Detection and characterization of plant viruses on major food crops in togo (West Africa)

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO (WEST AFRICA). Y. Mawuena Dieudonné GUMEDZOE,

Professor, Director of Research University of Lome/ Faculty of Agriculture/

Plant Virology & Biotechnology Lab (LVBV).

B.P.1515 LOME TOGO FAX (228) 2218595

E-mail : [email protected] of the academies: AAS, TWAS

DETECTION AND CHARACTERIZATION OF

PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO (WEST AFRICA).

SEMINAR PRESENTED AT

IITA

IBADAN, NIGERIA

16th MAY 2008



MAIN HEADLINESÌ. INTRODUCTION

ÌI. LVBV AND OBJECTIVES

ÌII.PLANT VIRUSES DIAGNOSTIC TECHNIQUES

ÌV. COLLABORATIVE PROJECTS ON: COWPEA VIRUSES CASSAVA BEGOMOVIRUSES

`V. OTHER PLANT VIRUSES UNDER STUDIES

`VI. CONCLUSION AND PERSPECTIVES



I. INTRODUCTION (1)PLANT VIRUSES :

• DEFINITION :

Submicroscopic,

ìntracellular particles and

òbligate parasites consisting

òf nucleic acid and protein.



I. INTRODUCTION (2)• ECONOMIC IMPORTANCE:

• 1) Responsible of major diseases and significant economic losses to

several agricultural and horticultural crops around the world.



I. INTRODUCTION (3)

ECONOMIC IMPORTANCE

2) IN AFRICA VIRUS DISEASES OF CROPS RANK SECOND AFTER FUNGI

CONTROL MEASURES

NO DIRECT CHEMICAL METHODS AVAILABLE TO CONTROL THEM


PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO (WEST AFRICA).II. LVBV AND OBJECTIVES(1)

LVBV: The Plant Virology and Biotechnology Laboratory, 1987, with the following objectives:

Develop diagnostic tools for the identification of plant pathogens especially plant viruses

Detect, identify and characterize plant viruses in Togo and elsewhere in Africa

Train students (MSc. and Ph.D levels) and technicians in plant pathology and biotechnology

Establish collaboration with other research

centers working in the same fields.



II. LVBV AND OBJECTIVES(2) DURING THIS SEMINAR ARE:

TO PRESENT RESULTS OBTAINED ON THE IDENTIFICATION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO USING A COLLABORATIVE APPROACH,

THE LESSONS LEARNT FROM THIS COLLABORATIVE APPROACH,

THE NEED FOR CONCERTED EFFORTS AMONG SCIENTISTS, IN ORDER TO FULLY EXPLOIT THE EXISTING EXPERTISE, KNOWLEDGE, FACILITIES, AND OPPORTUNITIES.

DETECTION AND CHARACTERIZATION PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO.III.PLANT VIRUSES DIAGNOSTIC TECHNIQUES(1)

TWO BROAD CATEGORIES:

A) BIOLOGICAL STUDIES SYMPTOMATOLOGY TRANSMISSION TESTS

B) INTRINSIC PROPERTIES OF VIRUS PARTICLES;



III. VIRUSES DIAGNOSTIC TECHNIQUES (2)

B) INTRINSIC PROPERTIES OF VIRUS PARTICLES

ELECTRON MICROSCOPY

DETECTION AND CHARACTERIZATION PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO.

III. DIAGNOSTIC TECHNIQUES (3) SEROLOGICAL AND MOLECULAR TECHNIQUES

(COAT PROTEIN AND NUCLEIC ACID)

COAT PROTEIN (SEROLOGICAL TESTS)DOUBLE DIFFUSION IN GEL ELISA TEST

1. Coating ELISA plates with appropriate dilute antisera2. Puting Antigens in place (Crushing samples in buffer and use the extracts)3. Add conjugated antisera4. Add susbtrat and stop the reaction when the yellow color appears

III. DIAGNOSTIC TECHNIQUES(4)DIFFERENTS STEPS OF THE DAS-ELISA TEST

Healthy Samples

Infected Samples

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO


III.DIAGNOSTIC TECHNIQUES(5)

MOLECULAR TESTS `(VIRAL NUCLEIC ACID) DOT-BLOT / `HYBRIDISATION ASSAYS / `PCR (amplification of specific `DNA Fragments)

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO.

