Overview of the PVY management in France by the seed potato sector

Presentacin por Ana Ponce de Len, Comit Nord/FN3PT anne.poncedeleon@comitenordplant.fr

Glais1,2, L.; Boquel3,5, S.; Khelifa4,5, M. ; Ponce de Leon5, A. ; Deveaux5, V. ; Le Hingrat1, Y.

XXV Congreso ALAP

Potato virus Y (PVY): main characteristics

Potato virus Y
- Potyviridae family, type-member of Potyvirus genus - Filamentous and flexuous particle (700 nm x 11 nm)
100 nm

Genome
- Positive single strand RNA of almost 10 Kb - Coding for 9 viral proteins
VPg 5
P1 HC-Pro P3 CI VPg NIa NIb CP

(A)n

Vectors
- Transmission in a non persistent manner by at least 70 aphids species
(including Myzus persicae)

Host range (500 plant species)

Cultivated Solanacea Ornemental plants Weeds
XXV Congreso ALAP

El PVY causa perdidas de rendimiento

Ensayos Comit Nord, 2010

-52%

-39%

Semente con 0% virus

Semente con 0% virus

XXV Congreso ALAP

PVY: main characteristics

Biological
Tobacco Potato Strain groups

Serological
PVYO
PVYN
type-O
P1 HC

Molecular
P3 CI NIa NIb CP

Mosaic

Mosaic and/or necrosis

type-N

vein necrosis, leaf deformation Mosaic and/or necrosis

Variants

PVYN-W PVYN:O

type-O

PVYNTN
PTNRD

type-N
XXV Congreso ALAP
(Schubert et al., 2007; Lorenzen et al., 2006 et 2008; Glais et al., non publi)

Historic and distribution of PVY

1935 1931 O N South of America 1958 1978
Russia Tunisia Lebanon Japan Oceania

1984

N
France

Hungary Poland

NTN N-W

Today in USA
100% 80%

in Europe
100% 80%

PVYO YN-W

PVYO
60% 40% 60% 40%

YN-W
20% 20%

YNTN

YNTN
0%

PVYN

0%

PVYN

- PVYN and its variants (PVYN-W, PVYNTN) in relation with PVY unceasant evolution are widespread in most potato growing areas in the world (independently of the closed systems) - The issue for the seed potato sector in France is to maintain a low level of PVY (as a virus complex) with a strong attention on PTNRD: thanks to specific management PVY incidence is very low in France

Main viral programs engaged by the french seed potato growers

Laurent Glais

To control the sanitary quality of seed potatoes.

PVY

To describe the biological, serological and molecular diversity of natural PVY populations.

To identify viral molecular determinants involved in PVY pathogenicity.

DISEASE

To identify the aphid vectors of PVY in natural conditions. To evaluate the aphid locomotive and feeding behaviours allowing PVY spreading. To evaluate the ability to transmit PVY to potato plants of different aphid species.
Sbastien BOQUEL

Aphids

To find solutions to protect the crop Potato XXV Congreso ALAP

DIAGNOSTIC

To develop serological and/or molecular diagnostic tools to identify and to detect PVY populations specifically.

Identification of PVY pathogenicity determinants

Necrosis symptoms on tobacco plants
N605 O139 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Clone 11 Clone 12 Clone 13 Clone 14 Tribodet et al., 2005 Faurez et al., 2012 p1

HC-Pro

P3

CI

NIa

NIb

CP

+ + + + + + + +

Involvement of two PVY genomic regions (HC-Pro, NIa). Three amino acid residues are crucial in the induction of tobacco vein necrosis symptom on tobacco plants.

Three amino acid residues are required and sufficient to confer necrotic properties of a PVY isolate with a non-necrotic HCPro sequence.
Other determinants in other parts of the PVY genome are involved in the tobacco pathogenicity. XXV Congreso ALAP

Identification of PVY pathogenicity determinants

Necrosis symptoms on potato tubers
N605 O139 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Clone 11 Clone 12 Clone 13 Clone 14 p1

HC-Pro

P3

CI

NIa

NIb

CP

+ + + + + + + +

In progress

XXV Congreso ALAP

Analyse of the PVY serological diversity

P1 HC P3 CI NIa NIb CP

98

267

Impact on the PVY antigenic properties :

27

- isolates escape to the serological detection - false serological typing (mutations) - some isolates are detected with both monoclonal antibodies against PVYN or PVYO isolates

29

: amino acid sequence of O-type : amino acid sequence of N-type : Mutation point

What is the serological variability of PVY isolates ?

