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.

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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)

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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)

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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

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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


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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/unit minimum significant difference in % at 5% 20 33% 30 24% 50 20% 75 15% 100 2%


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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 markersMAS

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 experimentsintensity - costs (suppl. plants, ……)-No control of pathogen variation - infrastructures


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Disease resistance as Selection objective

Evaluation in field / nursery experiment under natural

infection


Indirect evaluation of resistance :-Morphological markers-Biochemical markers-Molecular markersMAS


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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 populationRobusta (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?


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Score at leaf levelend of July (mid-infection)

Score at plant levelend of August??(end of infection)

Score 1-8 Score INBO Score INRA Other?

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Score at leaf level 1-8end of July (mid-infection)


Score INBO Score INRA Other?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



8 = joined uredinia covering more than 75% of the leaf area

400 to 600 trees per day

High repetability


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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

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Towards common protocols for poplar resistance to bacterial canker


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. 108 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=3 GI=4

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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)

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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 Towards common protocols to assess.

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