PLANT VIRUSES :DETECTION AND CHARACTERIZATION

WHY ? FIRST STEPS FOR EPIDEMIOLOGICAL STUDIES IN

ORDER TO DEVELOP STRATEGY TO CONTROL THEM.

DIFFICULTIES : MORE DIFFICULT TO IDENTIFY AND CHARACTERIZE THAN OTHER PLANT PATHOGENS,

REQUIRED ELABORATE AND EXPENSIVE EQUIPMENT AND REAGENTS, LACK OF EXPERTISE, INADEQUATE FUNDING AND FACILITIES IN SSA COUNTRIES.

III. DIAGNOSTIC TECHNIQUES(6)

WHY IS IT SO IMPORTANT TO DETECT ANDCHARACTERIZE PLANT VIRUSES ?

1) They represent the first steps for epidemiological studies in order to develop control measures

2) During diseases surveys rapid diagnosis is needed

3) Important for plant quarantine and seed certification and also for plant breedimg programmes4)Detection and characterization represent a vital task for plant virologists working in SSA countries.


PLANT VIRUSES :DETECTION AND CHARACTERIZATION

WHY ? FIRST STEPS FOR EPIDEMIOLOGICAL STUDIES IN

ORDER TO DEVELOP STRATEGY TO CONTROL THEM.

DIFFICULTIES : MORE DIFFICULT TO IDENTIFY AND CHARACTERIZE THAN OTHER PLANT PATHOGENS,

REQUIRED ELABORATE AND EXPENSIVE EQUIPMENT AND REAGENTS, LACK OF EXPERTISE, INADEQUATE FUNDING AND FACILITIES IN SSA COUNTRIES.

III.DIAGNOSTIC TECHNIQUES(5bis)

DIFFICULTIES ENCOUNTERED DURING PLANT VIRUSES DETECTION / CHARACTERIZATION ?

1) Plants viruses are more difficult to identify than other plant pathogens2) They require elaborate and expensive equipments and reagents3) In most SSA countries there is a lack of critical scientific expertise4) Inadequate research funding5) Research facilities and other items (irregular power and water supplies) are not always available.


PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO (WEST AFRICA).III. DIAGNOSTIC TECHNIQUES(7)

1) DETECTION AND CHARACTERIZATION PLAY A CRITICAL ROLE IN THE MANAGEMENT OF THESE VIRUS DISEASES

2) THEY REQUIRE DIFFERENT METHODS (BIOLOGICAL, SEROLOGICAL AND MOLECULAR TECHNIQUES) WHICH ARE NOT ALWAYS AVAILABLE IN MOST SSA COUNTRIES THEREFORE WE NEED TO JOINT OUR EFFORTS TOGETHER IN COLLABORATIVE PROJECTS


IV. COLLABORATIVE PROJECTS(1)

We are reporting here the results obtained during two collaborative projects:

1) A PILOT PROJECT ON COWPEA VIRUSES IDENTIFICATION WHICH HAVE INVOLVED SCIENTISTS FROM DEVELOPED AND DEVELOPING COUNTRIES AND ONE CENTRE OF CGIAR) (IITA).




2) IDENTIFICATION AND CHARACTERIZATION OF CASSAVA BEGOMOVIRUSES INVOLVING OUR LABORATORY AND TWO PARTNERS (CIRAD-BIOS AND IITA)


IV. COLLABORATIVE PROJECTS(3) COWPEA VIRUSES IDENTIFICATION

A) OBJECTIVES OF THE PROJECT

1) TO MAKE AVAILABLE SEROLOGICAL

DIAGNOSTIC TESTS FOR NARS.

