Emerging New Poleroviruses and Tospoviruses Affecting Emerging New Poleroviruses and Tospoviruses Affecting Vegetables in Asia and Breeding for ResistanceVegetables in Asia and Breeding for ResistanceVegetables in Asia and Breeding for Resistance Vegetables in Asia and Breeding for Resistance

Cherry A. Relevante, Sirirat Cheewachaiwit, Jutharat Chuapong, Mongkul Stratongjun, Venus E.Cherry A. Relevante, Sirirat Cheewachaiwit, Jutharat Chuapong, Mongkul Stratongjun, Venus E.Cherry A. Relevante, Sirirat Cheewachaiwit, Jutharat Chuapong, Mongkul Stratongjun, Venus E. Cherry A. Relevante, Sirirat Cheewachaiwit, Jutharat Chuapong, Mongkul Stratongjun, Venus E. Salutan, Dick Peters, Conrado H. Balatero and Simon Jan de Hoop Salutan, Dick Peters, Conrado H. Balatero and Simon Jan de Hoop

International Seminar on Emerging Infectious Diseases of Food Crops in Asia

Tokyo University of Agriculture, Tokyo, Japan, October 19-23, 2012

October 21, 2012Outline of Presentation

Overview of the genus PolerovirusCurrent status of cucurbit-infecting Poleroviruses in AsiaIdentification and characterization of Poleroviruses in the Philippines and ThailandPhilippines and ThailandOverview of Tospoviruses affecting vegetables in AsiaIdentification and characterization of a new Tospovirus speciesIdentification and characterization of a new Tospovirus species infecting tomato and pepper in Thailand Breeding for Polerovirus resistance in cucurbitsBreeding for Tospovirus resistance in vegetablesConclusions and recommendations

Polerovirus – emerging as potential threat

F il L t i id

g g pto cucurbit production

Family: LuteoviridaeGenus: Polerovirus (Potato leaf roll virus

as type species)as type species)Particle: isometric, non enveloped,

25-30 nm diameterG li (+) RNA 5 7 kbGenome: linear (+) ssRNA, 5.7 kbTransmission: aphids (Myzus persicae,

Aphis gossypii) in circulative, non‐propagative mannerp g yp ) , p p gNot mechanically transmittedDistribution: worldwide

Current members of the genus PolerovirusThere are now 13 formally accepted virus species in the genus Polerovirus:

Beet chlorosis virus (BChV)Beet mild yellowing virus (BMYV)Beet western yellows virus (BWYV)Beet western yellows virus (BWYV)Carrot red leaf virus (CtRLV)Cereal yellow dwarf virus-RPS (CYDV-RPS)Cereal yellow dwarf virus-RPV (CYDV-RPV)Cereal yellow dwarf virus-RPV (CYDV-RPV)Chickpea chlorotic stunt virus (CpCSV)Cucurbit aphid-borne yellows virus (CABYV)Melon aphid-borne yellows virus (MABYV)Melon aphid borne yellows virus (MABYV)Potato leafroll virus (PLRV)Sugarcane yellow leaf virus (ScYLV)Tobacco vein distorting virus (TVDV)Tobacco vein distorting virus (TVDV)Turnip yellows virus (TuYV)

At least 3 tentative species:p

Suakwa aphid borne yellows virus (SABYV)Cotton leaf roll dwarf virus (CLRDV)( )Wheat yellow dwarf virus (WhYDV-GPV)

http://ictvonline.org / virus Taxonomy.asp?version =2011

Symptoms of Polerovirus in cucurbit crops

CABYV in cushaw (Cucurbita moschata)

Xiang et al. 2008

Omar & Bagdady 2012

CABYV in squash (Cucurbita pepo)

Omar & Bagdady 2012

Lecoq et al. 1992

CABYV in melon (Cucumis melo)

Symptoms of Polerovirus in cucurbit crops

CABYV in bittergourd (Momordica charantia)

CABYV in cucumber (Cucumis sativus)

