylene-mediated cross-

talk between calcium-dependent protein kinase and MAPK signaling

controls stress responses in plants. Proceedings of the National academy

of Sciences of the United States of America, 102(30), 10736-10741.

Luna, E., Pastor, V., Robert, J., Flors, V., Mauch-Mani, B., & Ton, J. (2010).

Callose deposition: A multifaceted plant defense response. Molecular

Plant-Microbe Interactions, 24(2), 183-193.

MacKenzie, D. J., McLean, M. A., Mukerji, S., & Green, M. (1997). Improved

RNA extraction from woody plants for the detection of viral pathogens by

reverse transcription-polymerase chain reaction. Plant Disease, 81(2),

222-226.

Maki, L. R., Galyan, E. L., Chang-Chien, M.-M., & Caldwell, D. R. (1974). Ice

nucleation induced by Pseudomonas syringae. Applied Microbiology,

28(3), 456-459.

Malinovsky, F. G., Fangel, J. U., & Willats, W. G. T. (2014). The role of the cell

wall in plant immunity. Frontiers in Plant Science, 5, 1-12.

Mammarella, N. D., Cheng, Z., Fu, Z. Q., Daudi, A., Bolwell, G. P., Dong, X., &

Ausubel, F. M. (2014). Apoplastic peroxidases are required for salicylic

acid-mediated defense against Pseudomonas syringae. Phytochemistry,

112, 110-121.

Mansvelt, E. L., & Hattingh, M. J. (1987). Scanning electron microscopy of

colonization of pear leaves by Pseudomonas syringae pv. syringae.

Canadian Journal of Botany, 65(12), 2517-2522.

192

Mansvelt, E. L., & Hattingh, M. J. (1989). Scanning electron microscopy of

invasion of apple leaves and blossoms by Pseudomonas syringae pv.

syringae. Applied and Environmental Microbiology, 55(2), 533-538.

Marco, M. L., Legac, J., & Lindow, S. E. (2005). Pseudomonas syringae genes

induced during colonization of leaf surfaces. Environmental

Microbiology, 7(9), 1379-1391.

Marín‐Rodríguez, M. C., Orchard, J., & Seymour, G. B. (2002). Pectate lyases,

cell wall degradation and fruit softening. Journal of Experimental Botany,

53(377), 2115-2119.

Martín-Sanz, A., de la Vega, M. P., Murillo, J., & Caminero, C. (2013). Strains of

Pseudomonas syringae pv. syringae from pea are phylogenetically and

pathogenically diverse. Phytopathology, 103(7), 673-681.

McCann, H. C., Rikkerink, E. H. A., Bertels, F., Fiers, M., Lu, A., Rees-George,

J., Andersen, M. T., Gleave, A. P., Haubold, B., Wohlers, M. W.,

Guttman, D. S., Wang, P. W., Straub, C., Vanneste, J., Rainey, P. B., &

Templeton, M. D. (2013). Genomic analysis of the kiwifruit pathogen

Pseudomonas syringae pv. actinidiae provides insight into the origins of

an emergent plant disease. PLoS Pathogens, 9(7), e1003503.

McCormick, K. M., & Hollaway, G. J. (1999). First report of bacterial blight in

fenugreek (Trigonella foenum-graecum) caused by Pseudomonas syringae

pv. syringae. Australasian Plant Pathology, 28(4), 338-338.

McHale, L., Tan, X., Koehl, P., & Michelmore, R. W. (2006). Plant NBS-LRR

proteins: Adaptable guards. Genome Biol 7(4), 212.

Melotto, M., Underwood, W., Koczan, J., Nomura, K., & He, S. Y. (2006). Plant

stomata function in innate immunity against bacterial invasion. Cell,

126(5), 969-980.

Meyers, B. C., Kozik, A., Griego, A., Kuang, H., & Michelmore, R. W. (2003).

Genome-wide analysis of NBS-LRR–encoding genes in Arabidopsis. The

Plant Cell, 15(4), 809-834.

Michel, K., Abderhalden, O., Bruggmann, R., & Dudler, R. (2006).

Transcriptional changes in powdery mildew infected wheat and

Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell

death at infection sites. Plant Molecular Biology, 62(4-5), 561-578.

Milli, A., Cecconi, D., Bortesi, L., Persi, A., Rinalducci, S., Zamboni, A.,

Zoccatelli, G., Lovato, A., Zolla, L., & Polverari, A. (2012). Proteomic

analysis of the compatible interaction between Vitis vinifera and

Plasmopara viticola. Journal of Proteomics, 75(4), 1284-1302.

193

Misas-Villamil, J. C., Kolodziejek, I., Crabill, E., Kaschani, F., Niessen, S.,

Shindo, T., Kaiser, M., Alfano, J. R., & van der Hoorn, R. A. L. (2013).

Pseudomonas syringae pv. syringae uses proteasome inhibitor syringolin

A to colonize from wound infection sites. PLoS Pathogens, 9(3),

e1003281.

Mo, Y. Y., & Gross, D. C. (1991). Plant signal molecules activate the syrB gene,

which is required for syringomycin production by Pseudomonas syringae

pv. syringae. Journal of Bacteriology, 173(18), 5784-5792.

Mohr, P. G., & Cahill, D. M. (2003). Abscisic acid influences the susceptibility of

Arabidopsis thaliana to Pseudomonas syringae pv. tomato and

Peronospora parasitica. Functional Plant Biology, 30(4), 461-469.

Mohr, P. G., & Cahill, D. M. (2007). Suppression by ABA of salicylic acid and

lignin accumulation and the expression of multiple genes, in Arabidopsis

infected with Pseudomonas syringae pv. tomato. Funct Integr Genomics,

7(3), 181-191.

Mohr, T. J., Liu, H., Yan, S., Morris, C. E., Castillo, J. A., Jelenska, J., &

Vinatzer, B. A. (2008). Naturally occurring nonpathogenic isolates of the

plant pathogen Pseudomonas syringae lack a type iii secretion system and

effector gene orthologues. Journal of Bacteriology, 190(8), 2858-2870.

Mollet, C., Drancourt, M., & Raoult, D. (1997). rpoB sequence analysis as a

novel basis for bacterial identification. Molecular Microbiology, 26(5),

1005-1011.

Monteil, C. L., Bardin, M., & Morris, C. E. (2014). Features of air masses

associated with the deposition of Pseudomonas syringae and Botrytis

cinerea by rain and snowfall. The ISME Journal, 8(11), 2290-2304.

Monteiro, S., Barakat, M., Piçarra-Pereira, M. A., Teixeira, A. R., & Ferreira, R.

B. (2003). Osmotin and thaumatin from grape: A putative general defense

mechanism against pathogenic fungi. Phytopathology, 93(12), 1505-1512.

Moragrega, C., Llorente, I., Manceau, C., & Montesinos, E. (2003). Susceptibility

of European pear cultivars to Pseudomonas syringae pv. syringae using

immature fruit and detached leaf assays. European Journal of Plant

Pathology, 109(4), 319-326.

Morris, C. E., Monteil, C. L., & Berge, O. (2013). The life history of

Pseudomonas syringae: Linking agriculture to earth system processes.

Annual Review of Phytopathology, 51(1), 85-104.

Morris, C. E., Sands, D. C., Vanneste, J. L., Montarry, J., Oakley, B., Guilbaud,

C., & Glaux, C. (2010). Inferring the evolutionary history of the plant

pathogen Pseudomonas syringae from its biogeography in headwaters of

rivers in North America, Europe, and New Zealand. mBio, 1(3), e00107-

00110.

194

Morris, C. E., Sands, D. C., Vinatzer, B. A., Glaux, C., Guilbaud, C., Buffière,

A., Yan, S., Dominguez, H., & Thompson, B. M. (2008). The life history

of the plant pathogen Pseudomonas syringae is linked to the water cycle.

The ISME Journal, 2(3), 321-334.

Morris, C.E., Kinkel, L.L., Xiao, K., Prior, P., & Sands, D.C. (2007). Surprising

niche for the plant pathogen Pseudomonas syringae. Infection, Genetics

and Evolution, 7(1), 84-92.

Müller, B., & Sheen, J. (2007). Arabidopsis cytokinin signaling pathway. Science

Signaling, 2007(407), cm5-cm5.