2) PRODUCE ANTISERA (POLY-AND MONOCLONAL

TO BlCMV AND CAMV (POTY)

3) DEVELOP SEROLOGICAL STANDARDIZED DIAGNOSTIC TOOLS (COWPEA VIRUSES).

4) SHARE EXPERIENCES IDEAS AND MATERIALS AMONGST SCIENTISTS.

5)EXTEND TO OTHER PLANT VIRUSES.


IV COLLABORATIVE PROJECTS(4)B) ANTISERA AND PLANT MATERIALSUSED DURING OUR STUDIES WERE FROM VRS OR IITA.

C) METHODS

1)SURVEYS FOLIAR SAMPLES COLLECTEDTHROUGHOUT THE COUNTRY ON

COWPEAS AND WEEDS (FARMERS’FIELDS/ EXPERIMENTAL PLOTS).

2) IDENTIFICATION OF VIRUSES

SEROLOGY (DOUBLE DIFFUSION IN AGAROSE, ELISA AND PCR).



D) RESULTS IDENTIFICATION OF

VIRUSES 63.12 % samples reacted to

serological tests. Cowpea viruses (6) detected and present in various agro-ecological zones :- BlCMV(Potyvirus)(serotypes)- CAMV (Potyvirus)(serotypes)- CMeV (Carmovirus)- CMMV (Carlavirus)- CPMV (Comovirus)- SBMV-CS (Sobemovirus) CPMV (WIDESPREAD). MIXED INFECTIONS.


IV. COLLABORATIVE PROJECTS : COWPEA VIRUSES(6) D) RESULTS (continued)

BlCMV/ CAMV and their serotypes were identified on cowpea/ weeds.Acanthospermum hispidum (serotypes F, G / CAMV B / BlCMV) Cassia occidentalis (serotypes A,B/ BlCMV) Passiflora foetida (serotypes C,D/ CAMV) Desmodium tortuosum (serotypes C,D/CAMV) Abutilon mauritianum (serotypes C, E/CAMV) Centrosema pubescens (serotypes C,F,G/ CAMV) Pupalia lappacea (serotypes: C,D,E,F/CAMV).

IV. COLLABORATIVE PROJECTS (7)

2) REACTIONS OF COWPEA CULTIVARS ANDOTHER PLANT SPECIES TO COWPEA VIRUSES :

SCREENING OF COWPEA CULTIVARS USING CLONED VIRUS ISOLATES OF CAMV, CPMV, CMeV AND SBMV REVEALED THAT THE FOLLOWING COWPEA CULTIVARS WERE RESISTANT : IT82E-32, IT83S-818, TVX1850-01E, TVU410 AND TVU 645.

THE USE OF THESE RESISTANT COWPEA CULTIVARS COULD REDUCE LOSSES CAUSED BY THESE VIRUSES IN COWPEA GROWING AREAS.




IV COLLABORATIVE PROJECTS(8)

CASSAVA BEGOMOVIRUSES IDENTIFICATION

A) Objectives1) TO MAKE AVAILABLE SEROLOGICAL

DIAGNOSTIC TESTS FOR NARS

2) TO IDENTIFY AND CHARACTERIZE CASSAVA BEGOMOVIRUSES IN TOGO AND ELSEWHERE IN AFRICA (BY USING TAS-ELISA WITH MONOCLONAL AND POLYCLONAL ANTIBODIES, AND PCR)

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO (WEST AFRICA).

IV. COLLABORATIVE PROJECTS (9) Cassava BegomovirusesA) Objectives continued)3) Analysis of taxonomic identity of

the different components of these Begomoviruses (using PCR and sequencing) which will give insight into the genetic drift within these virus population in Togo.

