Session 2: Genome-Informed Diagnostics - In-field Detection of Bacterial Plant Pathogens

biosecurity built on science
Plant Biosecurity Cooperative Research Centre
James Stack
Professor and Director
In-field Detection of Bacterial Plant Pathogens
Genome-Informed Diagnostics

James Stack
PBCRC2002 & PBCRC2156
• 2002: Develop & validate laboratory and field
diagnostic protocols for plant pathogenic bacteria to
the pathovar level of discrimination
• 2156: Deploy validated field diagnostic protocols for
plant pathogenic bacteria to the pathovar level of
discrimination
• Increase national capability in plant bacteriology

James Stack
PBCRC 2002: Genome-based, bioinformatics-informed
diagnostics
• Developed & validated multiple laboratory and field
• Validated Pan-Genome Pipeline
• Increased national capability in plant bacteriology:
9 scientists trained & mentored in plant bacteriology
(Australia, New Zealand, U.S.)

James Stack
Other 2002/2156 Team Presentations – Don’t miss:
• Sarah Thompson: Metagenomic discovery of differential
diagnostic loci in CLos
• Jacqui Morris: Microflora analyses of the Australian eggplant
psyllid
• Rachel Mann: Complex diagnostics – keeping up with Ralstonia
solanacearum
• Toni Chapman: Genome-informed diagnostics – Xanthomonas
citri subsp citri
• Rebecca Roach: Identification of Xanthomonas species causing
bacterial leaf spot in Australia

James Stack
PBCRC 2156: Field-deployable genome-based,
bioinformatics-informed diagnostic protocols
• Developed & validated multiple field-deployable
• Validated Concept-to-Practice Pipeline
• Engaged end-user communities in field testing and
validation of protocols: laboratory and in-field end-
user training in new technologies and protocols

James Stack
PBCRC 2002/2156: Diverse Bacterial targets
• Xanthomonas citri subsp. citri: Gram negative, citrus
pathogen
• Candidatus Liberibacter solanacearum: Unculturable
bacterium, arthropod vector, potato pathogen
• Ralstonia solanacearum: Gram negative, potato
pathogen (other hosts)
• Rathayibacter toxicus: Gram positive, nematode
vector, annual ryegrass (other hosts)
• Pseudomonas syringae pv. actinidiae: Gram negative

James Stack
Bacterial Pathovar Diagnostics Team:
Xanth Liberibact Pseudo Rathay Erwinia Total
Genomes
sequenced
80 10 2 10 102
Target #
80 30 70 39 50 269
New taxa
sequenced
12 2 2 16
Psylid
Mitogenomes
sequenced
14
(5 species)
14
170 20 4 70 31248
R. sol
200 214

biosecurity built on scienceScience Exchange - August 2016
• Who are you?
• Where did you come from?
• How did you get here?
• When did you get here?
• Have you been here before? (prior
entry)
• Are you travelling alone? (vector)
Outbreak response:
You can’t answer those questions from
symptoms alone
Plant Biosecurity: Smart Surveillance
Pictures: J. Stack lab
How much can we determine in the field?
What do we want to know?
Particularly difficult with bacterial pathogensCan genome-informed diagnostics help?

Whoa Dude!
Look what I
found.

• Who are you: What pathogen is this?
• For most bacterial pathogens, the level
of discrimination is at the sub-specific
level
• For biosecurity, we need to know the
race, strain, or even the population
What do we want to know?
Can be very difficult for bacterial
pathogens using traditional technologies –
Almost impossible in the field

LAMP Workshop – La Trobe University
24 October 2016
Discriminating peppers from cows
No characteristics in common
No special training
required

24 October 2016
Discriminating sheep from cows
Shape, 4 legs, head, tail, 2 eyes, 2 ears
No special equipment
required

24 October 2016
Discriminating cows from cows
Color, weight, height, markings
Many fewer discriminating characteristics

Many distinguishing features
Discriminating bacteria from fungi

No distinguishing features
Discriminating bacteria from bacteria
Almost impossible in the field