Mur, L. A. J., Brown, I. R., Darby, R. M., Bestwick, C. S., Bi, Y.-M., Mansfield,

J. W., & Draper, J. (2000). A loss of resistance to avirulent bacterial

pathogens in tobacco is associated with the attenuation of a salicylic acid-

potentiated oxidative burst. The Plant Journal, 23(5), 609-621.

Mur, L. A. J., Kenton, P., Atzorn, R., Miersch, O., & Wasternack, C. (2006). The

outcomes of concentration-specific interactions between salicylate and

jasmonate signaling include synergy, antagonism, and oxidative stress

leading to cell death. Plant Physiology, 140(1), 249-262.

Mur, L. A., Kenton, P., & Draper, J. (2005). In planta measurements of oxidative

bursts elicited by avirulent and virulent bacterial pathogens suggests that

H2O2 is insufficient to elicit cell death in tobacco. Plant, Cell &

Environment, 28(4), 548-561.

Murray, G., & Brennan, J. (2009). Estimating disease losses to the Australian

wheat industry. Australasian Plant Pathology, 38(6), 558-570.

Naito, K., Ishiga, Y., Toyoda, K., Shiraishi, T., & Ichinose, Y. (2007). N-terminal

domain including conserved flg22 is required for flagellin-induced

hypersensitive cell death in Arabidopsis thaliana. Journal of General

Plant Pathology, 73(4), 281-285.

Najafi, P. H. G., & Taghavi, S. M. (2014). Discrimination of Pseudomonas

syringae pv. syringae isolates from different hosts in Iran using

pathogenicity tests and RAPD. International Journal of AgriScience, 4(1),

16-27.

Naseem, M., Kaltdorf, M., Hussain, A., & Dandekar, T. (2013). The impact of

cytokinin on jasmonate-salicylate antagonism in Arabidopsis immunity

against infection with Pst DC3000. Plant Signaling & Behavior, 8(10),

e26791.

Natalini, E., Rossi, M., Barionovi, D., & Scortichini, M. (2006). Genetic and

pathogenic diversity of Pseudomonas syringae pv. syringae isolates

associated with bud necrosis and leaf spot of pear in a single orchard.

Journal of Plant Pathology, 88(2), 219-223.

195

Navarro, L., Bari, R., Achard, P., Lisón, P., Nemri, A., Harberd, N. P., & Jones, J.

D. G. (2008). DELLAs control plant immune responses by modulating the

balance of jasmonic acid and salicylic acid signaling. Current Biology,

18(9), 650-655.

Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M.,

Voinnet, O., & Jones, J. D. G. (2006). A plant miRNA contributes to

antibacterial resistance by repressing auxin signaling. Science, 312(5772),

436-439.

Navarro, L., Zipfel, C., Rowland, O., Keller, I., Robatzek, S., Boller, T., & Jones,

J. D. G. (2004). The transcriptional innate immune response to flg22:

Interplay and overlap with Avr gene-dependent defense responses and

bacterial pathogenesis. Plant Physiology, 135(2), 1113-1128.

Neu, H. C. (1992). The crisis in antibiotic resistance. Science, 257(5073), 1064-

1073.

Noble, D. H., Cother, E. J., Hailstones, D. L., Flack, M., Oxspring, L., & Hall, B.

(2006). Characterisation of Pseudomonas syringae strains associated with

a leaf disease of leek in Australia. European Journal of Plant Pathology,

115(4), 419-430.

Nomura, K., Melotto, M., & He, S.-Y. (2005). Suppression of host defense in

compatible plant–Pseudomonas syringae interactions. Current opinion in

plant biology, 8(4), 361-368.

Nühse, T. S., Bottrill, A. R., Jones, A. M., & Peck, S. C. (2007). Quantitative

phosphoproteomic analysis of plasma membrane proteins reveals

regulatory mechanisms of plant innate immune responses. The Plant

Journal, 51(5), 931-940.

O’Brien, H. E., Desveaux, D., & Guttman, D. S. (2011). Next-generation

genomics of Pseudomonas syringae. Current Opinion in Microbiology,

14(1), 24-30.

O'Brien, H., Thakur, S., Gong, Y., Fung, P., Zhang, J., Yuan, L., Wang, P., Yong,

C., Scortichini, M., & Guttman, D. (2012). Extensive remodeling of the

Pseudomonas syringae pv. avellanae type III secretome associated with

two independent host shifts onto hazelnut. BMC microbiology, 12(1), 141.

O'Donnell, P. J., Schmelz, E. A., Moussatche, P., Lund, S. T., Jones, J. B., &

Klee, H. J. (2003). Susceptible to intolerance – a range of hormonal

actions in a susceptible Arabidopsis pathogen response. The Plant

Journal, 33(2), 245-257.

Palmieri, M. C., Perazzolli, M., Matafora, V., Moretto, M., Bachi, A., & Pertot, I.

(2012). Proteomic analysis of grapevine resistance induced by

Trichoderma harzianum T39 reveals specific defence pathways activated

against downy mildew. Journal of Experimental Botany, 63(17), 6237-

6251.

196

Pattemore, D. E., Goodwin, R. M., McBrydie, H. M., Hoyte, S. M., & Vanneste,

J. L. (2014). Evidence of the role of honey bees (Apis mellifera) as vectors

of the bacterial plant pathogen Pseudomonas syringae. Australasian Plant

Pathology, 1-5.

Penninckx, I. A. M. A., Thomma, B. P. H. J., Buchala, A., Métraux, J.-P., &

Broekaert, W. F. (1998). Concomitant activation of jasmonate and

ethylene response pathways is required for induction of a plant defensin

gene in Arabidopsis. The Plant Cell Online, 10(12), 2103-2113.

Peters, B. J., Ash, G. J., Cother, E. J., Hailstones, D. L., Noble, D. H., & Urwin,

N. A. R. (2004). Pseudomonas syringae pv. maculicola in Australia:

Pathogenic, phenotypic and genetic diversity. Plant Pathology, 53(1), 73-

79.

Petersen, M., Brodersen, P., Naested, H., Andreasson, E., Lindhart, U., Johansen,

B., Nielsen, H. B., Lacy, M., Austin, M. J., Parker, J. E., Sharma, S. B.,

Klessig, D. F., Martienssen, R., Mattsson, O., Jensen, A. B., & Mundy, J.

(2000). Arabidopsis MAP kinase 4 negatively regulates systemic acquired

resistance. Cell, 103(7), 1111-1120.

Pezet, R., Perret, C., Jean-Denis, J., Tabacchi, R., Gindro, K., & Viret, O. (2003).

Delta-viniferin, a resveratrol dehydrodimer: One of the major stilbenes

synthesized by grapevine leaves. J Agric Food Chem, 51, 5488-5492.

Pham, J., & Desikan, R. (2012). Modulation of ROS production and hormone

levels by AHK5 during abiotic and biotic stress signaling. Plant Signaling

& Behavior, 7(8), 893-897.

Pieterse, C. M. J., Pierik, R., & Van Wees, S. C. M. (2014). Different shades of

JAZ during plant growth and defense. New Phytologist, 204(2), 261-264.

Pieterse, C. M. J., Van der Does, D., Zamioudis, C., Leon-Reyes, A., & Van

Wees, S. C. M. (2012). Hormonal modulation of plant immunity. Annual

Review of Cell and Developmental Biology, 28(1), 489-521.

Pirrung, M. C., Biswas, G., & Ibarra-Rivera, T. R. (2010). Total synthesis of

syringolin A and B. Organic Letters, 12(10), 2402-2405.

Plant Health Australia. (2013). Notifiable plant pests from Australian state and

territory legislation. Retrieved from

http://www.planthealthaustralia.com.au/wp-

content/uploads/2013/06/Notifiable-plant-pests-from-Australian-state-

and-territory-legislation-May-2013.pdf.

Prokič, A., Gašič, K., Ivanovič, M., Kuzmanovič, N., Ševič, M., Pulawska, J., &

Obradovič, A. (2012). Detection and identification methods and new tests

as developed and used in the framework of cost873 for bacteria

pathogenic to stone fruits and nuts. Journal of Plant Pathology, 94(1), S1.

127-S121. 133.

197

Qi, M., Wang, D., Bradley, C. A., & Zhao, Y. (2011). Genome sequence analyses

of Pseudomonas savastanoi pv. glycinea and subtractive hybridization-

based comparative genomics with nine Pseudomonads. PloS One, 6(1),

e16451.

