Effects of the Plant Pathogen Pseudomonas syringae ...
Transcript of Effects of the Plant Pathogen Pseudomonas syringae ...
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
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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
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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:
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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
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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
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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.
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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
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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
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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
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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;
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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.,
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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
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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
eb e
t al
., 2
005
Sar
kar
& G
utt
man
, 2
00
4;
Hw
ang
et
al.,
200
5
1. F
orw
ard
str
and
pri
mer
(+
), r
ever
se s
tran
d p
rim
er (
-), P
CR
pri
mer
(p
) an
d s
equ
enci
ng p
rim
er (
s)
Fu
nct
ion
Pat
ho
var
id
enti
fica
tion
ML
ST
Seq
uen
ce
TG
GC
CG
AG
AA
CC
AG
TT
CC
GC
G
CG
GC
TT
CG
TC
CA
GC
TT
GT
TC
A
CC
GG
CS
GA
RC
TG
CC
ST
GG
CG
AR
TG
CA
CS
GG
BC
TS
TT
CA
CC
GT
GT
GR
TT
GG
CR
TC
GA
AR
AT
CG
A
CC
TC
BT
GC
GA
GT
CG
AA
GA
TC
AC
C
CT
GR
TC
GC
CA
AG
AT
GC
CG
AC
CG
AA
GA
TC
AC
GG
TG
AA
CA
TG
CT
GG
CT
TG
TA
VG
GR
CY
GG
AG
AG
CA
TT
TC
CB
GC
RG
CV
GA
RG
TS
AT
CA
TG
AC
TT
GT
CY
TT
GG
TC
TG
SG
AG
CT
GA
A
AG
GT
GG
AA
GA
CA
TC
AT
CC
GC
AT
G
YG
AA
GG
CG
AR
AT
YG
RA
AT
CG
CC
GA
TG
TT
GC
CT
TC
CT
GG
AT
CA
G
Pri
mer
s1
LA
PS
ps
LA
P2
7p
s
ga
pA
+26
4p
ga
pA
+31
2s
ga
pA
-87
4p
s
glt
A+
17
4p
glt
A+
51
3s
glt
A-1
130
s
glt
A-1
192
p
gyr
B+
27
1p
s
gyr
B-1
022
ps
rpo
D+
17
4p
rpo
D+
36
4s
rpo
D-1
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
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.
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.
72
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).
74
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,
75
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.
83
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).
88
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).
91
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.
92
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).
96
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
thet
ase
C (
syp
C),
an
d c
oro
naf
acta
te l
igas
e (c
fl).
Au
tho
rs
Th
is s
tud
y,
der
ived
fro
m
Fei
l et
al.
(2
00
5)
Th
is s
tud
y,
der
ived
fro
m
Zh
ang
et
al. (1
99
5)
Th
is s
tud
y,
der
ived
fro
m
Am
rein
et
al.
(20
04)
Th
is s
tud
y,
der
ived
fro
m
Sch
olz
-Sch
roed
er e
t al
.
(20
03
)
Th
is s
tud
y,
der
ived
fro
m
Ber
esw
ill
et a
l. (
19
94
)
Gen
Ba
nk
Acc
ess
ion
CP
00
00
75
U2
51
30
AJ5
48
82
6
AF
28
62
16
S7
39
73
Ta
rget
Len
gth
(bp
)
18
6
25
6
22
2
27
4
50
7
Seq
uen
ce (
5’→
3’)
AC
AC
GC
TC
TT
CT
TC
GG
TG
AT
CC
AG
GA
AA
TC
CT
CA
AC
CA
GA
TA
TC
GT
CT
CT
GC
GC
GT
AT
TG
GA
GA
AG
TC
GA
AA
CC
GA
TG
GA
AA
CC
AC
GG
CA
AA
CT
AT
CC
AG
CA
TG
AA
TG
AG
GT
GG
TG
TT
CG
TG
GC
CA
AG
GG
TT
AC
TT
GA
AC
CG
TT
TG
TG
TG
CA
GA
TT
CG
TC
AC
GA
GC
AC
CT
AT
AG
CG
TC
AG
AC
TG
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.
127
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
128
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
131
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
164
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
168
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
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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