session 2: genome-informed diagnostics - in-field detection of bacterial plant pathogens

biosecurity built on science

Plant Biosecurity Cooperative Research Centre

James StackProfessor and Director

In-field Detection of Bacterial Plant PathogensGenome-Informed Diagnostics




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




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.)




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




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




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




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 taxasequenced

12 2 2 16

PsylidMitogenomes 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

biosecurity built on scienceLAMP 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 earsNo 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


LAMP Workshop – La Trobe University24 October 2016


Why rapid is important




highimpact

Impactthreshold

lowimpact

diseasedetection

Critical response point

Time

Dis

ease

sev

erit

y




Localspread

outbreak

EradicationContainment

Containment

Containment – Eradication Window

Increasedspread




Localspread

outbreak

EradicationContainment

Containment

Containment – Eradication WindowContainment to Management

Dis

ease

sev

erit

y

Time

Impact

Critical response point

Early detection

DetectionLimit

SMART SurveillanceBetter 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




Why accurate is important




No unnecessaryaction taken

Appropriate regulatory action

taken

Neg

ative

Positi

ve

True

val

ue

Negative Positive

Diagnosis

- Consequence -

- Trade interrupted -

Costly mitigation actions taken unnecessarily

Biosecurity breached- incursion results -






Kiwifruit Pathogen

Non-Pathogenic

Highly virulent? New?


Psa Diagnostics


• 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!

Hypotheti

cal p

rotein

PsyB728a

HopAA1HrpW

1Eff

ector lo

cus p

rotein

HopM1

AvrE1

Hypotheti

cal p

rotein

Hypotheti

cal p

rotein

HrpRHrpSHrpA2En

donuclease

HrpBHrcJ HrpDHrpEHrpFHrpGHrcC HrpTHrpVHyp

othetica

l pro

tein

Hypotheti

cal p

rotein

HrcU HrcT HrcS HrcR HrcQb

HrcQa

HrpPHrpOHrcN HrpQHrcV HrpJSig

ma 70HrpK1AvrB

3HopX1HopZ3Hyp

othetica

l pro

teintRNA-Le

uqueA

Hypotheti

cal p

rotein

Psa NZ V-13

Psy ESC10

Psy ESC11

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

Global outbreak

NZ LV strains

Japanese

strains

Korean

strains

Bacterial Pathovar DeterminantsEffector Gene-based diagnostics

Low virulent strains

HIGH virulent strains

Multiplex endpoint PCR-based diagnosticsGenome 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




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

SpecificitySensitivity

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/ThermocyclerField deployable assays for identification of Pseudomonas syringae pv. actinidiae

Ladd

er

1 23 4 5 6 7 1213 14 15 16 18 19 21 22 8 9Global outbreak/Psa V

Ladd

erPsa Psa LV

hopZ3

hopZ5

Many non Psa strainsMany Psa strains

Isothermal amplification (LAMP) based diagnostics/ThermocyclerField deployable assays for identification of Pseudomonas syringae pv. actinidiae

Alternative Visualization technologies:

■ SYBR Green

ESC-10

Psa 7 Psa 9

Psa 1

2

Psa

14

Psa 1

5

Psa 8

ESC-11

■ Lateral flow device

Internal controlPositive samples

hopZ3 + LOOP

Field deployable assays for identification of Pseudomonas syringae pv. actinidiae

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

ESC-10

Psa 7

Psa 9

Psa 1

2Psa

14Ps

a 15


Sensitivity

hopZ3/RPA primers-probe

1 ng100 pg10 pg1 pg100 fg

1 fg10 fgH 2

O

Psa V

1 ng100 pg10 pg1 pg100 fg1 fg10 fgH 2

O

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




All good in the lab – does it work in the field?

Field Validation & End-user TrainingAustralia - 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

controlpositive



16S bacteria general

H2O HopZ3+C

In the orchard – all good!

NO Psa in Victoria orchard!Science Exchange - August 2016


Victoria Kiwi Orchard – Psa?Biosecurity Staff Training



Biosecurity Staff Technology Training



Victoria Kiwi Orchard – Psa?Biosecurity Staff Training






Dude! This is so cool!

Even Forrest Gump can do this



Field Validation & End-user TrainingNew Zealand - February 2016

New Zealand - February 2016Field Validation & End-user Training





End-User Workshop – 23 October 2016




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.)




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

AcknowledgementsPlant Biosecurity: Smart Surveillance





THE PBCRC Team





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 MeetingCrystal 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 MeetingCrystal City, VA

9-10 March 2016

NextGen NPDN: A National Network with Global Implications

Smart Spore Traps

Imagine all that in a drone!

Moving advanced diagnostics to the field

LAMP isothermal technologies in use

inoculation infection colonizationreproductiondispersal

time

Path

ogen

pop

ulat

ion

critical actionpoint

diagnosticsymptoms

PCR detection limit

104

103

102

101

disease spread

More time to respond &

prevent spread