Quigley, N. B., & Gross, D. C. (1994). Syringomycin production among strains

of Pseudomonas syringae pv. syringae: conservation of the syrB and syrD

genes and activation of phytotoxin production in plant signal molecules.

Molecular Plant-Microbe Interactions, 7, 78-90.

Ramel, C., Baechler, N., Hildbrand, M., Meyer, M., Schädeli, D., & Dudler, R.

(2012). Regulation of biosynthesis of syringolin A, a Pseudomonas

syringae virulence factor targeting the host proteasome. Molecular Plant-

Microbe Interactions, 25(9), 1198-1208.

Ramel, C., Tobler, M., Meyer, M., Bigler, L., Ebert, M.O., Schellenberg, B., &

Dudler, R. (2009). Biosynthesis of the proteasome inhibitor syringolin A:

The ureido group joining two amino acids originates from bicarbonate.

BMC Biochemistry, 10(1), 26.

Randhawa, P., & Civerolo, E. (1985). A detached-leaf bioassay for Xanthomonas

campestris pv. pruni. Phytopathology, 75(9), 1060-1063.

Rasmussen, J. B., Hammerschmidt, R., & Zook, M. N. (1991). Systemic

induction of salicylic acid accumulation in cucumber after inoculation

with Pseudomonas syringae pv. syringae. Plant Physiology, 97(4), 1342-

1347.

Reuveni, M. (1998). Relationships between leaf age, peroxidase and β-1,3-

giucanase activity, and resistance to downy mildew in grapevines. Journal

of Phytopathology, 146(10), 525-530.

Richardson, H. J., & Hollaway, G. J. (2011). Bacterial blight caused by

Pseudomonas syringae pv. syringae shown to be an important disease of

field pea in south eastern Australia. Australasian Plant Pathology, 40(3),

260-268.

Robert, N., Ferran, J., Breda, C., Coutos-Thévenot, P., Boulay, M., Buffard, D.,

& Esnault, R. (2001). Molecular characterization of the incompatible

interaction of Vitis Vinifera leaves with Pseudomonas syringae pv. pisi:

Expression of genes coding for stilbene synthase and class 10 PR protein.

European Journal of Plant Pathology, 107(2), 249-261.

Robert-Seilaniantz, A., Grant, M., & Jones, J. D. (2010). Hormone crosstalk in

plant disease and defense: More than just jasmonate-salicylate

antagonism. Annual Review of Phytopathology.

Robert-Seilaniantz, A., Navarro, L., Bari, R., & Jones, J. D. G. (2007).

Pathological hormone imbalances. Current Opinion In Plant Biology,

10(4), 372-379.

198

Rozhavin, M. A. (1983). Biological properties of melanin (Pseudomonas

aeruginosa). Zhurnal mikrobiologii, epidemiologii, i immunobiologii(1),

45-47.

Saitou, N., & Nei, M. (1987). The neighbour-joining method: A new method for

reconstructing phylogenetic trees. Molecular biology and evolution, 4,

406 - 425.

Samedov, A. N., Mogilevskaya, M. I., Karagezov, T. G., Khudaverdieva, S. R., &

Aliev, L. A. (1988). Detection of the causal agents of grape bacteriosis in

Aspheron (Azebaijan SSR, USSR). Izvestiya Akademii Nauk

Azerbaidzhanskoi SSP Senya Bioloicheskikh Nauki, 6, 18-23.

Sarkar, S. F., & Guttman, D. S. (2004). Evolution of the core genome of

Pseudomonas syringae, a highly clonal, endemic plant pathogen. Applied

and Environmental Microbiology, 70(4), 1999-2012.

Sarkar, S. F., Gordon, J. S., Martin, G. B., & Guttman, D. S. (2006). Comparative

genomics of host-specific virulence in Pseudomonas syringae. Genetics,

174(2), 1041-1056.

Sasaki‐Sekimoto, Y., Taki, N., Obayashi, T., Aono, M., Matsumoto, F., Sakurai,

N., Suzuki, H., Hirai, M. Y., Noji, M., & Saito, K. (2005). Coordinated

activation of metabolic pathways for antioxidants and defence compounds

by jasmonates and their roles in stress tolerance in Arabidopsis. The Plant

Journal, 44(4), 653-668.

Sawada, H., Suzuki, F., Matsuda, I., & Saitou, N. (1999). Phylogenetic analysis

of Pseudomonas syringae pathovars suggests the horizontal gene transfer

of argK and the evolutionary stability of hrp gene cluster. Journal of

Molecular Evolution, 49(5), 627-644.

Schellenberg, B., Ramel, C., & Dudler, R. (2010). Pseudomonas syringae

virulence factor syringolin A counteracts stomatal immunity by

proteasome inhibition. Molecular Plant-Microbe Interactions, 23(10),

1287-1293.

Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the

comparative CT method. Nature Protocols, 3(6), 1101-1108.

Scholz-Schroeder, B. K., Hutchison, M. L., Grgurina, I., & Gross, D. C. (2001).

The contribution of syringopeptin and syringomycin to virulence of

Pseudomonas syringae pv. syringae strain B301D on the basis of sypA

and syrB1 biosynthesis mutant analysis. Molecular Plant-Microbe

Interactions, 14(3), 336-348.

Scholz-Schroeder, B. K., Soule, J. D., & Gross, D. C. (2003). The sypA, sypS, and

sypC synthetase genes encode twenty-two modules involved in the

nonribosomal peptide synthesis of syringopeptin by Pseudomonas

syringae pv. syringae B301D. Molecular Plant-Microbe Interactions,

16(4), 271-280.

199

Schulze-Lefert, P., & Robatzek, S. (2006). Plant pathogens trick guard cells into

opening the gates. Cell, 126(5), 831-834.

Scortichini, M. (2002). Bacterial canker and decline of European hazelnut. Plant

Disease, 86(7), 704-709.

Scortichini, M., Marchesi, U., Dettori, M. T., & Rossi, M. P. (2003). Genetic

diversity, presence of the syrB gene, host preference and virulence of

Pseudomonas syringae pv. syringae strains from woody and herbaceous

host plants. Plant Pathology, 52(3), 277-286.

Scortichini, M., Rossi, M. P., Loreti, S., Bosco, A., Fiori, M., Jackson, R. W.,

Stead, D. E., Aspin, A., Marchesi, U., Zini, M., & Janse, J. D. (2005).

Pseudomonas syringae pv. coryli, the causal agent of bacterial twig

dieback of Corylus avellana. Phytopathology, 95(11), 1316-1324.

Segre, A., Bachmann, R. C., Ballio, A., Bossa, F., Grgurina, I., Iacobellis, N. S.,

Marino, G., Pucci, P., Simmaco, M., & Takemoto, J. Y. (1989). The

structure of syringomycins A1, E and G. FEBS letters, 255(1), 27-31.

Seo, H. S., Song, J. T., Cheong, J.-J., Lee, Y.-H., Lee, Y.-W., Hwang, I., Lee, J.

S., & Choi, Y. D. (2001). Jasmonic acid carboxyl methyltransferase: A

key enzyme for jasmonate-regulated plant responses. Proceedings of the

National Academy of Sciences, 98(8), 4788-4793.

Shapiro, A. D., & Zhang, C. (2001). The role of NDR1 in avirulence gene-

directed signaling and control of programmed cell death in Arabidopsis.

Plant Physiology, 127(3), 1089-1101.

Sheard, L. B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T. R.,

Kobayashi, Y., Hsu, F.-F., Sharon, M., Browse, J., He, S. Y., Rizo, J.,

Howe, G. A., & Zheng, N. (2010). Jasmonate perception by inositol

phosphate-potentiated COI1-JAZ co-receptor. Nature, 468(7322), 400-

405.

Shevchik, V. E., Boccara, M., Vedel, R., & Hugouvieux-Cotte-Pattat, N. (1998).

Processing of the pectate lyase pelI by extracellular proteases of Erwinia

chrysanthemi 3937. Molecular Microbiology, 29(6), 1459-1469.

Sorensen, K. N., Kim, K. H., & Takemoto, J. Y. (1996). In vitro antifungal and

fungicidal activities and erythrocyte toxicities of cyclic

lipodepsinonapeptides produced by Pseudomonas syringae pv. syringae.

Antimicrobial Agents and Chemotherapy, 40(12), 2710-2713.

Sørensen, S. O., Pauly, M., Bush, M., Skjøt, M., McCann, M. C., Borkhardt, B.,

& Ulvskov, P. (2000). Pectin engineering: Modification of potato pectin

by in vivo expression of an endo-1,4-β-D-galactanase. Proceedings of the

National Academy of Sciences, 97(13), 7639-7644.

