SECURE
QLK5-CT-2002-01813
SECURE (QLK5-CT-2002-01813)
SECURE
StEm Canker of oilseed rape: molecular
tools and mathematical modelling to
deploy dUrable REsistance.
SECURE (QLK5-CT-2002-01813)
SECURE
What is Oilseed rape?
What is stem canker?
Current methods of control?
The need for durable resistance
Project aims?
Who benefits from SECURE?
SECURE (QLK5-CT-2002-01813)
What is oilseed rape (OSR)?
Agricultural crop grown
throughout most of Europe, SE
and W Australia and in Canada
and the USA.
OSR typically grown in Europe is
a “Winter” type - sown in autumn
and harvested the following
summer.
Crop is grown primarily for seed
production – seeds are crushed
to produce oil (edible, industrial)
SECURE (QLK5-CT-2002-01813)
What is “stem canker”?
Globally important
disease of oilseed
rape
Caused by fungus
called Leptosphaeria
maculans
Infects in autumn
Major damage at
harvest
SECURE (QLK5-CT-2002-01813)
Basal stem
canker at end
of the growing
season
What is “stem canker”?
3. Fungus
grows down
through leaf
to…
1. Airborne
spores land on
young crop in
autumn
2. Spores infect to
cause leaf lesions
4. infect
the base
of the
stem
5. Cankers grow during
late spring/early summer
and can “girdle” stem
SECURE (QLK5-CT-2002-01813)
Current methods of control
Fungicide (Chemical control)


Environmentally unfriendly
Expensive for the farmer (time/money)
Cultivation techniques

Limited in usefulness
Host resistance?
SECURE aimed to increase our understanding of
host resistance and the best way to use this
resource.
SECURE (QLK5-CT-2002-01813)
What is host resistance?
Like us, plants and fungi have genes

Genes control all processes of living things
Some of these genes (resistance genes)
help fight attack by diseases – fungi,
viruses, bacteria.
However, diseases also have genes
(avirulence genes) which correspond and
interact with those in the plant
SECURE (QLK5-CT-2002-01813)
What is host resistance? - continued
Interaction between specific corresponding
resistance and avirulence genes, good for
the plant:


Plant recognises presence of fungus
Resistance mechanisms prevent infection
However, in the “war” of the genes,
sometimes the plant resistance genes are
overcome!
SECURE (QLK5-CT-2002-01813)
What is host resistance? - continued
The fungus mutates to loose the function of
the avirulence gene




No interaction with the plant resistance gene
The plant no longer recognises the presence
of the fungus
No resistance response
Infection of the plant!!
SECURE (QLK5-CT-2002-01813)
The need for durable resistance
Limited number of plant resistance genes
can be used in breeding programmes
Most are overcome in 3-5 years
However, some resistance genes remain
effective for 5-10 years – why?
“Durable resistance”
SECURE (QLK5-CT-2002-01813)
The need for durable resistance –
cont’d
Better understanding of interactions allows
“smart” use of available resistance
Allows development of breeding strategies to
maximise durability of resistance.
Oilseed rape flowers
in springtime
SECURE (QLK5-CT-2002-01813)
Aims of SECURE
Develop a mechanistic model of the life cycle of
Leptosphaeria maculans (pathogen)
Study molecular mechanisms which generate
virulent isolates of the pathogen
Analyse effects of genetic background and
environment on resistance durability
Construct models and disseminate strategies for
deployment of durable resistance
SECURE (QLK5-CT-2002-01813)
Who benefits from SECURE
People within the European Union


Reduced pesticide input
Reduced environmental impact
Global agricultural industry


Breeding of improved types of oilseed rape
Sustainable oilseed rape production
Global scientific community


