SECURE

1. SECURE QLK5-CT-2002-01813 SECURE (QLK5-CT-2002-01813)

2. SECURE • StEm Canker of oilseed rape: molecular tools and mathematical modelling to deploy dUrable REsistance.

3. SECURE • What is Oilseed rape? • What is stem canker? • Current methods of control? • The need for durable resistance • Project aims? • Who benefits from SECURE?

4. 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)

5. What is “stem canker”? • Globally important disease of oilseed rape • Caused by fungus called Leptosphaeria maculans • Infects in autumn • Major damage at harvest Basal stem canker at end of the growing season

6. What is “stem canker”? 3. Fungus grows down through leaf to… 1. Airborne spores land on young crop in autumn 4. infect the base of the stem 2. Spores infect to cause leaf lesions 5. Cankers grow during late spring/early summer and can “girdle” stem

7. 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.

8. 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

9. 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!

10. 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!!

11. 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”

12. 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

13. 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

14. 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

15. Visit the website www.secure.rothamsted.ac.uk/

16. Methods developed during SECURE • Ascospore shower inoculation protocol

17. Methods developed during SECURE • Green-Fluorescent Protein expressing isolates of L. maculans developed • Stem of oilseed rape cv. Darmor viewed under white light with visible necrosis at leaf scars of inoculated leaves. • 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

18. Ascopores Latent period Petiole Canker development Phoma spot visible Canker severity Results from Workpackage 1Development of a life cycle model for Leptosphaeriamaculans Developed during the first two years (2003-04) Stepwise model Validated with data collected during 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.

19. Results from Workpackage 2Effects 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, Bernard-Samain S, Balesdent MH, Rouxel T (2007) Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeriamaculans 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 AvrLm1avirulence gene of the DothideomyceteLeptosphaeriamaculans. Molecular Microbiology 60:67-80 • Gout L, Kuhn ML, Vincenot L, Bernard-Samain S, Cattolico L, Barbetti M, Moreno-Rico O, Balesdent MH, Rouxel T (2007) Genome structure impacts molecular evolution at the AvrLm1avirulence locus of the plant pathogen Leptosphaeriamaculans. Environmental Microbiology 9:2978-2992

20. Results from Workpackage 2 continuedEffects 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 Leptosphaeriamaculans (phoma stem canker of oilseed rape). European Journal of Plant Pathology, 114: 77-89

21. Results from Workpackage 3 Stachowiak et al., 2006.  Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe. European Journal of Plant Pathology 114: 67-75. Race structure of Leptosphaeriamaculans population characterised across Europe

22. Results from Workpackage 3 continued Multi-site evaluation of resistance (2002-2005):

23. Results from Workpackage 3 continued Multi-site evaluation of resistance (2002-2005): *2005 data only **2003 and 2005 data only

24. Results from Workpackage 3 continued 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 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 Leptosphaeriamaculans in Brassicanapus. New Phytologist, 170, 129-141

25. Results from Workpackage 3 continued 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. GFP (Green Fluorescent Protein) – expressing isolates of L. maculans developed GFP – expressing isolates used to These results were published in the following peer-reviewed publication: Huang et al., 2009. Quantitative resistance to symptomless growth of Leptosphaeriamaculans(phoma stem canker) in Brassicanapus (oilseed rape). Plant Pathology, 58, 314-323.

26. Results from Workpackage 4 Modelling investigated the merits of pyramiding vs. sequential deployment of resistance genes?

27. 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

28. SECURE For more details on the SECURE project, please see the website: www.secure.rothamsted.ac.uk

29. SECURE consortium