200

Spoel, S. H., Koornneef, A., Claessens, S. M. C., Korzelius, J. P., Van Pelt, J. A.,

Mueller, M. J., Buchala, A. J., Métraux, J. P., Brown, R., Kazan, K., Van

Loon, L. C., Dong, X., & Pieterse, C. M. J. (2003). NPR1 modulates

cross-talk between salicylate- and jasmonate-dependent defense pathways

through a novel function in the cytosol. The Plant Cell Online, 15(3), 760-

770.

Spoel, S. H., Mou, Z., Tada, Y., Spivey, N. W., Genschik, P., & Dong, X. (2009).

Proteasome-mediated turnover of the transcription coactivator NPR1

plays dual roles in regulating plant immunity. Cell, 137(5), 860-872.

Stachelhaus, T., & Marahiel, M. A. (1995). Modular structure of genes encoding

multifunctional peptide synthetases required for non-ribosomal peptide

synthesis. FEMS Microbiology Letters, 125(1), 3-14.

Stein, T., & Vater, J. (1996). Amino acid activation and polymerization at

modular multienzymes in nonribosomal peptide biosynthesis. Amino

Acids, 10(3), 201-227.

Studholme, D. J. (2011). Application of high-throughput genome sequencing to

intrapathovar variation in Pseudomonas syringae. Molecular Plant

Pathology, 12(8), 829-838.

Suhita, D., Raghavendra, A. S., Kwak, J. M., & Vavasseur, A. (2004).

Cytoplasmic alkalization precedes reactive oxygen species production

during methyl jasmonate- and abscisic acid-induced stomatal closure.

Plant Physiology, 134(4), 1536-1545.

Summermatter, K., Sticher, L., & Metraux, J. P. (1995). Systemic responses in

Arabidopsis thaliana infected and challenged with Pseudomonas syringae

pv. syringae. Plant Physiology, 108(4), 1379-1385.

Sun, H., Hu, X., Ma, J., Hettenhausen, C., Wang, L., Sun, G., Wu, J., & Wu, J.

(2014). Requirement of ABA signalling-mediated stomatal closure for

resistance of wild tobacco to Alternaria alternata. Plant Pathology, 63(5),

1070-1077.

Sun, T.-p., & Gubler, F. (2004). Molecular mechanism of gibberellin signaling in

plants. Annual Review of Plant Biology, 55(1), 197-223.

Sundin, G. W., & Bender, C. L. (1993). Ecological and genetic analysis of copper

and streptomycin resistance in Pseudomonas syringae pv. syringae.

Applied and Environmental Microbiology, 59(4), 1018-1024.

Taguchi, F., Takeuchi, K., Katoh, E., Murata, K., Suzuki, T., Marutani, M.,

Kawasaki, T., Eguchi, M., Katoh, S., & Kaku, H. (2006). Identification of

glycosylation genes and glycosylated amino acids of flagellin in

Pseudomonas syringae pv. tabaci. Cellular Microbiology, 8(6), 923-938.

201

Taguchi, F., Yamamoto, M., Ohnishi-Kameyama, M., Iwaki, M., Yoshida, M.,

Ishii, T., Konishi, T., & Ichinose, Y. (2010). Defects in flagellin

glycosylation affect the virulence of Pseudomonas syringae pv. tabaci

6605. Microbiology, 156(1), 72-80.

Tamura, K., Nei, M., & Kumar, S. (2004). Prospects for inferring very large

phylogenies by using the neighbor-joining method. Proceedings of the

National Academy of Sciences of the United States of America, 101(30),

11030-11035.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011).

MEGA5: Molecular evolutionary genetics analysis using maximum

likelihood, evolutionary distance, and maximum parsimony methods.

Molecular biology and evolution, 28, 2731 - 2739.

Tanaka, N., Che, F.-S., Watanabe, N., Fujiwara, S., Takayama, S., & Isogai, A.

(2003). Flagellin from an incompatible strain of Acidovorax avenae

mediates H2O2 generation accompanying hypersensitive cell death and

expression of PAL, Cht-1, and PBZ1, but not of LOX in rice. Molecular

Plant-Microbe Interactions, 16(5), 422-428.

Tanaka, Y., Sano, T., Tamaoki, M., Nakajima, N., Kondo, N., & Hasezawa, S.

(2006). Cytokinin and auxin inhibit abscisic acid-induced stomatal closure

by enhancing ethylene production in Arabidopsis. Journal of

Experimental Botany, 57(10), 2259-2266.

Tayeb, L. A., Ageron, E., Grimont, F., & Grimont, P. A. D. (2005). Molecular

phylogeny of the genus Pseudomonas based on rpoB sequences and

application for the identification of isolates. Research in Microbiology,

156(5–6), 763-773.

Thakur, P. B., Vaughn-Diaz, V. L., Greenwald, J. W., & Gross, D. C. (2013).

Characterization of five ECF sigma factors in the genome of

Pseudomonas syringae pv. syringae B728a. PloS One, 8(3), e58846.

Thilmony, R., Underwood, W., & He, S. Y. (2006). Genome-wide transcriptional

analysis of the Arabidopsis thaliana interaction with the plant pathogen

Pseudomonas syringae pv. tomato DC3000 and the human pathogen

Escherichia coli O157:H7. The Plant Journal, 46(1), 34-53.

Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K.,

He, S. Y., Howe, G. A., & Browse, J. (2007). JAZ repressor proteins are

targets of the SCFCOI1

complex during jasmonate signalling. Nature,

448(7154), 661-665.

Thomas, M. D., Langston-Unkefer, P. J., Uchytil, T. F., & Durbin, R. D. (1983).

Inhibition of glutamine synthetase from pea by tabtoxinine-β-lactam.

Plant Physiology, 71(4), 912-915.

202

Thomidis, T., Tsipouridis, C., Exadaktylou, E., & Drogoudi, P. (2005). Note:

Comparison of three laboratory methods to evaluate the pathogenicity and

virulence of ten Pseudomonas syringae pv.syringae strains on apple, pear,

cherry and peach trees. Phytoparasitica, 33(2), 137-140.

Thomma, B. P. H. J., Eggermont, K., Tierens, K. F. M.-J., & Broekaert, W. F.

(1999). Requirement of functional ethylene-insensitive 2 gene for efficient

resistance of Arabidopsis to infection by Botrytis cinerea. Plant

Physiology, 121(4), 1093-1101.

Thomma, B. P. H. J., Penninckx, I. A. M. A., Cammue, B. P. A., & Broekaert, W.

F. (2001). The complexity of disease signaling in Arabidopsis. Current

Opinion in Immunology, 13(1), 63-68.

Timperio, A. M., D'Alessandro, A., Fagioni, M., Magro, P., & Zolla, L. (2012).

Production of the phytoalexins trans-resveratrol and delta-viniferin in two

economy-relevant grape cultivars upon infection with Botrytis cinerea in

field conditions. Plant Physiology and Biochemistry, 50(1), 65-71.

To, J. P. C., & Kieber, J. J. (2008). Cytokinin signaling: Two-components and

more. Trends in Plant Science, 13(2), 85-92.

Toffolatti, S., Venturini, G., Maffi, D., & Vercesi, A. (2012). Phenotypic and

histochemical traits of the interaction between Plasmopara viticola and

resistant or susceptible grapevine varieties. BMC Plant Biology, 12(1),

124.

Ton, J., & Mauch-Mani, B. (2004). β-amino-butyric acid-induced resistance

against necrotrophic pathogens is based on ABA-dependent priming for

callose. The Plant Journal, 38(1), 119-130.

Ton, J., Flors, V., & Mauch-Mani, B. (2009). The multifaceted role of ABA in

disease resistance. Trends in Plant Science, 14(6), 310-317.

Torres, M. A. (2010). ROS in biotic interactions. Physiologia Plantarum, 138(4),

414-429.

Torres, M. A., Dangl, J. L., & Jones, J. D. G. (2002). Arabidopsis gp91phox

homologues AtrbohD and AtrbohF are required for accumulation of

reactive oxygen intermediates in the plant defense response. Proceedings

of the National Academy of Sciences, 99(1), 517-522.

Torres, M. A., Jones, J. D. G., & Dangl, J. L. (2006). Reactive oxygen species

signaling in response to pathogens. Plant Physiology, 141(2), 373-378.