Increased knowledge
Modelling of novel system
SECURE (QLK5-CT-2002-01813)
Visit the website
www.secure.rothamsted.ac.uk/
SECURE (QLK5-CT-2002-01813)
Methods developed during
SECURE
Ascospore shower inoculation protocol
SECURE (QLK5-CT-2002-01813)
Methods developed during
SECURE
Green-Fluorescent Protein expressing
isolates of L. maculans developed
a. Stem of oilseed rape cv. Darmor
viewed under white light with visible
necrosis at leaf scars of inoculated
leaves.
b. The same stem viewed under
fluorescent light, showing hyphae of
GFP-expressing transformed L.
maculans isolate ME24 indicating
initial infection of stem through the
petiole
SECURE (QLK5-CT-2002-01813)
Results from Workpackage 1
Development of a life cycle model for
Leptosphaeria maculans
Developed during the first two years
Ascopores
(2003-04)
Stepwise model
Validated with data collected during Latent
period
SECURE
Published in: Evans et al. (2007).
Development and validation of a
model to describe the progress phoma
stem canker epidemics in England and
France. Proceedings of the 12th
International Rapeseed Congress,
Wuhan, China, 26-30 March 2007, 4:
161-164.
SECURE (QLK5-CT-2002-01813)
Petiole
Phoma spot
visible
Canker development
Canker
severity
Results from Workpackage 2
Effects of pathogen variation at Avr loci on
durability of resistance
Cloning and functional analysis studies done on
effector genes AvrLm1 and AvrLm6
Three publications to date with more to come:



Fudal I, Ross S, Gout L, Blaise F, Kuhn ML, Eckert MR, Cattolico L, BernardSamain S, Balesdent MH, Rouxel T (2007) Heterochromatin-like regions as
ecological niches for avirulence genes in the Leptosphaeria maculans genome:
Map-based cloning of AvrLm6. Molecular Plant-Microbe Interactions 20:459-470
Gout L, Fudal I, Kuhn ML, Blaise F, Eckert M, Cattolico L, Balesdent MH, Rouxel
T (2006) Lost in the middle of nowhere: the AvrLm1 avirulence gene of the
Dothideomycete Leptosphaeria maculans. Molecular Microbiology 60:67-80
Gout L, Kuhn ML, Vincenot L, Bernard-Samain S, Cattolico L, Barbetti M, MorenoRico O, Balesdent MH, Rouxel T (2007) Genome structure impacts molecular
evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria
maculans. Environmental Microbiology 9:2978-2992
SECURE (QLK5-CT-2002-01813)
Results from Workpackage 2 continued
Effects of pathogen variation at Avr loci on
durability of resistance
Fitness studies using Near Isogenic Isolates (NIIs) for
AvrLm1/avrLm1, AvrLm4/avrLm4, AvrLm6/avrLm6
Ascospore shower inoculation method used
Results suggested a fitness cost was associated with
evolution from avirulence to virulence at both AvrLm1 and
AvrLm4 loci.
There was a difference in the strength of the effect
Results for AvrLm4 work published in:

