Pests and seed health testing. General introduction on detection of ...

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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

Main pests and their incidence on seed infestation

Viruses Bacteria

Fungi Nematodes

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

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

Cellular organisms

Main genus: Agrobacterium, Clavibacter, Xanthomonas, Pseudomonas, Erwinias, Acidovorax, Ralstonia

Seed health testing: – species and pv.

Bacteria

Pseudomonas

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

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 %

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

Alternaria dauci

Peronospora valerianellae

Fusarium / soybean

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

Phoma / beet

Sclerotinia / cabbage Alternaria / carrot

Localization of fungi in seeds Alternaria / Brassicae seeds

Photos S Pochon University of Angers

Size of pathogens

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

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

Seed health testing

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 100Taux de semences contaminées fusarioses (%)

(Analyses SNES sur Gélose)Fa

culté

Blé durBlé tendre

Fig: J.A. Fougereux, FNAMS

sans prétraitement

R2 = 0,8477

0 20 40 60 80 100

% alternaria dauci

Fig: B. Meriaux, FNAMS

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

Damages caused by seed transmitted pests : – Symptoms on roots,

stems, leaves causing a decrease of yield

– Toxin production

0.0 15.00

1.00 0

100000

120000

140000

160000

180000

200000

LotNaturellement

Contaminé

Contaminationà 10 000 sp

vivantes/grain

Contaminationà 1000 sp

vivantes/grain

Contaminationà 10 000 sp

mortes/grain

Lot Sain

riés r

% d'épis contaminés àN+1 sur Brains surl'Authion% d'épis contaminés àN+1 sur Bourges

Nb spores / grain à N+1

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

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

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

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

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

Grinding for extraction No isolation Identification: – Serological tests – RT-PCR – Indexing/biotests

Detection of viruses

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

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

Extraction – Soaking

Isolation – By concentration

Identification – Morphology – PCR

Detection of nematodes: Ditylenchus dipsaci

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

Example of Ditylenchus/alfalfa seeds

On the importance of performance criteria of methods

One method have risks of false negative results and a lower recovery of nematodes

Ditylenchus/alfalfa seeds

Thank you for your attention

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