(Schubert et al., 2007; Oshima et al, 2008 ; Glais et al., non published) (Dhar et al., 1997 ; Chikh Ali et al. 2007 ; Karasev et al., 2009)

XXV Congreso ALAP

Analyse of the PVY serological diversity

P1 HC P3 CI NIa NIb CP

98

267

27

To improve academic knowledge of serological diversity within/between PVY strain groups and variants (number and frequency of PVY antigenic structure ) To develop reliable serological diagnostic tools to allow right control of the potato seeds.

29

: amino acid sequence of O-type : amino acid sequence of N-type : Mutation point

To set up a PVY monoclonal antibody library linked to the actual knowledge of PVY epitopic diversity (77 Mabs). Analyse of the Mab reactivity with a collection of 148 european and non european PVY isolates.
XXV Congreso ALAP

(Schubert et al., 2007; Oshima et al, 2008 ; Glais et al., non published) (Dhar et al., 1997 ; Chikh Ali et al. 2007 ; Karasev et al., 2009)

Reactivity of 77 monoclonal antibodies

The 77 MAbs are distributed in 3 main groups according to the average percentage of detected serotype-N or O PVY isolates.

Nber of detected PVYO isolates (54)

50

40

30

80% correspond to polyvalent Mabs.

17% are close to Mabs specific to serotype-N isolates 3% are close to Mabs specific to serotype-O isolates
0 10 20 30 40 50 60 70 80 90

20

10

Nber of detected PVYN isolates (94)

MAb polyvalent : Y123 : 4C3 MAb anti-PVYO : Mab2 : Mab YO/C : Mabs of the library MAb anti-PVYN : Mab YN INRA/FN3PT : Mab YN-Sw : Mab YN-1F5

Almost all MAbs different reactivity.

present

77 MAbs defined 75 reactivity groups = at least 75 antigenic sites on PVY particles. XXV Congreso ALAP

The PVY serological diversity is very large

Impact of these knowledge on the PVY diagnosis

To identify molecular determinants of PVY pathogenicity To describe the serological PVY diversity

To set up new reliable diagnostic tools for the identification and detection of PVY isolates
Molecular tools are developed with non-neutral determinants link to the PVY pathogenicity (PCRq, SNP). Selection of cocktails of monoclonal antibodies allowing the detection of specific PVY populations.

The developed assays are transferred to the french organisations in charge of the seed potato certification:
XXV Congreso ALAP Each year production of ELISA reagents for the seed laboratories (>1,2 millions ELISA tests)

Vector propensity of aphids to transmit

Potato virus Y (PVY) to potato
Sbastien BOQUEL

Objective 1: Evaluate aphid vector activity Objective 2: Evaluate aphid vector efficiency

PhD funding by the Comit Nord Plants de Pomme de Terre (France) Supervisors: P. Giordanengo et A. Ameline Post Doctoral Research funded by SIPRE at Agriculture and Agri-Food Canada

What is vector propensity?

Represent the probability of a vector, having had the opportunity to acquire a virus on an infected plant, will transmit it to a healthy one. This concept includes two important notions: Vector activity Behaviours adapted to virus transmission Vector efficiency Vectors ability to acquire and inoculate viral particles

Orientation
Probing, Feeding Virus acquisition Aphid becoming infectious

Orientation
Probing, Feeding

Virus inoculation

Irwin & Ruesink 1986

Vector activity
Vector activity involves 2 behavioural parameters: Probing behaviour (needful for virus acquisition/inoculation) and... locomotive behaviour (needful for virus spreading)

Behaviours adapted to non-persistent virus transmission: Vector which: Lands on an infected plant Realizes intracellular punctures Moves Initiates a quick probe

Irwin & Ruesink 1986

Vector efficiency
Represents the probability of obtaining an infection with a given virus, using a given vector under a given set of conditions
Measured by a relative efficiency factor (REF)
No. infected plants by the tested species REF M. persicae: benchmark (value = 1) tested species =
van Hoof 1980 M. persicae M. euphorbiae A. fabae A. pisum 1 0.58 0.41 0.28 van Harten 1983 1 0.1 0.1 0.05 Piron 1986 1 0.2 0.14 0.17 Harrington 1989 1 0.91 0.9 0.45

No. infected plants by M. persicae

de Bokx 1990 1 0.07 0.07 0.11 Sigvald 1996 1 0.1 0.7 Verbeek 2010 1 0.00 0.04 0.07

R. padi
S. avenae B. brassicae

0.04
0.00 0.00

0.02
-

0.16
0.02 -

0.28
0.01 0.00

0.14
0.02 0.00

0.4
0.01 0.01

0.01
0.00 0.00

Large heterogeneity within the same species

Aphids Populations, clones Virus Strains and/or viral isolates Viral concentrations Plants Species, varieties, phenological stages Set of conditions Acquisition periods, humidity, etc.
Sylvester 1954 ; van Harten 1983

13

Vector propensity of aphids

Colonizing aphids
Vector activity Short probing delay Intracellular punctures Landing on plants Interplant movements Vector efficiency

Non-colonizing aphids
Vector activity Short probing delay Intracellular punctures. Landing on plants Interplant movements Vector efficiency Middle to bad vectors

Good vectors

Sedentary behaviour with good ability to transmit viruses

Restless behaviour with middle/bad ability to transmit viruses

Non colonizing aphids represent a more important risk in PVY epidemiology

La aplicacin de aceite mineral es el unico mtodo de control de la diseminacin del PVY utililizado hoy en Francia

Cual es el modo de accin del aceite mineral?

