1. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols to assess disease resistance Catherine Bastien, INRA (P1) Marijke Steenackers, INBO (P4)

2. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Assessment of Disease resistance/tolerance (P1 INRA/P4 INBO) STEP 1 : ask for Species x Pathogen and send the questionnaire (Aug 09 – CB,MS) The synthesis is focused on disease resistance (fungi, bacteria, virus) but if important could include insect resistance…. STEP 2 : collect full information (especially protocols used) ( End Sept 09 – CB, MS) STEP 3 : synthesis of general information on assessment of disease resistance in tree breeding programs ( End Oct 09 – CB,MS) STEP 4: synthesis by Species x pathogen to agree on common protocols ( End Nov 09 – CB,MS + partners concerned) STEP 5: preparation of assessment guidelines by Species x pathogen (2 pages) ( End 09 – CB,MS + partners concerned)

3. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Assessment of Disease resistance/tolerance STEP1 Answers : 16 / 28 partners no interest in disease resistance=3 Broadleaves Conifers Different situations: sympatric host/pathogen situations, new pests on autochtoneous species, pests on exotic species Important selection criterion in case of clonal selection Important in northern conifers in northern and continental conditions

4. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Disease Resistance Selection objective Evaluation on genetic experiments when attacks occur Methods: age, sampling, scoring Advantages / limits Methods : age, sampling, scoring Advantages / limits Poplars : Melampsora sp, Xanthomonas Wild cherry : Blumerellia, Pseudomonas syringae Ash : bacterial canker, chalara ? Scots pine : Lophodermium, Melampsora p. Norway spruce : Heterobasidion annuosum Larches : Lachnellula Agreement on protocols + some individual expertises

5. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Evaluation on genetic experiments when attacks occur Advantages : (1) evaluation of genetic variability for disease resistance (2) evaluation of effect of indirect selection (3) no specific costs Limits : - no control of homogeneity of infection - repeatability not known - timing of scoring attacks could be not optimal  biais Recommendations : - adapted to high level of natural infection (>25%) - identify extremes to evaluate the discrimination power of % - if possible, use a quantitative method of scoring - control presence of the pathogen on samples collected in the experiment to valid observations made on symptoms Methods : often % of infected trees (0/1) or (0,1,2) scores Precision is determined by nb of trees observed per prov/progeny/clone ! Effectives in field trial are optimized for quantitative observations … Nb of ind/unitminimum significant difference in % at 5% 2033% 3024% 5020% 7515% 100 2%

6. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Disease resistance as Selection objective Evaluation in field / nursery experiment under natural infection Evaluation in specific experiment under artificial infection Indirect evaluation of resistance : -Morphological markers -Biochemical markers -Molecular markers  MAS Under testing … Advantages - close to economic impact - control of homogeneity and - scoring integrating all resistance level of infection mechanisms - control of pathogen variation - limited extra costs - control of environmental conditions - non destructive Limits -dependant from natural infection - size of experiments intensity - costs (suppl. plants, ……) -No control of pathogen variation - infrastructures

7. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Disease resistance as Selection objective Evaluation in field / nursery experiment under natural infection Evaluation in specific experiment under artificial infection Indirect evaluation of resistance : -Morphological markers -Biochemical markers -Molecular markers  MAS

8. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols for poplar resistance to Mlp Evaluation in field/nursery experiment under natural infection Experimental design : - Randomized design (I or C blocks, 1-or n-tree-plot) -Reference clones to control genetic variation of pathogen population Robusta (0), Ogy (1), Candicans (2), Brabantica (3), Unal (4), Rap (5), 87B12 (6), Beaupré (7), Hoogvorst (8) - Standard clones (R, S) to optimize genotype ranking (To be defined according to the hybrid formula) Optimal age for evaluation : 1-2 Score INRA Control of pathogen population : collection of infected Robusta leaves early September and isolation of 60-100 uredosores. Then qualify pathotype in qualitative laboratory test  Reference platform to share?

9. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols for poplar resistance to Mlp Evaluation in field/nursery experiment under natural infection Score at leaf level end of July (mid-infection) Score at plant level end of August??(end of infection) Score 1-8Score INBOScore INRAOther?

10. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols for poplar resistance to Mlp Evaluation in field/nursery experiment under natural infection Score at leaf level 1-8 end of July (mid-infection) Score at plant level end of August??(end of infection) Score INBOScore INRAOther? 1 = no uredinia 2 = few uredinia difficult to detect 3 = uredinia easy to detect but not joined 4 = joined uredinia covering less than 10% of the leaf area 5 = joined uredinia covering between 10% and 25% of the leaf area 6 = joined uredinia covering between 25% and 50% of the leaf area 7 = joined uredinia covering between 50% and 75% of the leaf area 8 = joined uredinia covering more than 75% of the leaf area  400 to 600 trees per day High repetability

11. C. BASTIEN – INRA Orléans F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols for poplar resistance to Mlp Evaluation in field/nursery experiment under natural infection Score at plant level end of August??(end of infection) Score INRA 1 = 0 uredinia on the plant (qualitative Resistance) 2 = few uredinia hard to detect 3 = many uredinia per leaf but no decoloration neither necrotic zones 4 = many leaves infected but still green. Limited decolorated or necrotic zones, no defoliation 5 = numerous leaves highly infected with decoloration and necrotic areas, significant defoliation but between ½ and 1/3 of green leaves on the top of the plant. 6 = Most of leaves have decolorations and necrotic areas, important defoliation (30%-50%), less than ¼ of remaining leaves are still green. 7 = Important defoliation (>50%), no more significant elongation, only few remaining leaves on the top Score INBO 0 = 0 uredinia on the plant (qualitative Resistance) 0,5 = few uredinia hard to detect 1 = slight infection of the leaves up to 25% of total tree height 1.5 = slight infection of the leaves up to 50% of total tree height 2 = infection of the leaves up to 75% of total tree height 2,5 = infection of the leaves up to 75% of total tree height and beginning of black discoloration 3 = more than 75% of the leaves infected + black discoloration of the lower 25% leaves + beginning of leaf shrivelling 3,5 = more than 75% of the leaves infected + black discoloration of the lower 50% leaves + up to 25% of leaf shedding 4 = black discoloration of the lower 50% leaves and shrivelling of up to 75% of the leaves and + up to 50% of leaf shedding 4,5 = up to 75% of leaf shedding 5 = more than 75% of leaf shedding  400 to 600 trees per day High repetability

12. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols for poplar resistance to bacterial canker Evaluation in specific experiment under artificial infection Experimental design : - Randomized design (I or C blocks, 1-or n-tree-plot), between 5 to 8 trees / clone - Standard clones (R, S) to optimize genotype ranking (To be defined) Artificial inoculation: - 1-year after plantation, in Sept-Oct - 2 infection spots per tree on main stem - measure of stem diameter at inoculation at medium height between the 2 points ? - Strain ? Spm, other… - conc. 10 8 cells/ml Scoring system: - 1 and 2 years after inoculation, at the spot level - one quantitative measure : Canker Length (L) - one qualitative score: Girdling index (0-5) L G.I GI=1 GI=2 GI=3GI=4

13. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols to assess disease resistance 1 : Complete the list of Species x Pathogen in Leuven – Sept 09 ? 2 : collect full information (especially protocols-scoring systems) ( End Sept 09 – CB, MS) 3: synthesis by Species x pathogen to agree on common protocols ( End Nov 09 – CB,MS + partners concerned ) Agreement needed on : scoring scales standard clones (S++, S, R, R++) 6: finalization of of assessment guidelines per Species x pathogen before End 09 – CB,MS + partners concerned 4: preparation of assessment guidelines per Species x pathogen according to a standard format for disease assessment to define before End October 09 – CB,MS 5: synthesis of general information on assessment of disease resistance in tree breeding programs ( End Nov 09 – CB,MS)

14. F O O D A G R I C U L T U R E E N V I R O N M E N T Protocols for disease resistance traits workshopLeuven Sept 2009 Towards common protocols to assess disease resistance Perspectives Needs of reference platform(s) when genetic variation of pathogen is important ? Access to strains? Identification of pathogen populations ? Needs of methodological research for some important diseases? Contacts with pathologists? ……………………………………………?