Trdá, L., Fernandez, O., Boutrot, F., Héloir, M.-C., Kelloniemi, J., Daire, X.,

Adrian, M., Clément, C., Zipfel, C., Dorey, S., & Poinssot, B. (2014). The

grapevine flagellin receptor VvFLS2 differentially recognizes flagellin-

derived epitopes from the endophytic growth-promoting bacterium

Burkholderia phytofirmans and plant pathogenic bacteria. New

Phytologist, 201(4), 1371-1384.

203

Tsuda, K., & Katagiri, F. (2010). Comparing signaling mechanisms engaged in

pattern-triggered and effector-triggered immunity. Current Opinion in

Plant Biology, 13(4), 459-465.

Van der Does, D., Leon-Reyes, A., Koornneef, A., Van Verk, M. C., Rodenburg,

N., Pauwels, L., Goossens, A., Körbes, A. P., Memelink, J., Ritsema, T.,

Van Wees, S. C. M., & Pieterse, C. M. J. (2013). Salicylic acid suppresses

jasmonic acid signaling downstream of SCFCOI1

-JAZ by targeting GCC

promoter motifs via transcription factor ORA59. The Plant Cell Online,

25(2), 744-761.

Van Loon, L. C. (1997). Induced resistance in plants and the role of pathogenesis-

related proteins. European Journal of Plant Pathology, 103(9), 753-765.

Vassilev, V., Lavermicocca, P., Di Giorgio, D., & Iacobellis, N. S. (1996).

Production of syringomycins and syringopeptins by Pseudomonas

syringae pv. atrofaciens. Plant Pathology, 45(2), 316-322.

Vicente, J. G., & Roberts, S. J. (2007). Discrimination of Pseudomonas syringae

isolates from sweet and wild cherry using rep-PCR. European Journal of

Plant Pathology, 117(4), 383-392.

Vick, B. A., & Zimmerman, D. C. (1984). Biosynthesis of jasmonic acid by

several plant species. Plant Physiology, 75(2), 458-461.

Vierstra, R. D. (2009). The ubiquitin–26S proteasome system at the nexus of

plant biology. Nature Reviews Molecular Cell Biology, 10(6), 385-397.

Vinatzer, B. A., Teitzel, G. M., Lee, M.-W., Jelenska, J., Hotton, S., Fairfax, K.,

Jenrette, J., & Greenberg, J. T. (2006). The type III effector repertoire of

Pseudomonas syringae pv. syringae B728a and its role in survival and

disease on host and non-host plants. Molecular Microbiology, 62(1), 26-

44.

Voigt, C. A. (2014). Callose-mediated resistance to pathogenic intruders in plant

defense-related papillae. Frontiers in Plant Science, 5, 1-6.

Waite, H., Whitelaw-Weckert, M., & Torley, P. (2014). Grapevine propagation:

Principles and methods for the production of high-quality grapevine

planting material. New Zealand Journal of Crop and Horticultural

Science, 1-18.

Wang, D., Pajerowska-Mukhtar, K., Culler, A. H., & Dong, X. (2007). Salicylic

acid inhibits pathogen growth in plants through repression of the auxin

signaling pathway. Current Biology, 17(20), 1784-1790.

Wang, K. L.-C., Li, H., & Ecker, J. R. (2002). Ethylene biosynthesis and

signaling networks. The Plant Cell Online, 14(suppl 1), S131-S151.

204

Wang, N., Lu, S.-E., Records, A. R., & Gross, D. C. (2006a). Characterization of

the transcriptional activators salA and syrF, which are required for

syringomycin and syringopeptin production by Pseudomonas syringae pv.

syringae. Journal of Bacteriology, 188(9), 3290-3298.

Wang, N., Lu, S.-E., Wang, J., Chen, Z. J., & Gross, D. C. (2006b). The

expression of genes encoding lipodepsipeptide phytotoxins by

Pseudomonas syringae pv. syringae is coordinated in response to plant

signal molecules. Molecular Plant-Microbe Interactions, 19(3), 257-269.

Wang, Y., Li, J., Hou, S., Wang, X., Li, Y., Ren, D., Chen, S., Tang, X., & Zhou,

J.-M. (2010). A Pseudomonas syringae ADP-ribosyltransferase inhibits

Arabidopsis mitogen-activated protein kinase kinases. The Plant Cell,

22(6), 2033-2044.

Warcup, J. H., & Talbot, P. H. B. (1981). Host-pathogen index of plant diseases

in South Australia. Host-pathogen index of plant diseases in South

Australia.

Wäspi, U., Blanc, D., Winkler, T., Rüedi, P., & Dudler, R. (1998). Syringolin, a

novel peptide elicitor from Pseudomonas syringae pv. syringae that

induces resistance to Pyricularia oryzae in rIce. Molecular Plant-Microbe

Interactions, 11(8), 727-733.

Wathugala, D. L., Hemsley, P. A., Moffat, C. S., Cremelie, P., Knight, M. R., &

Knight, H. (2012). The mediator subunit SFR6/MED16 controls defence

gene expression mediated by salicylic acid and jasmonate responsive

pathways. New Phytologist, 195(1), 217-230.

Weingart, H., & Volksch, B. (1997). Ethylene production by Pseudomonas

syringae pathovars in vitro and in planta. Applied and Environmental

Microbiology, 63(1), 156-161.

Wendehenne, D., Lamotte, O., Frachisse, J.-M., Barbier-Brygoo, H., & Pugin, A.

(2002). Nitrate efflux is an essential component of the cryptogein

signaling pathway leading to defense responses and hypersensitive cell

death in tobacco. The Plant Cell, 14(8), 1937-1951.

Whitelaw-Weckert, M. A., Whitelaw, E. S., Rogiers, S. Y., Quirk, L., Clark, A.

C., & Huang, C. X. (2011). Bacterial inflorescence rot of grapevine

caused by Pseudomonas syringae pv. syringae. Plant Pathology, 60(2),

325-337.

Whitesides, S., & Spotts, R. (1991). Frequency, distribution, and characteristics

of endophytic Pseudomonas syringae in pear trees. Phytopathology,

81(4), 453-457.

Wielgoss, A., & Kortekamp, A. (2006). Comparison of PR1 expression in

grapevine cultures after inoculation with a host-and a non-host pathogen.

Vitis, 45(1), 9-13.

205

Wilson, M., & Lindow, S.E. (1994). Ecological similarity and coexistence of

epiphytic ice-nucleating (ice+) Pseudomonas syringae strains and a non-

ice-nucleating (ice-) biological control agent. Applied and Environmental

Microbiology, 60(9), 3128-3137.

Wimalajeewa, D. L. S., & Flett, J. D. (1985). A study of populations of

Pseudomonas syringae pv. syringae on stonefruits in Victoria. Plant

Pathology, 34(2), 248-254.

Wu, L., Chen, H., Curtis, C., & Fu, Z. Q. (2014). Go in for the kill. Virulence,

5(7), 710-721.

Wu, S., Shan, L., & He, P. (2014). Microbial signature-triggered plant defense

responses and early signaling mechanisms. Plant Science, 228, 118-126.

Wuest, W. M., Krahn, D., Kaiser, M., & Walsh, C. T. (2011). Enzymatic timing

and tailoring of macrolactamization in syringolin biosynthesis. Organic

Letters, 13(17), 4518-4521.

Xin X., & He, S.Y., (2013). Pseudomonas syringae pv. tomato DC3000: A model

pathogen for probing disease susceptibility and hormone signaling in

plants. Annual Review of Phytopathology, 51, 473–98.

Xu, T. F., Zhao, X. C., Jiao, Y. T., Wei, J. Y., Wang, L., & Xu, Y. (2014). A

pathogenesis related protein, VpPR-10.1, from Vitis pseudoreticulata: An

insight of its mode of antifungal activity. PLoS ONE, 9(4).

Xu, G.W., & Gross, D. C. (1988). Evaluation of the role of syringomycin in plant

pathogenesis by using Tn5 mutants of Pseudomonas syringae pv. syringae

defective in syringomycin production. Applied and Environmental

Microbiology, 54(6), 1345-1353.

Yamamoto, S., Kasai, H., Arnold, D. L., Jackson, R. W., Vivian, A., &

Harayama, S. (2000). Phylogeny of the genus Pseudomonas: Intrageneric

structure reconstructed from the nucleotide sequences of gyrB and rpoD

genes. Microbiology, 146(10), 2385-2394.