Pictures: International Symposium
on Bacterial Canker of Kiwifruit
Pseudomonas syringae
Kiwifruit Pathogen
Non-Pathogenic

Pictures: International
Symposium on Bacterial
Canker of Kiwifruit
24 October 2016
Why rapid is important

Canker of Kiwifruit
24 October 2016
high
impact
Impact
threshold
low
impact
disease
detection
Critical response point
Time
Diseaseseverity

Canker of Kiwifruit
24 October 2016
Local
spread
Eradication
Containment
Containment
Containment – Eradication Window

Increased
spread
Canker of Kiwifruit
24 October 2016
Local
spread
Eradication
Containment
Containment
Containment – Eradication WindowContainment to Management

Diseaseseverity
Time
Impact
Critical response point
Early detection
Detection
Limit
SMART Surveillance
Better diagnostic
technology
Diagnostic Methods & Early Detection

What if it is not a sporulating fungus?
pathogen isolation & culture
1 – 2 days for most bacteria
Traditional Diagnostic Methods
10 - 14 days for Rathayibacter

Canker of Kiwifruit
24 October 2016
Why accurate is important

Canker of Kiwifruit
24 October 2016
No
unnecessary
action taken
Appropriate
regulatory action
taken
NegativePositive
Truevalue
Negative Positive
Diagnosis
- Consequence -
- Trade interrupted -
Costly mitigation actions
taken unnecessarily
Biosecurity breached
- incursion results -

Canker of Kiwifruit
24 October 2016

Pictures: International Symposium
on Bacterial Canker of Kiwifruit
Kiwifruit Pathogen
Non-Pathogenic
Highly virulent? New?

Psa Diagnostics
Grethel Busot
• Population level discrimination required for Psa
• Very high background noise – path & nonpath pop’ns

Pseudomonas syringae pathovar actinidiae
• Many pathovars of Pseudomonas syringae
• P. syringae occurs naturally on MANY plant species
• P. syringae occurs naturally in rain & snow
• P. syringae occurs naturally throughout the world
High potential for false positives!

PsyB728
Psa NZ V
Psy ESC1
Psy ESC1
T3SS pathogenicity island
CEL Hrp/hrc cluster EEL
Sunshine Coast, Australia – 5 may 2014 – Busot, Arif, & Stack
Pathogenic strain Psy
Pathogenic strain Psa
nonPathogenic strain
nonPathogenic strain

NGS platforms:
PacBio & Illumina MiSeq
• Couplets: inner ring
PacBio, adjacent ring
illumina
• Comparisons for errors:
NGS platforms,
assemblers, assembly
methods (De novo and
genome mapping)
• Comparative genomics:
Genomic variation as a
function of sequencing
and assembly methods

Identification of diagnostic sequences in P. syringae pv. actinidiae
Target selection

Globa
l
outbr
eak
NZ LV
strains
Japan
ese
strains
Korea
n
strains
Bacterial Pathovar Determinants
Effector Gene-based diagnostics
Low virulent strains
HIGH virulent strains

Multiplex endpoint PCR-based diagnostics
Genome informed identification of diagnostic sequences in Pseudomonas syringae pv. actinidiae
Hop S2
Hop 01
Hop Z5
Hop Z5
Hop Z3
All Psa strains
Low virulent Psa strains
High virulent Outbreak Psa strain
Determine assay sensitivity

James Stack
PBCRC2002 & PBCRC2156
• 2002: Develop & validate laboratory and field
• 2156: Deploy validated field diagnostic protocols for
plant pathogenic bacteria to the pathovar level of
discrimination
• Increase national capability in plant bacteriology
GOAL: in-field detection
• Isothermal amplification technologies
• Many desirable features for the field (no
heat cycling)
• Very sensitive, accurate and fast!