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN

TOGO (WEST AFRICA).IV. COLLABORATIVE PROJECTS(10)

A) Objectives(continued)4) Develop strategies to control these Cassava Begomoviruses (by using all available technologies),

5) Screen cassava cultivars and select which of them are resistant to these viruses.


IV. COLLABORATIVE PROJECTS (11)B) Materials and Methods1) Plant materials, antisera, primers,

reagents and virus isolates: Provided by our lab or by our

partners(Dr Schoenfelder, DSMZ GERMANY and Dr PETERSCHMITT, CIRAD-BIOS, France), or bought from different manufacturers

Reference sequences of the Begomoviruses CP gene are from GenBank databases.


Reference number DSMZ

Origin Description

PV-0421 Kenya ACMV-NG, Cassava infected leaves

PV-0423 Kenya EACMV, Cassava infected leaves

PV-0424 Sri Lanka ICMV, cassava infected leaves

PV-0872/Ca-161 Cameroun EACMV, Cassava infected leaves

IV. COLLABORATIVE PROJECTS/CASSAVA VIRUSES(12)TABLEAU 2 LIST OF POSITIVE CONTROLS USED


IV. COLLABORATIVE PROJECTS (13)B) Materials and Methods2) Surveys Cassava and weeds foliar samples

(showing mosaic or mottling symptoms ) were collected from farmers’ fields and experimental plots in major cassava production areas in Togo.


TOGO

IV. COLLABORATIVE PROJECTS (14)B) Materials and Methods3) Identification of cassava Begomoviruses

The diagnosis of the three cassava Begomoviruses (ACMV, EACMV and ICMV) was performed by using:

-Serological test (TAS-ELISA) with monoclonal and polyclonal antibodies

PCR with specific primers (as designed by Fauquet et al.2000 and 2001) targeting the coat protein.

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO (WEST AFRICA)

PRIMER SEQUENCE(5’ 3’) TARGET REGION

JSP001 ATGTCGAAGCGACCAGGAGAT 5’ACMV/EACMV CP

JSP002 TGTTTATTAATTGCCAATACT 3’ACMV CP

JSP003 CCTTTATTA ATT TGTCACTGC 3’EACMVCP

JSP012 GTCCATATAGGTAARGTNATG 5’ICMV CP

JSP013 CCTGCTCCTTGCTNGCYTART 3’ICMV CP

IV. COLLABORATIVE PROJECTS/CASSAVA VIRUSES(15)TABLEAU1 : SPECIFIC PRIMERS USED FOR THE IDENTIFICATION OF ACMV, EACMV, AND ICMV


TOGO

IV. COLLABORATIVE PROJECTS (16)B) Materials and Methods3) Identification of cassava

Begomoviruses (continued)Specific Primers are as follows:JSP001/JSP002 for ACMVJSP001/JSP003 for EACMVJSP012/JSP013 for ICMV

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGOIV. COLLABORATIVE PROJECTS (17)B) Materials and Methods (continued)4) Study of the molecular variability of

cassava Begomoviruses Sequencing: 52 and 62 isolates

respectively of ACMV and EACMV were sequenced and partial sequences of ICMV isolates were obtained and compared with sequences from the GenBank databases and included in a phylogenetic analysis.


TOGOIV. COLLABORATIVE PROJECTS (18)C) Results Molecular analysis by PCR of cassava foliarsamples using specific primers revealed

that ACMV (incidence rate of 73,59%) is not

the only Begomoviruses occurring in Togo. Two other Begomoviruses are also present,

EACMV and ICMV with incidence rates of 44,62 and 4,01 respectively.