Distribution of cucurbit-infecting Poleroviruses

1992: France

2007-2009: China** 1996: Philippines** 2005: Thailand

Current status of cucurbit-infecting Poleroviruses in Asia

Country Virus ReferenceCABYV Xiang et al., 2007

China

CABYV Xiang et al., 2007

MABYV Xiang et al., 2008

SABYV Shang et al., 2009

CABYV Knierim et al., 2010

CABYV R Knierim et al 2010

Taiwan

CABYV-R Knierim et al., 2010

MABYV Knierim et al., 2010

SABYV Knierim et al., 2010

Philippines CABYV Koster et al., 2006

Thailand CABYV Relevante et Thailand CABYVal.,2012

AVRDC data (Kenyon, 2012): CABYV, CABYV-R and SABYV are present in ( y , ) , pthe Philippines and Thailand

Current status of cucurbit-infecting Poleroviruses in Asia

Full-length genome sequence of CABYV, MABYV, SABYV and CABYV-R from Taiwan have been determined using a modified RT-PCR procedure:

• A general strategy to amplify the complete 5’ end of Poleroviruses by “poly-C-tailing” and a primer with 8 conserved nucleotides of the 5’ endpoly C tailing and a primer with 8 conserved nucleotides of the 5 end

• A general strategy to amplify the complete 3’ end of Poleroviruses by nested RT-PCR using primer pairs with 6 and 14 conserved nucleotides from the 3’ end of Poleroviruses (Kenyon 2012; Knierem et al. 2012)from the 3 end of Poleroviruses (Kenyon 2012; Knierem et al. 2012)

Current status of cucurbit-infecting Poleroviruses in Asia

One universal primer for detecting polerovirusesThree virus specific primers for detection of CABYV MABYV and SABYV

C M S C M &S

Three virus-specific primers for detection of CABYV, MABYV and SABYV.

C M S C,M &S

mRT-PCR products:

CABYV 700 bCABYV, 700 bpMABYV, 450 bpSABYV, 950 bp

Shang et al 2012Shang et al., 2012

Identification of Polerovirus in the Philippines

1996: An outbreak of an unknown disorder inbittergourd locally known as ‘Namamarako’bittergourd, locally known as Namamarako(maleness tendency) in bittergourd was firstreported in central Luzon

Infection has spread to the neighboringprovinces in succeeding years

A collaborative research between East-WestSeed Company & Wageningen University inth N th l d i iti t d i 2003the Netherlands was initiated in 2003

CABYV has been found associated with “Namamarako” affecting bittergourd based on aphid transmission, particle p , pmorphology, serology and molecular characterization

Namamarako symptoms in bittergourd caused by CABYV

A BA B

Younger leaves (A) show interveinalchlorosis, wrinkling, green vein bandingand thickening while older leaves (B) showchlorotic patches

Namamarako symptoms in bittergourd caused by CABYV

An NMKAn NMK--infectedinfected bittergourdbittergourd plant (left) and a healthyplant (left) and a healthyAn NMKAn NMK--infectedinfected bittergourdbittergourd plant (left) and a healthyplant (left) and a healthyAn NMKAn NMK infected infected bittergourdbittergourd plant (left) and a healthy plant (left) and a healthy plant (right)plant (right)An NMKAn NMK infected infected bittergourdbittergourd plant (left) and a healthy plant (left) and a healthy plant (right)plant (right)

Namamarako symptoms in bittergourd caused by CABYV

A bittergourd plant A bittergourd plant heavily infected by NMK characterized by stunted growth and

heavily infected by NMK characterized by stunted growth andstunted growth and reduced flower production, hence the

stunted growth and reduced flower production, hence the term ‘namamarako’ (maleness tendency)term ‘namamarako’ (maleness tendency)

Biological & molecular characterization of CABYV-PH

Isometric particles, Transmission tests:NMK i f ti ll t itt d25 nm in diameter NMK is preferentially transmitted by Aphis gossypii in a persistent manner and by grafting

Purified NMK virus extract 2.395

manner and by grafting

Infected NMK leaf material 0.982Control (healthy) 0.084

NMK CP: 170 amino acid mismatches out of 170CABYV CP: 199 amino acids ***96% homology***