Yao, Y. A., Wang, J., Ma, X., Lutts, S., Sun, C., Ma, J., Yang, Y., Achal, V., &

Xu, G. (2012). Proteomic analysis of Mn-induced resistance to powdery

mildew in grapevine. Journal of Experimental Botany, 63(14), 5155-5170.

Yasuda, M., Ishikawa, A., Jikumaru, Y., Seki, M., Umezawa, T., Asami, T.,

Maruyama-Nakashita, A., Kudo, T., Shinozaki, K., Yoshida, S., &

Nakashita, H. (2008). Antagonistic interaction between systemic acquired

resistance and the abscisic acid–mediated abiotic stress response in

Arabidopsis. The Plant Cell Online, 20(6), 1678-1692.

Yoo, S.-D., Cho, Y., & Sheen, J. (2009). Emerging connections in the ethylene

signaling network. Trends in Plant Science, 14(5), 270-279.

206

Young, A. (2008). Notes on Pseudomonas syringae pv. syringae bacterial

necrosis of mango (Mangifera indica) in Australia. Australasian Plant

Disease Notes, 3, 138-140.

Young, J. M. (2010). Taxonomy of Pseudomonas syringae. Journal of Plant

Pathology, 92, S1.5-S1.14.

Young, J. M., & Triggs, C. M. (1994). Evaluation of determinative tests for

pathovars of Pseudomonas syringae van Hall 1902. Journal of Applied

Bacteriology, 77(2), 195-207.

Yu, Y., Zhang, Y., Yin, L., & Lu, J. (2012). The mode of host resistance to

Plasmopara viticola infection of grapevines. Phytopathology, 102(11),

1094-1101.

Zeng, W., & He, S. Y. (2010). A prominent role of the flagellin receptor

FLAGELLIN-SENSING2 in mediating stomatal response to

Pseudomonas syringae pv. tomato DC3000 in Arabidopsis. Plant

Physiology, 153(3), 1188-1198.

Zhang, J., Shao, F., Li, Y., Cui, H., Chen, L., Li, H., Zou, Y., Long, C., Lan, L.,

Chai, J., Chen, S., Tang, X., & Zhou, J. M. (2007). A Pseudomonas

syringae effector inactivates MAPKs to suppress PAMP-induced

immunity in plants. Cell Host and Microbe, 1(3), 175-185.

Zhang, Y., Gao, M., Singer, S. D., Fei, Z., Wang, H., & Wang, X. (2012).

Genome-wide identification and analysis of the TIFY gene family in

grape. PLoS ONE, 7(9), e44465.

Zhang, Z., Li, Q., Li, Z., Staswick, P. E., Wang, M., Zhu, Y., & He, Z. (2007b).

Dual regulation role of GH3.5 in salicylic acid and auxin signaling during

Arabidopsis-Pseudomonas syringae interaction. Plant physiology, 145(2),

450-464.

Zhao, W., Jiang, H., Tian, Q., & Hu, J. (2015). Draft genome sequence of

Pseudomonas syringae pv. persicae NCPPB 2254. Genome

Announcements, 3(3), e00555-00515.

Ziadi, S. l., Poupard, P., Brisset, M. N., Paulin, J. P., & Simoneau, P. (2001).

Characterization in apple leaves of two subclasses of PR-10 transcripts

inducible by acibenzolar-S-methyl, a functional analogue of salicylic acid.

Physiological and Molecular Plant Pathology, 59(1), 33-43.

207

Appendix 1 DNA Extraction of P. syringae Using Qiagen

DNeasy Blood and Tissue Kit (Cat No./ID 69504)

Grow P. syringae cultures on king’s B (KB) agar for 48 hours at 25°C.

Aseptically collect one loopful of bacteria and emulsify in 180 μL of Buffer ATL

in a sterile 1.5 mL tube. Add 20 μL proteinase K and vortex. Incubate the samples

at 56°C for 10 mins with regular vortexing every 2-3 mins.

Add 200 μL Buffer AL to each sample tube and mix thoroughly by vortexing.

Add 200 μL molecular grade ethanol (96-100%) and mix by gentle pipetting

action.

Transfer the mixture into the DNeasy Mini spin column in a 2 mL collection tube

and centrifuge at 8 000 rpm for 1 min – discard the flow-through.

Add 500 μL Buffer AW1 to the DNeasy Mini spin column and centrifuge for 1

min at 8 000 rpm – discard the flow-through.

Add 500 μL Buffer AW2 to the DNeasy Mini spin column and centrifuge for 3

mins at 14 000 rpm – discard the flow through. To ensure that the DNeasy Mini

spin column membrane is dry, place the column in a new 2 mL collection tube

and centrifuge at 14 000rpm for 1 min.

Place the DNeasy Mini spin column in a sterile 1.5 mL tube and pipette 100 μL

Buffer AE (or nuclease-free water) directly onto the DNeasy membrane. Incubate

at room temperature for 1 min and centrifuge at 8 000 rpm for 1 min to elute.

For maximum DNA yield, pipette another 100 μL Buffer AE (or nuclease-free

water) directly onto the DNeasy membrane and incubate again for 1 min at room

temperature then centrifuge at 8 000 rpm for 1 min.

Carefully remove the DNeasy Mini spin column and store elute at 4°C for upto 7

days or at -20°C for long term storage.

208

Appendix 2 GenBank Accession Numbers for

P. syringae Isolates.

Isolate

GenBank Accession Numbers for rpoB and MLST sequencing

rpoB gapA gltA gyrB rpoD

P. s. syringae DAR72042 KJ170140 KP127641 KP136846 KP192345 KP229314

P. s. syringae DAR73915 KJ170141 KP127642 KP136847 KP192346 KP229315

P. s. syringae DAR77819 KJ170138 KP127636 KP136841 KP192340 KP229309

P. s. syringae DAR77820 KJ170147 KP127634 KP136839 KP192338 KP229307

P. s. syringae DAR82159 KJ170146 KP127628 KP136833 KP192332 KP229301

P. s. syringae DAR82160 KJ170148 KP127629 KP136834 KP192333 KP229302

P. s. syringae DAR82161 KJ170145 KP127630 KP136825 KP192334 KP229303

P. s. syringae DAR82162 KJ170135 KP127631 KP136836 KP192335 KP229304

P. s. syringae DAR82165 KJ170137 KP127632 KP136837 KP192336 KP229305

P. s. syringae DAR82166 KJ170143 KP127633 KP136838 KP192337 KP229306

P. s. syringae DAR82169 KJ170139 KP127637 KP136842 KP192341 KP229310

P. s. syringae DAR82170 KJ170136 KP127638 KP136843 KP192342 KP229311

P. s. syringae DAR82171 KJ170133 KP127639 KP136844 KP192343 KP229312

P. s. syringae DAR82440 KJ590780 KP127643 KP136848 KP192347 KP229316

P. s. syringae DAR82441 KJ590781 KP127645 KP136850 KP192349 KP229318

P. s. syringae DAR82442 KJ590782 KP127646 KP136851 KP192350 KP229319

P. s. syringae DAR82443 KJ590788 KP127652 KP136857 KP192356 KP229325

P. s. syringae DAR82444 KJ590789 KP127653 KP136858 KP192357 KP229326

P. s. syringae DAR82445 KJ590790 KP127654 KP136859 KP192358 KP229327

P. s. syringae DAR82446 KJ590791 KP127655 KP136860 KP192359 KP229328

P. s. syringae DAR82447 KJ590792 KP127656 KP136861 KP192360 KP229329

P. s. syringae DAR82448 KJ590793 KP127657 KP136862 KP192361 KP229330

P. s. syringae DAR82449 KJ590794 KP127658 KP136863 KP192362 KP229331

P. s. syringae DAR82450 KJ590795 KP127659 KP136864 KP192363 KP229332

P. s. syringae DAR82451 KJ590796 KP127660 KP136865 KP192364 KP229333

P. s. syringae DAR82452 KJ590797 KP127661 KP136866 KP192365 KP229334

P. s. syringae DAR82453 KJ590798 KP127662 KP136867 KP192366 KP229335

P. s. syringae BRIP34823 KJ590783 KP127647 KP136852 KP192351 KP229320

P. s. syringae BRIP34831 KJ590784 KP127648 KP136853 KP192352 KP229321

P. s. syringae BRIP34899 KJ590785 KP127649 KP136854 KP192353 KP229322

P. s. syringae BRIP38670 KJ590786 KP127650 KP136855 KP192354 KP229323

P. s. maculicola BRIP38817 KJ741399 KP127664 KP136869 KP192368 KP229337

P. s. striafaciens BRIP34832 KJ741398 KP127663 KP136868 KP192367 KP229336

P. s. tabaci BRIP34803 KJ741400 KP127665 KP136870 KP192369 KP229338

P. s. phaseolicola BRIP38811 KJ590787 KP127651 KP136856 KP192355 KP229324

P. s. mori BRIP34805 KJ741401 KP127666 KP136871 KP192370 KP229339

P. s. morsprunorum

DAR33419 KJ170149 KP127640 KP136845 KP192344 KP229313

Pseudomonas fragi (ST128) KJ939262 KP127644 KP136849 KP192348 KP229317

209

Appendix 3 Analysis of Molecular Variance Results

Using Arelquin Software.