Mixed infections were also recorded (ACMV+EACMV/39.45%; ACMV+ICMV/1.72% and EACMV+ICMV/1.29%)


IV. COLLABORATIVE PROJECTS (19)C) Results (continued)

Figure 13 : Taux d'incidence de trois Begomovirus infectant le manioc au Togo

31%

4%

1%

39%

2%

1%

22%ACMV seul

EACMV seul

ICMV seul

ACMV + EACMV

ACMV + ICMV

ACMV+EACMV+ICMV

VIRUS NON IDENTIFIES

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGOIV. COLLABORATIVE PROJECTS (20)C) Results (continued) Identification of ACMV by PCR


IV. COLLABORATIVE PROJECTS /Cassava Viruses (21)C) RESULTS (CONTINUED)

IDENTIFICATION OF ICMV BY PCR

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO.

EACMV/ICMV-TG

EACMV AF423178Uganda

EACMV AJ717534

Kenya

EACMV AY562424

Kenya

ICMV AJ314739Sri Lanka

EACMV EU155147

93 93 93 72

EACMV EU155148

96 96 96 71

EACMV EU155149

81 81 80 67

ICMV EU152125

61 61 61 92

TABLE NUCLEOTIDE SEQUENCE OF PAIRWISE COMPARISON OF EACMV/ICMV FROM GENBANK AND EU155147, EU1551478,EU155149 (EACMV) AND EU152125 (ICMV)FROM TOGO.(22)

DETECTION AND CHARACTERIZATION OF PLANT VIRUSES ON MAJOR FOOD CROPS IN TOGO`IV.COLLABORATIVE `PROJECTS (23)/

``C) Results `(continued)`Cassava `Viruses

`: Distribution `of `cassava/`Begomoviruse``s in Togo.


IV.COLLABORATIVE PROJECTS (24)D) LESSONS LEARNT FROM THESE COLLABORATIVE

PROECTS

1) ANTIBODIES PRODUCED, DISTRIBUTED TO COLLABORATORS

2) USE OF STANDARDIZED SEROLOGICAL TESTS


IV.COLLABORATIVE PROJECTS (25)

D) LESSONS LEARNT FROM THESE COLLABORATIVE PROECTS (continued)

3) WORKSHOPS (EXCHANGE OF IDEAS AND EXPERIENCES) AND TRAINING OF STUDENTS,

4) COLLABORATIVE APPROACH EXTENDED (FOOD CROPS VIRUSES)


IV. COLLABORATIVE PROJECTS D) LESSONS LEARNT FROM THESE PROJECTS(cont.)(26)

5) NETWORKING SCIENTISTS, CGIAR CENTRES, DEVELOPED,DEVELOPPING COUNTRIES.

6) KEY OF SUCCESS CGIAR

centres

NARS(universities, research centres indeveloping countries)

NARS (develope

dcountries)

7) OTHER COLLABORATIVE PROJECTS ON PLANT VIRUSES DETECTION / CHARACTERIZATION a) Cassava Begomoviruses (Fargette et al.1985, 1988) b) TSWV (CHO et al. 1989)

V. OTHER PLANT VIRUSES UNDER STUDIES IN OUR LAB

1) RICERICE YELLOW MOTTLE VIRUS (RYMV)

2) TOMATOES

TOMV, TSWV AND TYLCV

3) YAM POTYVIRUSESYAM MOSAIC VIRUS (YMV) AND YAM

MILD MOSAIC VIRUS (YMMV) IDENTIFIED


YMV

RYMV


VI. CONCLUSION AND PERSPECTIVES

A) CONCLUSION (1) Plant viruses are important economically in developing

countries that are heavily dependent on agricultural production for food security, employment and export earnings,

These viruses affect numerous crops in the tropics causing losses that are usually substantial and sometimes devastating,

Their identification and characterization represent a vital task for plant virologists working in tropical regions before developing any strategy to control them.


VI. CONCLUSION AND PERSPECTIVES

A) CONCLUSION (2)

The Plant Virology and Biotechnology Laboratory LVBV, in the frame of its objectives has detected and identified various viruses on the following crops:

Cowpeas: Six viruses identified and their geagraphic distribution established. Screening tests revealed cowpea cultivars resistant to them (their use could reduce

losses).