Other cucurbit species as alternative hosts to CABYV-PH

Watermelon (C lanatus) Zucchini (C pepo) Cucumber (C sativus)Watermelon (C. lanatus) Zucchini (C. pepo) Cucumber (C. sativus)

Snakegourd (T. anguina) Ridge gourd (L. acutangula) Waxgourd (B. hispida)

Common weeds as alternative hosts to CABYV-PH

Identification of Polerovirues in Thailand2004: A disorder in bittergourd, locally known as‘Mara Ba’ (crazy bittergourd) which is similar insymptomatology to NMK in the Philippines was firstsymptomatology to NMK in the Philippines was firstobserved in Ratchaburi province

2005-2006: An outbreak was observed in2005 2006: An outbreak was observed inRatchaburi including provinces in the centralpart (Chai Nat, Suphanburi, Kanchanaburi)

2007-2009: An outbreak was observed in ChiangMai (North Thailand)

A collaborative research between East-West Seedand Wageningen Univ was again initiated in 2008;with Kasetsart Univ in 2011

CABYV and MABYV have been foundCABYV and MABYV have been found associated with “Mara Ba” affecting bittergourd in Thailand based on aphid transmission and moleculartransmission and molecular characterization

Mara Ba symptoms in bittergourd caused by CABYV and MABYV

A BA B

Older leaves show (A) chlorotic patchesOlder leaves show (A) chlorotic patches while younger leaves (B) show interveinalchlorosis, wrinkling, green vein banding and thickeningand thickening

Molecular characterization of CABYV and MABYV in Thailand

CM1-3: ChiangmaigSP1-4 : Suphanburi

CBG-2: Chiangmai

Nucleotide sequence analysis of the cloned CP gene of CM1-3 SP1-4 and CBG-2 revealed 2 distinct polerovirus species:3, SP1-4 and CBG-2 revealed 2 distinct polerovirus species: CM1-3 and SP1-4 are closely related to CABYV while CBG-2 is closely related to MABYV

Tospovirus – rapidly emerging as a major p p y g g jconstraint in vegetable production

Family: BunyaviridaeGenus: Tospovirus (Tomato spotted wilt

as type species)as type species)Particle: spherical, enveloped, 80-120 nm diameterGenome: ssRNA, tripartite (8.9, 4.8, 2.9 kb)T i i th i i i t tTransmission: thrips in persistent,

propagative mannerMechanically transmitted by sapNot seed transmittedDistribution: worldwide

Geographic Distribution of TospovirusesPhylogeny based on nucleocapsid (N) protein amino acid sequence of tospoviruses (Hassani-Mehrabanet al 2011)

Cluster I (American)Alstroemeria necrotic streak virusTomato chlorotic spot viruset al. 2011) pGroundnut ringspot virusTomato spotted wilt virusZucchini lethal chlorosis virusChrysanthemum stem necrosis virusImpatiens necrotic spot virusMelon severe mosaic virus

Cluster II (Eurasian)Polygonium ringspot virusTomato yellow ring virusIris yellow spot virus

Cluster III (Asian)Tomato necrotic ring virusMelon yellow spot virusGroundnut ringspot virusTomato zonate spot virusCalla lily chlorotic spot virusCapsicum chlorosis virusW t l b d i i

0.1

Watermelon bud necrosis virusWatermelon silver mottle virusPeanut yellow spot virusPeanut chlorotic fan-spot virus

Distribution of Tospoviruses in AsiaAbout 75% of tospovirus species described globally

occur in Asia

GBNVTSWV

TSWVCNSV

CaCVTZSCTSWVINSV

TSWV

WSMoVMYSVIYSVTSWVTSWV

INSVMYSV

CaCVPCFVCCSV WSMoV

MYSVGBNV

TSWVGBNVTYRVIYSV

IYSVPYSVWBNVIYSVCaCV

TSWVGBNV

CaCVMYSVWSMoVTNRVCaCV GBNV

WSMoVTNRVIYSV

Thrips vector of known Tospoviruses in Asia

Thrips vector Tospovirus OccurrenceCerathripoides claratris Capsicum chlorosis virus Th il dCerathripoides claratris Capsicum chlorosis virus ThailandScirtothrips dorsalis Peanut yellow spot virus India

