Diagnostics and genomic diversity of Erwinia amylovora Rachel Mann Research Scientist DPI VIC biosecurity built on science Cooperative Research Centre for National Plant Biosecurity
“It is said that if you know your enemies and know yourself, you will not be imperiled in a hundred battles; if you do not know your enemies but do know yourself, you will win one and lose one; if you do not know your enemies nor yourself, you will be imperiled in every single battle.”
The Art of War Sun Tzu – 600BC
biosecurity built on science
The Enemy
Australian apple orchards predominantly have root stocks susceptible to E. amylovora biosecurity built on science
Know yourself Do we have E. amylovora or closely related species? We know we don’t want E. amylovora We know we have susceptible hosts We know it is an expensive and difficult (sometimes impossible) feat to eradicate a pathogen – 1997 E. amylovora incursion $20 million Our best line of defence is to not let E. amylovora in border detection and diagnostics
Distribution of plants in the family Rosaceae Source: Atlas of Living Australia 2012 http://biocache.ala.org.au
biosecurity built on science
Know yourself – Do we have E. amylovora?
Surveillance for E. amylovora In 2008 we sampled E. amylovora host plants across 104 sites 200 strains of bacteria were isolated -
Selective plating ambiguous Biochemical assays PCR 16S rDNA sequencing HR Pathogenicity assays on immature pear fruit
No E. amylovora identified
112 25 72 23050A 37 14 23048B E. billingiae Eb661 39 8 AG 16 2226A 38 9 BE66 38 14 94 49 21 E. persicina Cp2 99 10 E. rhapontici Nk4-18 BE56 DAR 61733 91 BE57 BE65 E. tasmaniensis Et1/99 E. amylovora 273 DAR 75964 95 73 E. pyrifoliae Ep16/96 83 23064A P. ananatis M471 49 13 94 P. agglomerans CE21 23091B 23093A Pantoea sp. EM486 23069A 23095D 99 23046A 23085C 23086A BE62 BE68 Enterobacteriaceae bacterium BFo-2 BE55 Pear ooze A S. proteamaculans PW172 94 11 6 Rahnella sp. Pv5 R. aquatilis KNOUC601 11 22 91 11 21 11 24
82
100
99
99 89
97
75
139 24 143 10 BE63 88 P. poae BCHCNZ318 138 19 23075A P. graminis PDD-32b-66 78 100 BE3 23063A AG 16 2226B 96 P. putida strain jvu23 72 72 AG 16 222C 23042A 23098D 23076A 23083A 49 10 P. syringae PNA29.1a 20795B 23094C 23074A 100 BE60 23095C 100 Curtobacterium sp. S121 C. flaccumfaciens 11.1 23046B 23048C 100 M. yunnanensis L7-617 100 23085B Paenibacillus sp. 9N4 100 AG 16 2268C D E. aurantiacum BVC2 100 149 22 Staphylococcus sp. SV3 AG 16 2268A D AG 16 2268B D 99 B. simplex CCMM B622 95 23079A 23097A 99 23013A B. licheniformis T10 93 AG 16 1909C 98 B. atrophaeus BPRIST036 B. subtilis SJ01 98 126 8 23052A 94 23023C 23044A
Erwinia
Pantoea
Serratia Rahnella
Pseudomonas
Curtobacterium
Micrococcus Paenibacillus Exiguobacterium Staphylococcus
Bacillus
20
biosecurity built on science
Know yourself – What's our bet line of defence?
Detection Molecular tests – more time efficient, sensitive and are culture independent Evaluation of E. amylovora specific PCR -
95 strains of bacteria, 8 common PCRs 7 out of 8 tests tested gave unsatisfactory results Both false negatives and false positives A number of the false positives were with bacteria isolated from our surveys
Powney et al (2011) The specificity of PCR-based protocols for detection of Erwinia amylovora. Australas Plant Pathol 40(1): 87-97
One primer set robust in this evaluation No diagnostic is perfect so reliance on one marker is not the best practice solution – like with other important pathogenic bacteria a multiplex approach is more desirable. biosecurity built on science
Know your enemy We know the host range We know there are host specific-groupings
Spiraeoideae-infecting strains (Apple strains)
- Spiraeoideae-infecting - Rubus-infecting
Do we know what causes host specificity? Do we know how much genetic diversity there is between strains?
