A ‘Genome to Paddock’ Approach to

Controlling Blackleg of Canola

Steve Marcroft, Marcroft Grains Pathology, Horsham,

Andrew Ware, SARDI,

Angela Van de Wouw, Phil Salisbury & Barbara Howlett,

the University of Melbourne

2

Canola in Australia: high intensity, long growing

season in temperate climate

2.4 m ha sown in 2012; large amount of

blackleg-infested stubble inoculum in 2013

High disease pressure

• Prolific sexual and asexual reproduction

• Large populations of windborne ascospores (inoculum)

– Fungal populations evolve very quickly

– Major gene resistance results in strong selection

pressure towards isolates virulent to that resistance

gene

– Frequency of virulent isolates in populations increases

– On Lower Eyre Peninsula blackleg resistance

‘overcome’ in 2003; prevented (averted) in 2012

Leptosphaeria maculans is a high risk

pathogen for ‘overcoming’ resistance

‘Breakdown’ of resistance in 2003 • in 2000 ‘sylvestris’ cultivars released with major seedling

genes, Rlm1 & LepR3 (no disease); cultivars sown extensively

Eyre Peninsula

2001 and 2002 2003

‘Sylvestris’ cultivars

2003: Beacon

Steve Marcroft

90% yield losses ($30 m) in Eyre Peninsula

Seed withdrawn from sale

2003: Sylvestris

Breakdown of disease resistance

• Increased frequency of isolates attacking cultivars with sylvestris- resistance, not new strain: – isolates collected before 1988 caused disease

– breakdown occurred in other areas where sylvestris cultivars sown extensively

• Why does virulence evolve so rapidly in the blackleg fungus?

Leptosphaeria maculans genome

• Rouxel & Balesdent, INRA & Howlett, Genoscope; URGI, France; Oliver (Perth)

• 12,500 genes; 45 Mb -closely related Stagonospora nodorum (37 Mb)

• Repetitive DNA: 36% genome (9% S. nodorum) – Degenerated transposable elements: truncated &

Class I LTR retrotransposons & Class II TIR DNA transposons

– Repeat induced point (RIP) mutations: occurs prior to meiosis; mutates multi copy DNA; transition from C:G to T:A – stop codons

• Nature Communications (2011)

8

Patchwork genome: gene –rich (GC) &

gene-poor, repeat-rich (AT) blocks

Gene-rich blocks (1 gene per 2.4 kb)

• ‘Invaded plastic’ genome: genes readily gained, lost or mutated

9

Repeat-rich blocks Gene poor (1 gene/ 30 kb) degenerated transposons. Contain only 3.5% of total genes but 20% of effector

(avirulence) genes-AvrLm1 (Rlm1)

ACTACTACTACTACTACTACT ACTACTACTACTACTACTACT

ACTACTACTACTACTACTACT ACTACTACTACTACTACTACT

ACTACTACTACTACTACTACT ACTACTACTACTACTACTACT Avr

ACTACTACTACTACTACTACT ACTACTACTACTACTACTACT

If repeats align correctly during meiosis:

Avirulence genes are located in repetitive regions of genome

Avr

Avr

Avr

or

Avirulence genes identical in parents and

progeny

ACTACTACTACTACTACTACT

OR

If repeats align incorrectly during meiosis

Option 1: Gene duplication and may then

undergo RIP mutation

Option 2: Deletion of Avirulence gene

ACTACTACTACTACTACTACT ACTACTACTACTACTACTACTACTACT--------------- Avr

--------ACTACTACTACTACTACTACT CTACT-------------- Avr

ACTACTACTACTACTACTACT ACTACTACTACTACTACTACTACTACT--------------- Avr Avr

Avirulence genes are located in repetitive regions of genome

Breakdown of Sylvestris resistance

• Sequenced AvrLm1 in isolates

– 137 collected before breakdown of sylvestris resistance (< 2004)

– 158 collected after breakdown (2004 and later)

• Eight fold increase in isolates with deletions of

AvrLm1 during and after resistance breakdown

• Blackleg fungal populations change rapidly

• Need to develop strategies to avoid resistance

breakdown

• Field and pot trials show that cultivars with different

resistance genes can be rotated to avoid resistance

breakdown

12

Staying Ahead of Blackleg

Kurt Lindbeck

Angela Van de Wouw, Barb

Howlett, Phil Salisbury,

Ravjit Khangura

Andrew Ware

Angela Van de Wouw

Steve Marcroft

Vicki Elliott

13

Resistance genotyping (grouping) of Australian lines and cultivars

• Use set of differential isolates

(different Avirulence genes) to

classify all National Variety Trial lines

according to resistance gene

complement (adult and seedling)

• Seven resistance groups:

– R genes previously identified (eg.