Cassava: Using TAS-ELISA and PCR, three Begomoviruses, ACMV, EACMV and ICMV were detected and characterized and their

geographic distribution established.


VI.CONCLUSION AND PERSPECTIVES

A) CONCLUSION (3)

Rice: Among major rice viruses identified throughout the world, only RYMV was identified so far in Togo

Tomatoes: The following viruses were detected, ToMV, TSWV and TYLCV

Yams: YMV and YMMV were identified.

Mixed infections were identified with variouscombinations of these viruses.


TOGO (WEST AFRICA).


B) PESRSPECTIVES(4)

Identification and molecular characterization of viruses infecting cowpea, cassava, rice tomatoes and yams should continue with our partners,

Screening plant cultivars resistant to the viruses under studies


TOGO (WEST AFRICA).VI.CONCLUSION AND PERSPECTIVES

B) PESRSPECTIVES (5)

Lessons learnt from collaborative project bring us to the conclusion that we emphasised on concerted efforts to fully exploit the existing expertise, knowledge, facilities and opportunities,

We appealing for networking existing laboratories and by creating centres of excellence in plant virology, biotechnology in Africa.


ACKNOWLEDGEMENTSTHE DIRECTOR GENERAL OF IITA

DRS BANDYOPADHYAY, RANAJIT , BI VROH ; AND THEIR COLLABORATORS

THE ORGANISERS OF THIS SEMINARALL THE IITA STAFF IN THEIR DIFFERENTS

COMPONENTS

ALL MY FRIENDS AND COLLEAGUES AND

MRS MARTINE ZANDJANAKOU



B) PERSPECTIVES(6)

We are involved in the training of students (MSc. and Ph.D; levels) and it is important to consider the following as high priorities :

Capacity building in plant virology, biotechnology and molecular biology,

Funding, Equipment and better environment for research activities.

MERCI DE VOTRE AIMABLE ATTENTION

THANK YOU FOR YOUR ATTENTION


TOGO (WEST AFRICA).


B) PESRSPECTIVES(4)

Identification and molecular characterization of viruses infecting cowpea, cassava, rice tomatoes and yams should continue with our partners,

Screening plant cultivars resistant to the viruses under studies



A) CONCLUSION (3)

Rice: Among major rice viruses identified throughout the world, only RYMV was identified so far in Togo

Tomatoes: The following viruses were detected, ToMV, TSWV and TYLCV

Yams: YMV and YMMV were identified.

Mixed infections were identified with variouscombinations of these viruses.


TOGO (WEST AFRICA).VI.CONCLUSION AND PERSPECTIVES

B) PESRSPECTIVES (5)

Lessons learnt from collaborative project bring us to the conclusion that the emphasis should put on concerted efforts to fully exploit the existing expertise, knowledge, facilities and opportunities,

We are appealing for networking existing laboratories and by creating centres of excellence in plant virology, biotechnology in Africa.


ACKNOWLEDGEMENTSTHE DIRECTOR GENERAL OF IITA

DRS BANDYOPADHYAY, RANAJIT , BI VROH ; AND THEIR COLLABORATORS

THE ORGANISERS OF THIS SEMINARALL THE IITA STAFF IN THEIR DIFFERENTS

COMPONENTS

ALL MY FRIENDS AND COLLEAGUES AT IITA AND

MRS MARTINE ZANDJANAKOU



B) PERSPECTIVES(6)

We are involved in the training of students (MSc. and Ph.D; levels) and it is important to consider the following as high priorities :

Capacity building in plant virology, biotechnology and molecular biology,

Funding, Equipment and better environment for research activities.

MERCI DE VOTRE AIMABLE ATTENTION

THANK YOU FOR YOUR ATTENTION

Detection and characterization of plant viruses on major food crops in togo (West Africa)

Technology

Transcript of Detection and characterization of plant viruses on major food crops in togo (West Africa)