Peanut chlorotic fanspot virus Taiwan

Groundnut bud necrosis virus India

Frankliniella occidentalis Tomato spotted wilt virus Israel

Thrips tabaci Iris yellow spot virus Israel, IndiaFrankliniella cephalica Tomato spotted wilt virus JapanFrankliniella schultzei Groundnut bud necrosis virus IndiaFrankliniella schultzei Groundnut bud necrosis virus IndiaThrips palmi Groundnut bud necrosis virus India

Watermelon silver mottle virus Japan, IndonesiaMelon yellow spot virus JapanMelon yellow spot virus Japan

Thrips palmi is considered the ‘F. occidentalis’ of the tropical and s b tropical Asiaof the tropical and sub-tropical Asia

Pappu et al 2009Pappu et. al. 2009

Diversity of Tospoviruses in IndiaThe world’s second largest producer of vegetables withan annual production ofan annual production of 87.53 million tons from 5.86 million hectares representing 14 4% to the world

Groundnut bud necrosis virus14.4% to the world production (FAO statistics, 2009).

GBNV - most widespread and damaging causing production problems to

Watermelon bud necrosis virus

solanaceous and legumes

WBNV – economically important pathogen ofimportant pathogen of watermelon

IYSV – caused epidemic in

Capsicum chlorosis virus

pmajor bulb and seed onions

CaCV – reported in tomato d i 2007Iris yellow spot virus

Kunkalikar et al. 2011/Plant Pathology 101:367-376and pepper in 2007 y p

Reported Tospoviruses in Thailand

WSMoVWSMoV MYSVMYSV CaCVCaCV TNRVTNRV

S ib t l 2012 P t d t th 2012 I t ti l C f T i lSeepiban et al. 2012. Paper presented at the 2012 International Conference on Tropical Plant Diseases, Chiangmai, Thailand

TospovirusesTospoviruses infecting cucurbits in infecting cucurbits in ThailandThailand

Year Virus Natural host2003 WSMoV Cantaloupe2003 MYSV M l2003 MYSV Melon2003 WSMoV Snake gourd2004 MYSV Cucumber2004 MYSV Cucumber2004 MYSV Watermelon2005 MYSV Sponge gourdp g g2005 WSMoV Watermelon2006 MYSV Wax gourd

2011-2012 Survey on types of thrips and tospovirusin central Thailand;in central Thailand;

MYSV and WSMoV Cucurbits T. palmi

TNRVTNRV –– a recently described a recently described TospovirusTospovirus species infecting species infecting tomato and pepper in Thailandtomato and pepper in Thailandp ppp pp

Hassani-Mehraban et al. 2011/Eur J Plant Pathol doi 10.1007/s10658-011-9771-9

Fi t b d i t t d l t d t t dFirst observed in tomato and pepper plants under protected cultivation in Chiangmai, Thailand in 2008Symptomatic tomato and pepper samples were characterized at the Laboratory of Virology, Wageningen University in the Netherlands

TNRVTNRV –– a recently described a recently described TospovirusTospovirus species infecting species infecting tomato and pepper in Thailandtomato and pepper in Thailandp ppp pp

0 10.1Hassani-Mehraban et al. 2011/Eur J Plant Pathol doi 10.1007/s10658-011-9771-9

TospovirusesTospoviruses in the Philippinesin the Philippines

Virus Crops OccurrencepWSMoV Watermelon central, south

and north LuzonMYSV Watermelon

MelonCucumber

central, southand north Luzon

CucumberIYSV Onion central Luzon

* Based on field surveys and positive reactions Based on field surveys and positive reactions in DAS-ELISA using specific polyclonal antisera

Plant Pathology Department, East-West Seed Company (2003 2010 unpublished data)Company (2003-2010 unpublished data)

TospovirusesTospoviruses infectinginfecting cucurbitscucurbits in the Philippinesin the Philippines

