Tospoviruses in Tomato and Pepper in ASEAN: Current Status &

Challenges

Channarong Seepiban, Ph.D.National Center for Genetic Engineering and

Biotechnology (BIOTEC), Pathum Thani, Thailand

Tospovirus

Enveloped RNA viruses Family: BunyaviridaeGenus: TospovirusVector: Thrips

NSs

proteinN

proteinS RNA

segment 5’ 3’

GN/GC

glycoprotein

NSm

proteinM RNA segment 5’ 3’

RNA-dependent RNA polymerase (RdRP) L RNA

segment 5’ 3’

Genome organization

Causes major losses in solanaceous crops, cucurbits, celery, lettuce, onion, legumes and ornamental crops

Geographic distribution of 29 Tospoviruses

Asia

Australia

North America

South America

Europe

GRSV

INSV

IYSV

TSWV

CaCV

CCSV

CSNV

GBNV

GYSV

HCRV

INSV

IYSV

LNRV

MVBaV

MYSV

PCFSV

PCSV

TNRV

TNSV

TSWV

TYRV

TZSV

WBNV

WSMoV

Africa

CaCV

INSV

IYSV

TSWV

CSNVINSV

IYSV

PolRSV

TSWV

GRSV

INSV

IYSV

MSMV

SVNV

TSWV

ANSV

BeNMV

CSNV

GRSV

INSV

IYSV

PNSV

TCSV

TSWV

ZLCV

IYSV clade (Europe & Middle East)

TSWV clade (America)

WSMoV clade (Asia)

Phylogenetic analysis of N proteins of 29 tospovirus species

GYSV clade (Asia)

SVNV clade (America)

(Oliver and Whitfield., 2016)

Transmission of tospoviruses

Frankliniella occidentalis

Frankliniella fusca

Frankliniella schultzei

Frankliniella bispinosa

Frankliniella zucchini

Frankliniella gemina

Frankliniella cephalica

Frankliniella intonsa

Thrips setosus

Thrips palmi

Thrips tabaci

Scirtothrips dorsalis

Ceratothripoides claratris

Dictyothrips betae

Neohydatothrips variabilis

- 15 Thrips vectors

- Vegetative propagation

- Seed transmission

Soybean vein necrosis virus (SVNV) (Groves et al., 2016)

T. palmi

C. claratris

Transmission of tospovirus by thrips vector

Acquisition

Transmission can only occur if the viruses are acquired from infected plants by larvae thrips

1# instar larva

2# instar larva

Transmission

Tospovirus infected tomato

Viruliferous thrips (Thrips that carry tospovirus)

Pupa

Tospoviruses in ASEAN

Thailand- WSMoV- MYSV- CaCV- TNRV

The Philippines- WSMoV- MYSV- IYSV

Indonesia- WSMoV- GBNV- TSWV

Status of tospoviruses in other countries is not known.

Capsicum chlorosis virus (CaCV)

- First report in Australia, later in several countries in Asia (Thailand, China, Taiwan, India)

- Tomato, Pepper, Peanut, Orchids- Thrips palmi, Ceratothripoides claratris, Frankliniella schultzei

Tomato necrotic ringspot virus (TNRV)

- First report in Thailand

- Tomato, Pepper - Thrips palmi, Ceratothripoides claratris

Watermelon silver mottle virus (WSMoV)

- First report in Taiwan, later in several countries in Asia (China, Japan, Thailand, The Philippines, Indonesia)

- Cucurbits, tomato, pepper- Thrips palmi

Thrips palmi

Groundnut bud necrosis virus (GBNV)- First report in India- Report in peanut in Indonesia (Damayanti and Naidu., 2009)

- Groundnut, tomato, mung bean, potato - Thrips palmi, Frankliniella schultzei, Scirtothrips dorsalis

- Worldwide distribution- Report in tomato and pepper in Indonesia (Damayanti and Naidu., 2009)

- Wide Host ranges (tomato, pepper, tobacco, lettuce, potato, beans, ornamentals)- Transmitted by several species of thrips (main vector is Frankliniella occidentalis)

Tomato spotted wilt virus (TSWV)

Why is it difficult to control Tospoviruses ?

Tospoviruses• Wide host ranges• Virus diversity• Evolution of new strains (resistance breaking strains)

Thrips• Polyphagous pest• Efficient tospovirus vector• Difficult to control by insecticide

Inappropriate cultural practice • Grow different plant varieties which can be serve as host for

tospoviruses and thrips• Year-round overlapping plantings

Limited host resistance against tospoviruses available for breeding• Sw-5 in tomato (S. peruvianum)• Tsw in pepper (C. chinense)

Tospoviruses infecting tomato in Thailand

Why is it difficult to control Tospoviruses ?

