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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Pests and seed health testing. General introduction on detection
of bacteria, viruses, fungi and nematodes in seeds
Valérie GRIMAULT, GEVES-SNES Phytopathology laboratory ISTA Seed Health Committee
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Main pests and their incidence on seed infestation
Viruses Bacteria
Fungi Nematodes
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Non cellular organisms: DNA or RNA with an external envelop: capsid
Seed health testing: -> species
What symptoms can be observed on mother plants in seed production? – Mosaics, stunting, necrosis
How are seeds contaminated – Contamination of mother plant
• Base seeds • Mechanical: contact, men i.e: TMV/tomato,
PMMoV/pepper • With vectors :
– Insects i.e aphids: BCMNV/bean, LMV/l
Viruses
LMV / lettuce
PeMV / pea
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Localisation in an infected pepper seed: in epidermis and parenchyma, not detected in embryo or endosperm
Example of localisation of PMMoV
Endosperm
Embryo
Epidermis
Endosperm
Embryo
Endosperm from Genda et al., 2005
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Cellular organisms
Main genus: Agrobacterium, Clavibacter, Xanthomonas, Pseudomonas, Erwinias, Acidovorax, Ralstonia
Seed health testing: – species and pv.
Bacteria
Pseudomonas
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
What symptoms can be observed on mother plants in seed production? – Cankers, chlorotic spots, necrosis,
watersoaked areas, wilting, rot… How are seeds contaminated ? – Contamination of mother plant
• By seeds • By woundings or natural openings
(stomates) • By watering or rain • By men during cultural practices
– Pollution • During harvest • During cleaning and sorting of seeds
Bacteria
Xanthomonas / cabbage
Acidovorax / corn salad
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
External and internal contamination of bean seeds by X. axonopodis pv. phaseoli and P. savastanoi pv. phaseolicola
Analysis of seed lots coming from Michigan, USA
% lots
Nb lots tested
% Conta-
Conta. I + E
Conta Ext. only
Conta. Int. only
1976 58 46
31 % 11 %
44 % 20 %
39 % 80 %
17 % 0 %
1977 30 34
23 % 12 %
57 % 25 %
43 % 67 %
0 % 8 %
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Pluricellular organisms
Mycelium: growing vegetative part
Reproduction : - Sexual/perfect : spores -Vegetative/imperfect: conidia (spores)
Conservation: - Sclerotes -Asexual reproduction ( chlamydospores)
Seed health testing: àgenus or species
Fungi
Alternaria dauci
Peronospora valerianellae
Fusarium / soybean
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
What symptoms can be observed on mother plants in seed production? – Necrosis, rot, sporulations,
damping off How are seeds contaminated ? – Contamination of mother plant
• Base seeds • Wounding • Wind • Watering or rain
– By men during cultural practices – Pollution
• During harvest • During cleaning and sorting of
seeds
Fungi
Phoma / beet
Sclerotinia / cabbage Alternaria / carrot
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Localization of fungi in seeds Alternaria / Brassicae seeds
Photos S Pochon University of Angers
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Size of pathogens
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Worms, small size (200-8000 x 10-40 µm) Parasites of plants: stylet Ectoparasites (soil): Pratylenchus, Xiphinema Endoparasites (enter in tissues) – Sedentary:
• Cysts: Heterodera, Globodera • Galls: Meloïdogyne
– Migrating: • Soil or tissues of plants :
Pratylenchus (roots), Ditylenchus (stems)
Nematodes
Ditylenchus dipsaci
LSV Rennes
Globodera
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Seed health testing: -> species What symptoms can be observed on mother plants in seed production? – Stunting of stems or leaves
How are seeds contaminated? – Contamination of mother plant
• Base seeds • Soil
– Pollution: • Soil • Harvesting • Sampling
Nematodes
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Seed health testing
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Infested seeds can constitute a disease focus and decrease commercial value of a culture – Damages caused by seed
transmitted pests : • Lower germination, damping off
Why testing for seed health? figure 1 : Impact des fusarioses sur la faculté germinative des semences de blé tendre et blé dur : une semence contaminée n'a qu'une chance sur quatre de générer une plantule normale
0
20
40
60
80
100
0 20 40 60 80 100Taux de semences contaminées fusarioses (%)
(Analyses SNES sur Gélose)Fa
culté
ger
min
ativ
e (
Blé durBlé tendre
Fig: J.A. Fougereux, FNAMS
sans prétraitement
R2 = 0,8477
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40
0 20 40 60 80 100
% alternaria dauci
% a
norm
aux
pour
ris
Fig: B. Meriaux, FNAMS
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Damages caused by seed transmitted pests : – Lower seed production,
abnormal seeds – Symptoms on stems,
petioles, fruits decreasing the visual quality of the product
Why testing for seed health?