W t l il ttl Melon yellow spot virus (MYSV)

Watermelon silver mottle virus (WSMoV)

TospovirusTospovirus infectinginfecting onionsonions in the Philippinesin the Philippines

Iris yellow spot virus (IYSV)y p ( )

66% of symptomatic onion leaf samples collected from Nueva Ecija (centralLuzon) were positive for IYSV in DAS-ELISA using specific polyclonalLuzon) were positive for IYSV in DAS ELISA using specific polyclonalantiserum kindly provided by Dick Peters (Laboratory of Virology, WageningenUniversity, the Netherlands)

Breeding for Polerovirus resistance gin cucurbits

Limited information on the genetic resistance of cucurbit-infectingPoleroviruses:Poleroviruses:

Dogimont et al. (1997) reported two complementary recessive g ( ) p p ygenes, cab-1 and cab-2, conferring resistance to CABYV in anIndian melon line PI 124112 Resistant genotypes identified: Faizabadi Phoont, 90625, PI 124112,

PI 124440, PI 255478, PI 282448 and PI 414723 (both fields and screening under controlled conditions using viruliferous Myzus persicae)screening under controlled conditions using viruliferous Myzus persicae)

Breeding for CABYV resistance in cucurbitsBreeding for CABYV resistance in cucurbits

So far no information about resistance b di f CABYV i A ibreeding for CABYV in Asia

In 2001, East-West Seed Companyp yPhilippines initiated the screening of bittergourd germplasm for CABYV resistanceresistance

Identified accession EW 1696 as a reliable source of resistance to CABYV. This line forms the basis of the East-West Seed Polerovirus resistancebreeding programsbreeding programs

Breeding for CABYV resistance in cucurbitsBreeding for CABYV resistance in cucurbits

THE CASE OF BITTERGOURD“B di f i t t ‘N k ’ th“Breeding for resistance to ‘Namamarako’ or the Cucurbit aphid-borne yellows virus (CABYV)”

Very difficult start - an example of a complex problem; cause of the disorder was unknown in the beginningSome farmers even blamed EW for the disorder – alleged that the disorder was associated with EW bittergourdvarieties (>80% market share)varieties (>80% market share)YET, this is a good example of how to deal with a complex problem; requires multi-disciplinary approach (pathology,problem; requires multi disciplinary approach (pathology, genetics, breeding, molecular biology)In the end, the breeder won the battle by developing and introducing “GALACTICA”

Breeding for CABYV resistance in Breeding for CABYV resistance in bittergourdbittergourd

APPROACHESAPPROACHES

Identification of Identification of Incorporation of the causal pathogen

resistance source

presistance to elite

breeding lines

Breeding for CABYV resistance and introduction of “Galactica”

2001 –

2003 to 2005 -

Line2006 to 2007 –2001

Identified EW1696

as R source

Line development using backcross

d

Controlled seedling

screeningInheritance

study

2007First test

hybrids with CABYV

i t

2008 –Launched GALACTICA

source and pedigree selection

resistance (HOT, RYT)

Breeding for CABYV resistance and introduction of “Galactica”

GalacticaGalaxy- check

Breeding for Tospovirus resistance in g pvegetables

Currently, only 2 single dominant R genes (Sw-5 and Tsw) are Currently, only 2 single dominant R genes (Sw-5 and Tsw) are available for tospovirus resistance breeding in tomato and pepper, respectively:Tomato:

available for tospovirus resistance breeding in tomato and pepper, respectively:Tomato:Tomato:

Sw-5 (derived from S. peruvianum): common in many commercial varieties Resistance breaking strains in Hawaii, South AfricaS 7 (d i d f S hil LA 1938) id t bl l l f

Tomato:Sw-5 (derived from S. peruvianum): common in many commercial varieties Resistance breaking strains in Hawaii, South AfricaS 7 (d i d f S hil LA 1938) id t bl l l fSw-7 (derived from S. chilense LA 1938): provide acceptable level of resistance to virulent strains of TSWV

Pepper:

Sw-7 (derived from S. chilense LA 1938): provide acceptable level of resistance to virulent strains of TSWV

