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

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

2. The synthesis is focused on disease resistance (fungi, bacteria, virus) but if important could include insect resistance…. STEP 1 : ask for Species x Pathogen and send the questionnaire (Aug 09 – CB,MS) 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

3. Answers : 16 / 28 partners no interest in disease resistance=3 Broadleaves Conifers • Important selection criterion in case of clonal selection • Important in northern conifers in northern and continental conditions • Different situations: sympatric host/pathogen situations, new pests on autochtoneous species, pests on exotic species 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

4. Disease Resistance Evaluation on geneticexperimentswhenattacksoccur Selectionobjective • Methods: age, sampling , scoring • Advantages / limits • Methods : age, sampling, scoring • Advantages / limits Agreement on protocols • Poplars : Melampsora sp, Xanthomonas • Wild cherry: Blumerellia, Pseudomonas syringae • Ash: bacterial canker, chalara ? • Scots pine: Lophodermium, Melampsora p. • Norway spruce: Heterobasidion annuosum C. BASTIEN – INRA Orléans • Larches: Lachnellula + some individual expertises 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

5. Evaluation on geneticexperimentswhenattacksoccur • Advantages: (1) evaluation of geneticvariability for diseaseresistance • (2) evaluation of effect of indirect selection • (3) no specificcosts • Limits : - no control of homogeneity of infection • - repeatability not known • - timing of scoringattackscouldbe not optimal  biais • Methods : often% of infectedtrees(0/1) or (0,1,2) scores • Precisionisdetermined by nb of treesobserved per prov/progeny/clone ! • Effectives in field trial are optimized for quantitative observations … • Nb of ind/unitminimum significantdifference in % at 5% • 20 33% • 30 24% • 50 20% • 75 15% • 100 2% • 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 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

6. Diseaseresistance as Selectionobjective • Indirectevaluation of resistance : • Morphological markers • Biochemicalmarkers • Molecular markersMAS Evaluation in field / nurseryexperimentundernatural infection Evaluation in specificexperimentunderartificialinfection Advantages - close to economic impact- control of homogeneity and - scoringintegrating all resistancelevel of infectionmechanisms - control of pathogen variation - limited extra costs - control of environmental conditions - non destructive Under testing … • Limits • dependantfromnatural infection - size of experimentsintensity - 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

7. Diseaseresistance as Selectionobjective • Indirectevaluation of resistance : • Morphological markers • Biochemicalmarkers • Molecular markersMAS Evaluation in field / nurseryexperimentundernatural infection Evaluation in specificexperimentunderartificialinfection 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

8. Evaluation in field/nursery experimentundernatural 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 optimizegenotyperanking(To bedefinedaccording to the hybrid formula) Control of pathogen population : collection of infected Robusta leavesearlySeptember and isolation of 60-100 uredosores. Thenqualifypathotype in qualitative laboratory test Referenceplatformto share? Optimal age for evaluation: 1-2 C. BASTIEN – INRA Orléans Score INRA 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

9. Evaluation in field/nursery experimentundernatural infection Score atplant levelend of August??(end of infection) Score atleaflevelend of July (mid-infection) C. BASTIEN – INRA Orléans Score 1-8 Score INBO Score INRA Other? 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

10. Evaluation in field/nursery experimentundernatural infection Score atplant levelend of August??(end of infection) Score atleaflevel 1-8end of July (mid-infection) 1 = no uredinia 2 = few uredinia difficult to detect 3 = uredinia easy to detect but not joined 4 = joined uredinia covering less than10% 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 Score INBO Score INRA Other?  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

11. Evaluation in field/nursery experimentundernatural infection Score atplant levelend of August??(end of infection) Score INBO Score INRA C. BASTIEN – INRA Orléans 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 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  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

12. L G.I Evaluation in specificexperimentunderartificial infection Experimental design : - Randomized design (I or C blocks, 1-or n-tree-plot), between5 to 8 trees / clone - Standard clones (R, S) to optimizegenotyperanking (To bedefined) Artificial inoculation: - 1-yearafter plantation, in Sept-Oct - 2 infection spots per tree on main stem - measure of stem diameterat inoculation at medium heightbetween the 2 points ? - Strain ? Spm, other… - conc. 108cells/ml Scoring system: - 1 and 2 yearsafter inoculation, at the spot level - one quantitative measure : CankerLength (L) - one qualitative score: Girdling index (0-5) GI=2 GI=3 GI=4 GI=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

13. 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++) 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) 6: finalization of of assessment guidelines per Species x pathogen before 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

14. 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? • ……………………………………………? 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