Pseudomonas / bean
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Damages caused by seed transmitted pests : – Symptoms on roots,
stems, leaves causing a decrease of yield
– Toxin production
Why testing for seed health?
27,00
60,00
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0.0
0.0 15.00
57.00
17.00
1.00 0
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LotNaturellement
Contaminé
Contaminationà 10 000 sp
vivantes/grain
Contaminationà 1000 sp
vivantes/grain
Contaminationà 10 000 sp
mortes/grain
Lot Sain
Nom
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/gra
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% d'épis contaminés àN+1 sur Brains surl'Authion% d'épis contaminés àN+1 sur Bourges
Nb spores / grain à N+1
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Knowing risky lots: allows to decide for seed treatments – Chemical – Disinfection (hot water, hypochlorite)
Introduction of a pest in a new region: quarantine conditions Use of healthy and tested seeds : warranty for – Governments – Seed companies – Growers
Why testing for seed health?
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Regulatory purposes: – Certification: % of infection – Quarantine: zero tolerance
• Phytosanitary certificates (imports/exports) • Phytosanitary passports
– IPPC: ISPM movement of seeds
Commercial purposes – Warranty for clients that seeds are healthy
Seed health testing for
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Official international methods: ISTA (International Seed Testing Association) methods compulsory for orange certificates: 30 SH methods ISTA seed health methods
International: ISHI-Veg (International Seed Health Initiative) ISF seed health methods
Published/internal/ validated methods
Analysis according to methods
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
General principles for pests detection : – Extraction
• Soaking, seed wash, hydratation (Pseudomonas, Xanthomonas, Peronospora, Tilletia, Ditylenchus,…)
• Grinding (viruses) • Direct growth on media (Ascochyta, Fusarium,
Alternaria, ...) • Tissue digestion (Ustilago) • Staining (Neotyphodium)
– Isolation • Soaking
– Identification
Seed health testing
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Sample size: – Minimum sample size generally – Defined according to epidemiological studies or
experience, growth conditions – Depends of host/pest and feasability – I.e: 5 000 Psp/bean; 30 000 Xcc/brassicas
Subsample size: – Maximum sample size generally – Depends of level of detection of the method: detection
of 1 infected seed in XX – I.e : 1 000 Psp/bean; 10 000 Xcc/brassicas
Sample and subsample size
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Grinding for extraction No isolation Identification: – Serological tests – RT-PCR – Indexing/biotests
Detection of viruses
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Soaking of seeds and isolation on semi-specific media – Identification
• Morphological • Pathogenicity tests • PCR tests
ELISA Immunofluorescence Moledular biology: bio-PCR, d-PCR Biotests: grow-out
Detection of bacteria
Xanthomonas campestris pv. campestris
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
With incubation (media, blotter) Without incubation – Direct observation of
seeds – Seed wash and
observation – Extraction and staining
of embryos and tissues – Observation of
symptoms on plantlets (grow out)
– Serology: « Immunoblot »
Detection of fungi
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Extraction – Soaking
Isolation – By concentration
Identification – Morphology – PCR
Detection of nematodes: Ditylenchus dipsaci
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Analytical sensitivity: level of detection Analytical specificity: risk of false positive and false negative results Repeatability: variation within a lab Reproducibility: variation between labs (or analysts) PM 7/98 (2) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity ISTA SHC Guidelines ISTA Seed Health Committee
What to check for setting up a SH testing method
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Example of Ditylenchus/alfalfa seeds
On the importance of performance criteria of methods
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
One method have risks of false negative results and a lower recovery of nematodes
Ditylenchus/alfalfa seeds
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ISTA Seminar ‘Molecular tools for seed quality and seed health’ June 2015 – Valérie GRIMAULT
Thank you for your attention