Estudio del efecto del aceite mineral sobre la aquisicin del PVY por Rhopalosiphum padi

Trabajos llevados por S.BOQUELen Canada bajo la supervisin de Yvan PELLETIER, en AAC, Fredericton (ver presentacin ALAP 2008)
Financiados por el Comit Nord

Transmision de los virus no persistentes

CS CA

Los estiletos transitan por via apoplasmica / inoculacin durante breves picaduras intracelulares

Adquisicin

Sondeo

Planta infectada

Retencin de las particulas virales al nivel del acrostilo

Localizado en las partes distales de los estiletos

Hyptesis :

CS CA

El aceite minerala forma una capa aceitosa al nivel de los estiletos de los pulgones inibiendo que las particulas virales se fijen al nivel del acroestilo

Plante infecte

Mtodo de deteccin del PVY en los estiletos del pulgn

Stylet -20C Corps
Dissection

Ethanol 95% Acquisition 4C

-20C

Extraction

Broyage des tissus

Zhang et al., Entomol. Exp. Appl. Soumis

Arn -20C

RT-PCR

Discusin
Parece confirmarse la hipotesis de la capa de aceita: Se detecta PVY en los cuerpos pero no en los estiletos para las modalidades tratatadas

Las particules virales que se encuentran el cuerpo no se fijan en el acrostilo

La dosis de 10l/ha es eficaz para una proteccion durante 8 dias (ensayos en laboratorio)

Corrobora los resultados de campo (12 l/ha/semana)

El aceite mineral penetra en la planta (estudios preliminarios sobre la relacin entre la concentracin de aceite mineral en la planta y la inibicin de la aquisicin)
24h

48h

48h

48h

72h

48h 72h

48h

Proyecto Vegephy

M.Khelifa

I Desarrollar formulaciones con aceites vegetales

Estudios de varios origenes Con diferentes composiciones fisico quimicas
POURCENTAGE DE VIROSE APRES PULVERISATION (24H AVANT L'INOCULATION DU PVY) VIRUS

II- Cual es el mecanismo de fitoproteccin?

Effect on vector population: Effect on vector devloppement upon the treated plant: Effect on virus Effect on virus propagation within plant --Effect on plant response ++ Effect on virus-vector interaction ++ -------

a
Pourcentage virose

60% 50% 40% 30% 20% 10% 0% temoins 733 20% 42%

ab b
32%

ab

ab

34% 24%

799

801

803

How the seed potato growers manage to reduce PVY rate

Environment parameters Aphids
- quantitative (species) - qualitative (demography)

- Landscape - Meteorology

Quality of seeds

Quality of tuber production

Production Choice of healthy prebasic material: Short multiplication scheme. The First tuber And also take into account for in vitro come from a National Variety other infection sources: Farm Crop Sanitary Collection Save Seed, Neighbouring crops, Importance of applying a low tolerance for PVY Weeds in certification: (Ver pres. G.CROUAU ALAP 2008) -Visual control during vegetation Cultural practices -Post Harvest Check of the direct Progeny by - Choice of variety ELISA test in each generation -Elimination of infected plants (early and repeated rogging), of -Visual control of the tubers: stricter volunteers requirements than EU regulation: max 0,1% of - Insecticids XXV Congreso ALAP tubers with necrosis - Mineral oil: only efficient treatment until now against PVY

Seed

Conclusin

En relacin a la complejidad de la diseminacin del PVY es esencial una relacin estrecha entre investigacin/organizacin de productores/servicios oficiales para tomar las buenas opciones en el manejo del cultivo y para elaborar la reglamentacin Colaboraciones deben hacerse a nivel internacional en un contexto de especializacin de los temas de investigacin con el objetivo de compartir experiencias y de reducir costes de cara a los riesgos sanitarios: que interacciones con LatinoAmrica? El objetivo final de la investigacin: semilla de alta calidad sanitaria para dar en consumo buenos rendimientos y buena calidad
Presentacin general Francia con enfoque a las actividades de la FN3PT el Miercoles a las 16h00 Visitenos: Stand MULTIPLANTA FN3PT con Marcio de Assis
XXV Congreso ALAP