Tospoviruses• Wide host ranges• Virus diversity• Evolution of new strains (resistance breaking strains)

- T. palmi is a polyphagous pestSolanaceae: pepper, tobacco, eggplant, potato Cucurbitaceae: cucumber, watermelon, melon, squash, pumpkin

- T. palmi was the vector of Asian tospoviruses such as WSMoV, CaCV, WBNV, CCSV, GBNV, TNRV and MYSV

- T. palmi has been reported in many countries in South and Southeast Asia (Thailand, The Philippines, Indonesia, Malaysia)

- T. palmi is resistant to most insecticides

The major vector in South East Asia: T. palmi

Why is it difficult to control Tospoviruses ?

Inappropriate cultural practice - Use of susceptible plant varieties- Grow different plant varieties which can be serve as host for tospoviruses and thrips in the same field resulting in virus accumulation

- Year-round overlapping plantings provide extensive reservoirs for Tospovirus and thrips

Tospovirus research in Thailand

Genetic diversity of tospovirusesinfecting tomato, pepper and cucurbits in Thailand

Production of highly specific monoclonal antibodies to CaCV, WSMoVand TNRV and Development of ELISA-based detection

2003-2005

Relationship between types of thrips, host plants and tospovirus infection in tomato, pepper and cucurbit production fields

Characterization of Tomato necrotic ringspot virus, a New Species of Tospovirus Infecting Tomato in Thailand

Survey of tospovirues infecting tomato, pepper and cucurbits in Thailand

Production of monoclonal antibodies and development of immunoassays for detection of tospoviruses

2011 2011-2013 2013-2016

Detection of tospoviruses by using ELISA

http://www.tbrcnetwork.org/antibodies/result.php?Antigen_subgroup[]=virus

Tospovirus PAbMYSV6

PAbA3

MAbL4E8

MAb5E7

MAb14H12

MAb18D11

MAb7B9

CaCV-NRA + + + - - - +

CaCV-AIT + + + - - - -

WSMoV + + + - - + -

TNRV + + - - + - -

MYSV + - - + - - -

Identification of tospoviruses by using RT-PCR

828 bp

846 bp

840 bp

828 bp

CaC

V

WSM

oV

TNR

V

MY

SV

CaCV N-gene specific primer

MYSV N-gene specific primer

TNRV N-gene specific primer

WSMoV N-gene specific primer

Total RNA Isolation

First strand cDNA synthesis

N Protein Gene Amplification

N protein Gene Sequencing

Identification of major thrips and tospovirus species in pepper, tomato and cucurbits in Thailand

Plant Major Tospovirus Major Thrips

Pepper TNRV Thrips palmi

TomatoTNRV

Ceratothripoides

claratrisCaCV

CucurbitsMYSV

Thrips palmiWSMoV

Tospovirus research in Thailand

Genetic diversity of tospovirusesinfecting tomato, pepper and cucurbits in Thailand

Production of highly specific monoclonal antibodies to CaCV, WSMoVand TNRV and Development of ELISA-based detection

2003-2005

Relationship between types of thrips, host plants and tospovirus infection in tomato, pepper and cucurbit production fields

Characterization of Tomato necrotic ringspot virus, a New Species of Tospovirus Infecting Tomato in Thailand

Survey of tospovirues infecting tomato, pepper and cucurbits in Thailand

Production of monoclonal antibodies and development of immunoassays for detection of tospoviruses

2011 2011-2013 2013-2016 2015-2017

Development of efficient inoculation protocol for TNRV

and CaCV resistance screening in tomato

APSA-BIOTEC project

Project Title: Development of efficient inoculation protocol for TNRV and CaCV resistance screening in tomato

Project Duration: 2 Years (1 Sep 2015-31 Aug 2017)

Project output: Standard protocol for screening of TNRV and CaCV-resistant tomato

Mechanical inoculation

Tospovirus-inoculated plant Mock inoculation

• Investigation of major tospoviruses and thrips vector species in particular area

Opportunities for Public-Private Partnerships

FUTURE

• Development of diagnostic techniques for identification of Tospoviruses (ELISA and RT-PCR)

• Searching for new tospovirus resistance genes in the germplasmTomato (S. lycopersicum, S. peruvianum, S. pimpinellifolium, S. chilense, S. arcanum, S. habrochaites, S. corneliomuelleri)Pepper (C. chinense, C. bacbatum)

• Development of reliable screening protocol for breeding programs of tomato and pepper

Acknowledgements

• Cluster and Research Program Department, National Science and Technology Development Agency (NSTDA), Thailand

• National Center for Genetic Engineering and Biotechnology, NSTDA, Thailand

Dr. Oraprapai GajanandanaDr. Orawan ChatchawankanphanichDr. Saengsoon CharoenvilaisiriMs. Nuchnard WarinMs. Kirana Yoohat

Thank you for your attention