Arlequin settings:

AMOVA

Standard AMOVA computations (haplotypic format)

No. Of permutations 10000

Compute Minimum Spanning Network (MSN) among

haplotypes

- Compute distance matrix

210

BIR AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"BIR_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 92.675 5.20804 Va

15.42

Within

populations 24 685.825 28.57604 Vb

84.58

-----------------------------------------------------------------

Total 25 778.500 33.78408

-----------------------------------------------------------------

Fixation Index FST : 0.15416

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00178

P(rand. value = obs. value) = 0.00000

P-value = 0.00178+-0.00038

211

Tyrosinase AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Tyrosinase_pos"

"Tyrosinase_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 92.403 5.76111 Va

16.77

Within

populations 24 686.097 28.58738 Vb

83.23

-----------------------------------------------------------------

Total 25 778.500 34.34850

-----------------------------------------------------------------

Fixation Index FST : 0.16773

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00297

P(rand. value = obs. value) = 0.00000

P-value = 0.00297+-0.00057

212

Tartaric acid AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"TA_pos"

"TA_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 66.369 19.38319 Va

23.59

Within

populations 26 753.667 28.98718 Vb

76.41

-----------------------------------------------------------------

Total 27 820.036 48.37037

-----------------------------------------------------------------

Fixation Index FST : 0.23581

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.05378

P(rand. value = obs. value) = 0.03782

P-value = 0.09160+-0.00298

213

Lactic acid AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"LA_pos"

"LA_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 52.876 4.36232 Va

12.88

Within

populations 26 767.160 29.50615 Vb

87.12

-----------------------------------------------------------------

Total 27 820.036 33.86847

-----------------------------------------------------------------

Fixation Index FST : 0.12880

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.05693

P(rand. value = obs. value) = 0.00050

P-value = 0.05743+-0.00242

214

Ice nucleation activity AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"INA_pos"

"INA_neg"

Computing conventional F-Statistics from haplotype frequencies

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 0.503 0.00285 Va

0.57

Within

populations 26 12.926 0.49715 Vb

99.43

-----------------------------------------------------------------

Total 27 13.429 0.50000

-----------------------------------------------------------------

Fixation Index FST : 0.00570

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00000

P(rand. value = obs. value) = 0.85614

P-value = 0.85614+-0.00340

215

Grapevine leaf pathogenicity AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Grapevine_patho"

"Grape_non_patho"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 67.988 3.72030 Va

11.40

Within

populations 26 752.048 28.92491 Vb

88.60

-----------------------------------------------------------------

Total 27 820.036 32.64521

-----------------------------------------------------------------

Fixation Index FST : 0.11396

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.01495

P(rand. value = obs. value) = 0.00010

P-value = 0.01505+-0.00120

216

Tobacco leaf hypersensitivity reaction AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"tobacco_HR_neg"

"tobacco_HR_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 71.568 6.22119 Va

17.44

Within

populations 24 706.932 29.45549 Vb

82.56

-----------------------------------------------------------------

Total 25 778.500 35.67669

-----------------------------------------------------------------

Fixation Index FST : 0.17438

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.02386

P(rand. value = obs. value) = 0.00000

P-value = 0.02386+-0.00152

217

Lemon pathogenicity AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Lemon_po"

"lemon_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 52.523 2.17711 Va

6.71

Within

populations 24 725.977 30.24906 Vb

93.29

-----------------------------------------------------------------

Total 25 778.500 32.42617

-----------------------------------------------------------------

Fixation Index FST : 0.06714

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.06307

P(rand. value = obs. value) = 0.00000

P-value = 0.06307+-0.00252

218

Syringopeptin production by inhibition of Bacillus megaterium AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Bmega_pos"

"Bmega_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 90.542 16.75828 Va

36.89

Within

populations 24 687.958 28.66493 Vb

63.11

-----------------------------------------------------------------

Total 25 778.500 45.42321

-----------------------------------------------------------------

Fixation Index FST : 0.36894

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00297

P(rand. value = obs. value) = 0.00287

P-value = 0.00584+-0.00076

219

Syringomycin production by inhibition of Geotrichum candidum AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Gcand_pos"

"Gcand_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 57.075 2.19502 Va

6.81

Within

populations 24 721.425 30.05937 Vb

93.19

-----------------------------------------------------------------

Total 25 778.500 32.25439

-----------------------------------------------------------------

Fixation Index FST : 0.06805

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.04683

P(rand. value = obs. value) = 0.00010

P-value = 0.04693+-0.00194

220

Syringomycin production by inhibition of Saccharomyces cerevisiae AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Scere_pos"

"Scere_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 105.655 6.00627 Va

17.64

Within

populations 24 672.845 28.03522 Vb

82.36

-----------------------------------------------------------------

Total 25 778.500 34.04149

-----------------------------------------------------------------

Fixation Index FST : 0.17644

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00079

P(rand. value = obs. value) = 0.00000

P-value = 0.00079+-0.00027

221

Chloramphenicol resistance AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Chloramphenicol_pos"

"Chloramphenicol_neg"

Computing conventional F-Statistics from haplotype frequencies

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 0.423 0.00752 Va

1.53

Within

populations 24 12.000 0.50000 Vb

98.47

-----------------------------------------------------------------

Total 25 12.423 0.49248

-----------------------------------------------------------------

Fixation Index FST : 0.01527

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.84040

P(rand. value = obs. value) = 0.15960

P-value = 1.00000+-0.00000

222

Ampicillin resistance AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"ampicillin_pos"

"Ampicillin_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 82.750 5.82405 Va

16.73

Within

populations 24 695.750 28.98958 Vb

83.27

-----------------------------------------------------------------

Total 25 778.500 34.81363

-----------------------------------------------------------------

Fixation Index FST : 0.16729

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00683

P(rand. value = obs. value) = 0.00000

P-value = 0.00683+-0.00082

223

Tetracycline resistance AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"Tet_neg"

"Tet_pos"

Computing conventional F-Statistics from haplotype frequencies

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 0.466 0.01709 Va

3.55

Within

populations 26 12.963 0.49858 Vb

96.45

-----------------------------------------------------------------

Total 27 13.429 0.48148

-----------------------------------------------------------------

Fixation Index FST : 0.03550

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.85663

P(rand. value = obs. value) = 0.14337

P-value = 1.00000+-0.00000

224

Syringolin A (sylC) gene presence AMOVA results =================================================================

AMOVA ANALYSIS MLST

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"sylC_neg"

"sylC_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 46.409 2.34965 Va

7.15

Within

populations 24 732.091 30.50379 Vb

92.85

-----------------------------------------------------------------

Total 25 778.500 32.85343

-----------------------------------------------------------------

Fixation Index FST : 0.07152

-----------------------------------------------------------------

Significance tests (1023 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.11926

P(rand. value = obs. value) = 0.00000

P-value = 0.11926+-0.01070

225

Syringopeptin (sypC) gene presence AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"sypC_Pos"

"sypC_Neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 37.079 0.84797 Va

2.74

Within

populations 26 782.957 30.11371 Vb

97.26

-----------------------------------------------------------------

Total 27 820.036 30.96168

-----------------------------------------------------------------

Fixation Index FST : 0.02739

-----------------------------------------------------------------

Significance tests (1023 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.21017

P(rand. value = obs. value) = 0.00000

P-value = 0.21017+-0.01233

226

Syringomycin (syrB) gene presence AMOVA results =================================================================

AMOVA ANALYSIS MLST

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"syrB_neg"