Pepper:Tsw (found in accessions of C. chinense ‘PI15225’ and ‘PI159236’): deployed into several commercial sweet and hot pepper varieties Strongly influenced by temperature & physiological conditions

Tsw (found in accessions of C. chinense ‘PI15225’ and ‘PI159236’): deployed into several commercial sweet and hot pepper varieties Strongly influenced by temperature & physiological conditionsStrongly influenced by temperature & physiological conditions Virulent strains in the field can overcome this gene

Strongly influenced by temperature & physiological conditions Virulent strains in the field can overcome this gene

Breeding for Tospovirus resistance in g pvegetables

Sw-5 and Sw-7 however, appear ineffective against South and S th t A i T iSw-5 and Sw-7 however, appear ineffective against South and S th t A i T iSoutheast Asian Tospoviruses

Resistance found in 60% of S. peruvianum accessions

Southeast Asian Tospoviruses

Resistance found in 60% of S. peruvianum accessionsResistance found in 60% of S. peruvianum accessions screened with Sw-5-breaking isolates of TSWV could be the best source of tospovirus resistance in tomato (Gordillo and

Resistance found in 60% of S. peruvianum accessions screened with Sw-5-breaking isolates of TSWV could be the best source of tospovirus resistance in tomato (Gordillo and Stevens, 2008; Hanson et al., 2009)Stevens, 2008; Hanson et al., 2009)

Identifying sources of resistance to Tospoviruses in Asia

Sources of resistance to GBNV/PBNV Groundnut varieties (ICGS 44 and ICGS 11) with GBNV resistance Groundnut varieties (ICGS 44 and ICGS 11) with GBNV resistance

released in India (Mandal et al., 2012) Peanut lines suitable for use in PBNV resistance breeding program

identified in Thailand (Pensuk et al., 2002)identified in Thailand (Pensuk et al., 2002) 2 accessions of S. peruvianum (L06138, L00671) identified as potential

sources for PBNV and CaCV resistance in AVRDC field trials in India and Southeast Asia (Hanson et al., 2009)

Sources of resistance to TSWV and CaCV Seedling screening for TSWV and CaCV resistance in tomato and pepper

i h i l i l ti i t AVRDC T i (Kusing mechanical inoculation now in progress at AVRDC, Taiwan (Kenyon,2012 personal communication)

Sources of resistance to Tospoviruses infecting cucurbitsSources of resistance to Tospoviruses infecting cucurbits limited information Sugiyama et al. (2009) reported cucumber accessions from Southeast

Asia with moderate resistance to MYSVAsia with moderate resistance to MYSV Resistance to MYSV is temperature dependent

Identifying sources of resistance to Tospoviruses in Thailand

East-West Seed developed screening protocols for WSMoV(watermelon) and MYSV (cucumber) using thrips inoculation:2011: Identified 1 long cucumber accession (EW18185) with resistance to MYSV; 3/141 watermelon inbred lines (BL-15, EW14778, KK-359) with highMYSV; 3/141 watermelon inbred lines (BL 15, EW14778, KK 359) with high level of resistance to WSMoV2012: identified 2/159 watermelon inbred lines (EW15633, EW15223) with high level of resistance to WSMoVlevel of resistance to WSMoV

Developed screening protocols for TNRV using mechanical inoculation:2012: identified 2 lines (PY6423, PY6424) with high level of resistance

Conclusions and recommendations

Poleroviruses and tospoviruses are rapidly emerging as potential th t t t bl d ti i A ithreats to vegetable production in Asia

More intensive surveys and diversity analysis of these viruses are y y ynecessary to enable effective control

Resistance breaking strains and the occurrence of different speciesResistance breaking strains and the occurrence of different species or strains in the field may complicate efforts being made to breed for durable resistance in vegetable crops

Good breeding strategies must be developed Requires comprehensive understanding of the virus-vector/virus-

host interactions as well as virus diversityhost interactions as well as virus diversity

Breeding for plants with insect resistance?

THANK YOU FOR YOUR ATTENTION!