Rubus-infecting strains
Need to be considered to in diagnostic design
biosecurity built on science
Know your enemy – Genetic diversity of E. amylovora
The Pan-genome of E. amylovora 12 Genomes of E. amylovora (we sequenced six for this project)
- 9 Spiraeoideae-infecting (very little genetic diversity) - 3 Rubus-infecting (more genetic diversity)
Powney et al (2011) Genome sequence of an Erwinia amylovora strain with pathogenicity restricted to Rubus plants. J Bacteriol 193(3): 785-786
5733 genes 3414 core gene shared by all strains Average E. amylovora genome has Phylogenetic tree based on core genome 3819 genes: 90% of each individual genome is core -> good for diagnostics Compared to similar types of analysis with other plant pathogenic bacteria E. amylovora is not a genetically diverse species biosecurity built on science
Know your enemy – Do we know what causes host specificity?
The Pan-genome of E. amylovora Plasmids
LPS
Rezzonico et al (2012) Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideaeinfective genotypes of Erwinia amylovora. Mol Plant Pathol
Hrp Pathogenicity island
T6SS variation
Mann et al (2012) Comparative analysis of the Hrp pathogenicity island of Rubus- and Spiraeoideaeinfecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element. Gene.
Putative secondary metabolite cluster (PKS &NRPS) Mann et al (in preparation) The pan-genome of E. amylovora
Arabinose biosynthesis cluster biosecurity built on science
Know your enemy – using the knowledge to better our defences
Diagnostic design –drawing on our knowledge Unique target identification pipeline
Ea core genome
vs
Function
E. tasmaniensis
Erwinia Ejp
Location
E. pyrifoliae
Ea-potential targets
NCBI database
Ea-specific targets
HGT
E. billingiae
Analyse for suitability as a diagnostic target
Primer design
66 regions of sequence collectively spanning 101561 bps Powney et al (2011) An analysis of the Erwinia amylovora pan-genome identifies novel chromosomal targets for molecular diagnostics Acta Hort 896
E. amylovora specific multiplex-PCR
Rubus-infecting E. amylovora specific multiplex-PCR
biosecurity built on science
Thank you For more information, please email rachel.mann@dpi.vic.gov.au
Dr Brendan Rodoni Dr Kim Plummer Dr Jo Luck
Prof Steven Beer Dr Tim Sawbridge Dr Brion Duffy Dr Theo Smits Jean Bonasera
biosecurity built on science
biosecurity built on science
Phylogenetic tree based on SNP variation in the Spiraeoideae-infecting strains
biosecurity built on science
% core
Pan-genome
Species
CDS
No. Genomes
type
Escherichia coli
44%
17
Open
Ralstonia solanacearum
48%
6
Unknown
Xanthomonas oryzae
63%
4
Open
Pseudomonas syringae
64%
19
Unknown
Streptococcus pneumoniae
74%
44
Open
Xanthomonas campestris
75%
5
Open
Listeria monocytogenes
80%
26
Open
Staphylococcus aureus
85-89%
17
Closed
Erwinia amylovora
90%
12
Open
Mycobacterium tuberculosis
98%
9
Closed
biosecurity built on science
biosecurity built on science
biosecurity built on science
ICE in the hprPAI of Erwinia sp
biosecurity built on science
Erwinia ancestor
E. billingiae
Pathoadaptation - Acquisition of Hrp T3SS cluster - Acquisition of ICE
Pathoadapted Erwinia ancestor
Loss of ICE due to genome rearrangement
E. tasmaniensis
- Deletion of AttL and related genes - Acquisition of clpB and genes encoding EF-hand domain proteins
Large-scale adaptations
E. piriflorinigrans
E. pyrifoliae
E. amylovora
In each strain individually: - Deletion of smaller regions in ICE - Modification of the EF-hand domain region
Adaptation due to reductive genomics Spiraeoideaeinfecting isolates
Rubus-infecting isolates
Evolution of hrpPAI region in pathoadapted Erwinia sp. biosecurity built on science