"syrB_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 39.455 0.84660 Va

2.68

Within

populations 24 739.045 30.79355 Vb

97.32

-----------------------------------------------------------------

Total 25 778.500 31.64014

-----------------------------------------------------------------

Fixation Index FST : 0.02676

-----------------------------------------------------------------

Significance tests (1023 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.24340

P(rand. value = obs. value) = 0.00000

P-value = 0.24340+-0.01351

227

Tyrosinase positive P. syringae from BIR affected grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Tyrosinase_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 86.042 5.56925 Va

17.29

Within

populations 22 586.000 26.63636 Vb

82.71

-----------------------------------------------------------------

Total 23 672.042 32.20561

-----------------------------------------------------------------

Fixation Index FST : 0.17293

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00822

P(rand. value = obs. value) = 0.00000

P-value = 0.00822+-0.00100

228

Tartaric acid positive P. syringae from BIR affected grapevine AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"TA_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 61.375 19.90417 Va

45.43

Within

populations 15 358.625 23.90833 Vb

54.57

-----------------------------------------------------------------

Total 16 420.000 43.81250

-----------------------------------------------------------------

Fixation Index FST : 0.45430

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.06010

P(rand. value = obs. value) = 0.11683

P-value = 0.17693+-0.00348

229

Lactic acid negative P. syringae from BIR affected grapevine AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"LA_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 43.217 3.62148 Va

12.69

Within

populations 17 423.625 24.91912 Vb

87.31

-----------------------------------------------------------------

Total 18 466.842 28.54059

-----------------------------------------------------------------

Fixation Index FST : 0.12689

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.08871

P(rand. value = obs. value) = 0.00347

P-value = 0.09218+-0.00284

230

Non-ice nucleation active P. syringae from BIR affected grapevine AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"INA_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 29.022 2.71667 Va

10.20

Within

populations 15 358.625 23.90833 Vb

89.80

-----------------------------------------------------------------

Total 16 387.647 26.62500

-----------------------------------------------------------------

Fixation Index FST : 0.10203

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.23733

P(rand. value = obs. value) = 0.05772

P-value = 0.29505+-0.00388

231

Grapevine leaf non-pathogenic P. syringae from BIR affected grapevine

AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Grape_non_patho"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 76.164 4.95980 Va

15.11

Within

populations 21 585.054 27.85969 Vb

84.89

-----------------------------------------------------------------

Total 22 661.217 32.81949

-----------------------------------------------------------------

Fixation Index FST : 0.15112

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00950

P(rand. value = obs. value) = 0.00000

P-value = 0.00950+-0.00099

232

Tobacco leaf hypersensitivity response negative P. syringae from BIR

affected grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"tobacco_HR_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 84.675 9.00412 Va

24.97

Within

populations 18 486.875 27.04861 Vb

75.03

-----------------------------------------------------------------

Total 19 571.550 36.05273

-----------------------------------------------------------------

Fixation Index FST : 0.24975

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00931

P(rand. value = obs. value) = 0.00069

P-value = 0.01000+-0.00096

233

Non-pathogenic to lemon P. syringae from BIR affected grapevine AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"lemon_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 61.704 3.47860 Va

11.11

Within

populations 21 584.339 27.82568 Vb

88.89

-----------------------------------------------------------------

Total 22 646.043 31.30428

-----------------------------------------------------------------

Fixation Index FST : 0.11112

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.03188

P(rand. value = obs. value) = 0.00010

P-value = 0.03198+-0.00189

234

Syringopeptin production by inhibition of Bacillus megaterium negative P.

syringae from BIR affected grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Bmega_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 104.819 23.08862 Va

50.40

Within

populations 16 363.625 22.72656 Vb

49.60

-----------------------------------------------------------------

Total 17 468.444 45.81519

-----------------------------------------------------------------

Fixation Index FST : 0.50395

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00673

P(rand. value = obs. value) = 0.00584

P-value = 0.01257+-0.00103

235

Syringomycin production by inhibition of Geotrichum candidum negative P.

syringae from BIR affected grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Gcand_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 29.037 0.37393 Va

1.51

Within

populations 24 586.425 24.43437 Vb

98.49

-----------------------------------------------------------------

Total 25 615.462 24.80830

-----------------------------------------------------------------

Fixation Index FST : 0.01507

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.28416

P(rand. value = obs. value) = 0.00069

P-value = 0.28485+-0.00454

236

Syringomycin production by inhibition of Saccharomyces cerevisiae negative

P. syringae from BIR affected grapevine AMOVA results ================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Scere_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 23.673 0.14007 Va

0.64

Within

populations 26 565.542 21.75160 Vb

99.36

-----------------------------------------------------------------

Total 27 589.214 21.89168

-----------------------------------------------------------------

Fixation Index FST : 0.00640

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.33931

P(rand. value = obs. value) = 0.00099

P-value = 0.34030+-0.00459

237

Chloramphenicol resistance in P. syringae from BIR affected grapevine

AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Chloramphenicol_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 33.441 0.25264 Va

0.86

Within

populations 33 958.730 29.05243 Vb

99.14

-----------------------------------------------------------------

Total 34 992.171 29.30507

-----------------------------------------------------------------

Fixation Index FST : 0.00862

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.31584

P(rand. value = obs. value) = 0.00020

P-value = 0.31604+-0.00490

238

Ampicillin resistance in P. syringae from BIR affected grapevine AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"ampicillin_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 91.602 7.21543 Va

20.13

Within

populations 20 572.625 28.63125 Vb

79.87

-----------------------------------------------------------------

Total 21 664.227 35.84668

-----------------------------------------------------------------

Fixation Index FST : 0.20129

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.00426

P(rand. value = obs. value) = 0.00000

P-value = 0.00426+-0.00065

239

Tetracycline resistance in P. syringae from BIR affected grapevine AMOVA

results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"Tet_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 25.509 0.14503 Va

0.51

Within

populations 41 1165.329 28.42265 Vb

99.49

-----------------------------------------------------------------

Total 42 1190.837 28.27762

-----------------------------------------------------------------

Fixation Index FST : 0.00513

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.50802

P(rand. value = obs. value) = 0.00010

P-value = 0.50812+-0.00488

240

Presence of syringolin A (sylC) gene in P. syringae from BIR affected

grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"sylC_neg"

"BIR_pos"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 52.525 4.01758 Va

13.03

Within

populations 18 482.625 26.81250 Vb

86.97

-----------------------------------------------------------------

Total 19 535.150 30.83008

-----------------------------------------------------------------

Fixation Index FST : 0.13031

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.04683

P(rand. value = obs. value) = 0.00050

P-value = 0.04733+-0.00199

241

Presence of syringopeptin (sypC) gene in P. syringae from BIR affected

grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"sypC_neg"

Distance method: Pairwise difference

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 17.851 0.60411 Va

2.76

Within

populations 19 426.625 22.45395 Vb

97.24

-----------------------------------------------------------------

Total 20 444.476 21.84984

-----------------------------------------------------------------

Fixation Index FST : 0.02765

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.57832

P(rand. value = obs. value) = 0.00386

P-value = 0.58218+-0.00439

242

Presence of syringomycin (syrB) gene in P. syringae from BIR affected

grapevine AMOVA results =================================================================

AMOVA ANALYSIS

=================================================================

---------------------------

Genetic structure to test :

---------------------------

No. of Groups = 1

[[Structure]]

StructureName = "New Edited Structure"

NbGroups = 1

#Group1

Group={

"BIR_pos"

"syrB_neg"

Computing conventional F-Statistics from haplotype frequencies

--------------------------

AMOVA design and results :

--------------------------

Weir, B.S. and Cockerham, C.C. 1984.

Excoffier, L., Smouse, P., and Quattro, J. 1992.

Weir, B. S., 1996.

-----------------------------------------------------------------

Source of Sum of Variance

Percentage

variation d.f. squares components of

variation

-----------------------------------------------------------------

Among

populations 1 0.345 0.01386 Va

2.97

Within

populations 21 10.089 0.48044 Vb

97.03

-----------------------------------------------------------------

Total 22 10.435 0.46659

-----------------------------------------------------------------

Fixation Index FST : 0.02970

-----------------------------------------------------------------

Significance tests (10100 permutations)

------------------

Va and FST : P(rand. value > obs. value) = 0.76495

P(rand. value = obs. value) = 0.02594

P-value = 0.79089+-0.00376