Huang Y-J, Li Z-Q, Evans N, Rouxel T, Fitt BDL, Balesdent M-H
(2006). Fitness cost associated with loss of the AvrLm4
avirulence function in Leptosphaeria maculans (phoma stem
canker of oilseed rape). European Journal of Plant Pathology,
114: 77-89
SECURE (QLK5-CT-2002-01813)
Results from Workpackage 3
Race structure of Leptosphaeria maculans
population characterised across Europe
Stachowiak et al., 2006. Frequency of
Avirulence Alleles in Field Populations
of Leptosphaeria maculans in Europe. European
Journal of Plant Pathology 114: 67-75.
SECURE (QLK5-CT-2002-01813)
Results from Workpackage 3 continued
Multi-site evaluation of resistance (2002-2005):
SECURE (QLK5-CT-2002-01813)
Results from Workpackage 3 continued
Multi-site evaluation of resistance (2002-2005):
Cultivar
Phom)
stem
canker
%inc
Phoma
stem
canker
G2
index
Apex
Darmor
Darmor-MX
64.6
59.5
23.8
2.94
2.13
0.69
68.7
52.6
50.8
Escort
Eurol
Eurol-MX
Falcon
Samourai
62.3
73.4
31.2
63.3
78.0
2.93
3.54
1.16
2.90
4.06
Shogun
Capitol
Sed (average)
80.3
77.7
2.08
<0.001
F test
SECURE (QLK5-CT-2002-01813)
Phoma stem Phoma stem
lesions
lesion
%inc
G2 index
Phoma leaf
lesions
%inc
Phoma leaf
lesion
% area
1.95
1.34
1.45
64.5
60.0
7.3
2.09
2.02
0.16
67.5
69.6
80.2
66.3
74.2
1.86
2.61
3.69
2.16
2.70
58.5
60.9
9.6
53.0
63.2
1.60
1.70
0.19
1.44
1.95
4.56
3.49
0.147
48.9
87.3
3.81
1.48
3.95*
0.195
68.4
63.0
2.72
2.22
1.50**
0.218
<0.001
<0.001
<0.001
<0.001
<0.001
*2005 data only **2003 and 2005 data only
Results from Workpackage 3 continued
Darmor MX (containg the Rlm6 resistance gene used to investigate
effect of temperature and leaf wetness on infection.
Rlm6 resistance found to be temperature sensitive
Results published in: Huang Y-J, Evans N, Li Z-Q, Eckert M, Chevre
A-M, Renard M, Fitt BDL (2006). Temperature and leaf wetness
duration affect phenotypic expression of Rlm6-mediated resistance
to Leptosphaeria maculans in Brassica napus. New
Phytologist, 170, 129-141
Symptoms on the leaves of Brassica napus Darmor (lacking Rlm6) and DarmorMX (carrying
Rlm6) inoculated with ascospores (without wounding, a,b,c,d,e; with 48 h wetness) or
conidia (after wounding, f,g; with 72 h wetness) of Leptosphaeria maculans carrying the
effector gene AvrLm6. (a) Large grey lesions on Darmor 11 d after inoculation at 15°C; (b)
no visible symptoms on DarmorMX 11 d after inoculation at 15°C; (c) small dark spots
(black arrows) and green islands (white arrows) on DarmorMX 18 d after inoculation at
15°C; (d) small dark spots (arrows) on DarmorMX 11 d after inoculation at 20°C; (e) large
grey lesions on DarmorMX 16 d after inoculation at 25°C; (f) large grey lesions on Darmor
16 d after inoculation at 25°C; (g) large grey lesions on DarmorMX 16 d after inoculation at
25°C. Bar, 5 mm
SECURE (QLK5-CT-2002-01813)
Results from Workpackage 3 continued
GFP (Green Fluorescent Protein) – expressing isolates
of L. maculans developed
GFP – expressing isolates used to
These results were published in the following peerreviewed publication: Huang et al., 2009. Quantitative
resistance to symptomless growth of Leptosphaeria
maculans (phoma stem canker) in Brassica napus
(oilseed rape). Plant Pathology, 58, 314-323.
SECURE (QLK5-CT-2002-01813)
a) Stem of oilseed rape cv. Darmor
viewed under white light with visible
necrosis at leaf scars of inoculated
leaves. b) the same stem viewed
under fluorescent light, showing
hyphae of GFP-expressing
transformed L. maculansisolate
ME24 indicating initial infection of
stem through the petiole.
Results from Workpackage 4
Modelling investigated the merits of
pyramiding vs. sequential deployment of
resistance genes?
SECURE (QLK5-CT-2002-01813)
Pyramiding vs. sequential
deployment of resistance genes?
Conclusions:
Fitness cost of virulence high – Pyramid
resistance genes
Fitness cost of virulence low – Sequential
deployment more beneficial
SECURE (QLK5-CT-2002-01813)
SECURE
For more details on the SECURE project,
please see the website:
www.secure.rothamsted.ac.uk
SECURE (QLK5-CT-2002-01813)
SECURE consortium
SECURE (QLK5-CT-2002-01813)