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)

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)

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

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

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%

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

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

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 uredosores. Then qualify pathotype in qualitative laboratory test  Reference platform to share?

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?

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

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

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 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

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)

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? ……………………………………………?