Rlm1, Rlm4 and Rlm9; LepR1-3)

– Unknown/new resistance genes

14

Marcroft et al (2012) Crop and

Pasture Science 63, 338-350

Monitoring blackleg virulence & efficacy of

resistance genes across Australia

Single isolates

• Genotype for Avr genes

• Assess disease on range of cultivars in glasshouse

Populations

• Assess disease in field sites and crops

• Genotype ascospores released from stubble onto tape

• Assess disease on range of cultivars in glasshouse infected with ascospores released from stubble

eastern Australia

Surveys of disease severity of cultivars (with

different resistance genes); blackleg monitoring

Internal stem infection

Western Australia

Monitoring blackleg virulence

Single isolates

• Genotype for Avr genes

• Assess disease on range of cultivars in glasshouse

Populations

• Assess disease in field sites and crops

• Genotype ascospores released from stubble onto tape (high throughput)

• Assess disease on range of cultivars in glasshouse infected with ascospores released from stubble

Monitoring virulence of blackleg populations • Stubble collected from different areas across

Australia, placed in wind tunnel & ascospores captured on tape

• DNA extracted and analysed by quantitative PCR for presence/absence of band for AvrLm1, AvrLm6

• Total number spores estimated by qPCR of ITS region

• Pyrosequencing assay for AvrLm4 based on SNP at base 358 leading to amino acid change G120 to R120

• Frequency virulence allele measured; risk resistance breakdown determined in each region

18 Van de Wouw et al. 2010 Plant Path; 2013 J App Microbiol

Single isolates

• Genotype Avirulence genes (molecular assay)

• Assess disease on range of cultivars in glasshouse

Populations

• Assess disease in field sites and crops

• Genotype ascospores released from stubble onto tape

• Assess disease on range of cultivars in glasshouse infected with ascospores released from stubble (high throughput)

Monitoring blackleg virulence & efficacy of

resistance genes across Australia

Monitoring virulence of blackleg populations

20

Resistant Susceptible

•Stubble of different cultivars collected from different regions across Australia

•Stubble wetted and placed above pots

•Ascospore Shower technique: ascospores released from stubble infect seedlings below & disease assessed at plant maturity

Rotation of cultivars

minimises disease

Garnet infected from

A/ Garnet stubble

B/ ATR-Cobbler stubble

A B

Avoiding ‘breakdown’ of resistance in 2012 • After 2008 Hyola 50 and other Group D cultivars sown

extensively on Eyre Peninsula

Eyre Peninsula

2011: Field and pot trials predicted

resistance breakdown of Group D

cvs. (eg. Hyola 50) in 2012

22

Hyola50 infected from Hyola50 stubble

Hyola50 infected from ATR-Cobbler stubble

Breakdown of Hyola 50 Resistance Feb 2012

Disease severity of cultivars from different

resistance groups: Eyre Peninsula field plots

0

20

40

60

80

100

Hyola444 ATR-Marlin ATR-Stingray CB-Telfer Thumper TT

Perc

enta

ge inte

rnal in

fection

Cultivar

Group

A Group

C Group

B

Group

E

October 2012

Group

D

Hyola 50: 90% disease severity

Eyre Peninsula October 2012

Group D Group E

Wagga Wagga NSW Oct 2012

Group D

Financial Savings to Eyre Peninsula

farmers and Seed Companies

• 60,000 ha sown to canola in 2012

• Assuming 50% yield losses if 30% of area sown to

Group D cultivars (eg. Hyola 50) and $500 /tonne

for canola (conservative estimates)

• Benefits

– Farmers have been saved losses of $18 million

– Group D cultivars can still be sown in other

regions, so breeding companies very supportive

of recommendations to avoid resistance

breakdown

Summary • Blackleg fungus (Leptosphaeria maculans) is a high risk

pathogen for breakdown of resistance in canola cultivars

• Resistance breakdown accounted for by location of

avirulence genes in repetitive regions of ‘patchwork’

genome of L.maculans brassicae

• Rotation of resistance genes in cultivars minimises

disease

• ‘Genome to paddock’ approach is used to monitor race

specific virulence of populations derived from stubble

across Australia

– estimates risk of breakdown of resistance

– averted breakdown of resistance in Eyre Peninsula in 2012

29 Rouxel

Balesdent

Blackleg Resistance Groups

Group A –Rlm1 and sylvestris resistance Group B – Rlm4 Group C* – Rlm2, Rlm3, Rlm9, none Group D* – Unknown (Hyola50) seedling resistance Group E* – Unknown (Thumper) seedling resistance Group F* – Unknown (Mustang) seedling resistance Group G – Juncea resistance

*Notes • Group C cultivars have any combination of Rlm2, 3, 9 or no R genes.

When releasing the resistance groups, only cultivars with MR rating or above will be given a resistance group.