Field deployable assays for identification of Psa
hop Z5 LAMP
hop Z3 LAMP
Loop-mediated isothermal amplification LAMP
Specificity
Sensitivity

Isothermal amplification (LAMP) based diagnostics/ Genie
Field deployable assays for identification of Pseudomonas syringae pv. actinidiae
With Loop primers Without Loop primers
LAMP: 6 primers

Isothermal amplification (LAMP) based diagnostics/Thermocycler
1 23 4 5 6 7 1213 14 15 16 18 19 21 22 8 9
Global outbreak/Psa VPsa Psa LV
hopZ3
hopZ5
Many non Psa strainsMany Psa strains

Isothermal amplification (LAMP) based diagnostics/Thermocycler
Alternative Visualization
technologies:
■ SYBR Green
■ Lateral flow device
Internal control
Positive samples
hopZ3 + LOOP

Recombinase Polymerase Amplification (RPA) coupled to Lateral
Flow Device
FAM-biotin/digoxigenine amplicons
Modified from Journal of Virological Methods, 2014;208:144–151

Isothermal amplification (RPA) based diagnostics coupled to LFD
Specificity

Sensitivity
hopZ3/RPA primers-probe
Psa V
hopZ5/RPA primers-probe
Psa V
Isothermal amplification (RPA) based diagnostics coupled to LFD
Differential sensitivity – hopZ3 and hopZ5

Isothermal amplification (RPA) based diagnostics
Multiplexing with RPA? - Yes

Canker of Kiwifruit
24 October 2016
All good in the lab – does it work in the field?

Field Validation & End-user Training
Australia - February 2016
Science Exchange - August 2016

Victoria Kiwi Orchard
In & Out

Victoria Kiwi Orchard – Psa?Samples: fruit, leaves, twigs

Tailgate Diagnostics

Moving advanced diagnostics to the fieldField-deployable technologies are here
LAMP and RPA isothermal technologies in use

SYBR Green visualization

Lateral Flow Device visualization
control
positive

16S bacteria
general
H2O HopZ3+C
In the orchard – all good!
NO Psa in Victoria orchard!

Victoria Kiwi Orchard – Psa?Biosecurity Staff Training

Biosecurity Staff Technology Training

24 October 2016
Dude! This is so
cool!
Even Forrest Gump can do this

New Zealand - February 2016

End-User Workshop – 23 October 2016

Acknowledgements

THE PBCRC Team

James Stack
Genome-Informed
Diagnostics
Thank you &Have a nice
day!

Field-Deployable Detection and Diagnostics - Rathayibacter toxicus
NZ Plant & Food 22 February 2016
Sample Prep

Genomic analyses of the select agent Rathayibacter toxicus
APS Annual Meeting - Tampa – 31 July 2016

NPDN National Meeting
Crystal City, VA
9-10 March 2016
What’s next? Where will this technology lead us?
• The FERA SMART spore trap combines:
 automated loop-mediated isothermal amplification
(LAMP) analysis
 to identify pathogens and measure spore loads
 a weather station and a communication capability
(both satellite and mobile phone network)
 sends diagnostic and weather data to a central
facility.
• Collaboration between OptiGene Ltd, Fera, The
University of Hertfordshire, Bayer Crop Science and
Frontier Agriculture.
Smart, sophisticated, in-field pathogen detection with
wireless communication.

NPDN National Meeting
Crystal City, VA
9-10 March 2016
NextGen NPDN: A National Network with Global Implications
Automated, rapid detection of DNA from airborne spores as a network of samplers to
inform growers
Three new projects starting April 2015: Potato pathogens (TSB); Beet Pathogens (TSB);
Arable pathogens (HGCA) (+ AgriTech catalyst application pending)
Miniature Virtual Impactor (patented), which
samples at high flow rate into liquid
(incubation media or extraction buffer)
Future Work:
Isothermal DNA
amplification
9
Smart Spore Traps
Imagine all that in a drone!
Moving advanced diagnostics to the field
LAMP isothermal technologies in use

inoculation infection colonization reproduction dispersal
time
Pathogenpopulation
critical action
point
diagnostic
symptoms
PCR detection limit
104
103
102
101
disease spread
More time to respond
& prevent spread

Session 2: Genome-Informed Diagnostics - In-field Detection of Bacterial Plant Pathogens

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Session 2: Genome-Informed Diagnostics - In-field Detection of Bacterial Plant Pathogens