detectionandidentificationofthripsdetection and … and identification of thrips-borne...

Detection and identification of thripsDetection and identification of thripsDetection and identification of thripsDetection and identification of thrips--borne tospoviruses by serological and borne tospoviruses by serological and p y gp y gnucleic acid toolsnucleic acid tools

ShyiShyi‐‐Dong YehDong Yehyy gg

Department of Plant PathologyDepartment of Plant PathologyDepartment of Plant PathologyDepartment of Plant Pathology

National Chung Hsing UniversityNational Chung Hsing University

Plant PathologyPlant PathologyNCHUNCHU

Th di k tt d iltTh di k tt d ilt

TospovirusesTospoviruses

The disease known as spotted wilt was The disease known as spotted wilt was first found in Australia in 1915. first found in Australia in 1915. ((BrittlebankBrittlebank 1919 J Agric Victoria 17:2131919 J Agric Victoria 17:213 235)235)((BrittlebankBrittlebank, 1919. J. Agric. Victoria 17:213, 1919. J. Agric. Victoria 17:213--235)235)

This disease was associated with aThis disease was associated with aThis disease was associated with a This disease was associated with a virus then named as virus then named as Tomato spotted Tomato spotted wilt viruswilt virus (TSWV) in 1930(TSWV) in 1930wilt viruswilt virus (TSWV) in 1930. (TSWV) in 1930.

((SamulSamul et al., 1930. Aust. et al., 1930. Aust. CouncCounc. Sci. Ind. Bull. 44:1. Sci. Ind. Bull. 44:1--46)46)

According to the properties of its particle morphology, host According to the properties of its particle morphology, host range, and transmission by thrips, TSWV was classified as the range, and transmission by thrips, TSWV was classified as the type member of “tomato spotted wilt virus group” in 1970.type member of “tomato spotted wilt virus group” in 1970.

This virus group was later designated as the genusThis virus group was later designated as the genus TospovirusTospovirus


This virus group was later designated as the genus This virus group was later designated as the genus TospovirusTospovirusbelonging to the family belonging to the family BunyaviridaeBunyaviridae by the “International by the “International Committee on Taxonomy of Virus” (ICTV) in 1991Committee on Taxonomy of Virus” (ICTV) in 1991Committee on Taxonomy of Virus (ICTV) in 1991.Committee on Taxonomy of Virus (ICTV) in 1991.

TospovirusTospovirus is the only genus that infects plants in the familyis the only genus that infects plants in the family((FranckiFrancki et al., 1991. Arch. et al., 1991. Arch. VirolVirol. Suppl. 2:450). Suppl. 2:450)

TospovirusTospovirus is the only genus that infects plants in the family is the only genus that infects plants in the family BunyaviridaeBunyaviridae, which is a large family that includes viruses of , which is a large family that includes viruses of the generathe genera OrthobunyavirusOrthobunyavirus HantavirusHantavirus NairovirusNairovirus andandthe genera the genera OrthobunyavirusOrthobunyavirus, , HantavirusHantavirus, , NairovirusNairovirus, and , and PhlebovirusPhlebovirus that usually infect vertebrates and transmitted that usually infect vertebrates and transmitted by mosquitoes ticks phlebotomies flies and otherby mosquitoes ticks phlebotomies flies and otherby mosquitoes, ticks, phlebotomies flies, and other by mosquitoes, ticks, phlebotomies flies, and other arthropods.arthropods. (Elliot et al., 2000. J. Gen. Virol. 7:501(Elliot et al., 2000. J. Gen. Virol. 7:501--522)522)

Persistently transmitted by thrips.Persistently transmitted by thrips.

Currently, Currently, TospovirusTospovirus consists of consists of Cu e t y,Cu e t y, ospovi usospovi us co s sts oco s sts o19 species.19 species.


Asian type (12 species)Asian type (12 species)

WSMoVWSMoV TaiwanTaiwan, Japan, Japan Watermelon, Other cucurbits, Calla lilyWatermelon, Other cucurbits, Calla lily ,,TomatoTomatoTospovirusTospovirus DistributionDistribution Host cropsHost crops

yp ( p )yp ( p )

PBNVPBNV India, SouthIndia, South--East AsiaEast Asia Peanut, TomatoPeanut, Tomato

WBNVWBNV IndiaIndia WatermelonWatermelon

CaCVCaCV TaiwanTaiwan, Australia, , Australia, ThailandThailand

Capsicum, Tomato, Capsicum, Tomato, Gloxinia, Orchid, Calla lilyGloxinia, Orchid, Calla lily

CCSVCCSV TaiwanTaiwan Calla lilyCalla lilyyy

MYSVMYSV TaiwanTaiwan, Japan, Thailand, Japan, Thailand Melon, Watermelon, Melon, Watermelon, Physalis minimaPhysalis minima

TZSVTZSV ChinaChina Tomato, Chili pepperTomato, Chili pepper

IYSVIYSV Brazil, Israel, Netherland, Brazil, Israel, Netherland, Slovenia, USASlovenia, USA

Onion, Iris, Leek, Amaryllis, GarlicOnion, Iris, Leek, Amaryllis, Garlic

TYRVTYRV IranIran Tomato, Chrysanthemum, Gazania, Potato,Tomato, Chrysanthemum, Gazania, Potato,TYRVTYRV IranIran Tomato, Chrysanthemum, Gazania, Potato, Tomato, Chrysanthemum, Gazania, Potato, SoybeanSoybean

PolRSVPolRSV ItalyItaly BuckwheatBuckwheat

PCFVPCFV T iT i PPPCFVPCFV TaiwanTaiwan PeanutPeanut

PYSVPYSV India, ThailandIndia, Thailand PeanutPeanut


EuroEuro‐‐American type (7 species)American type (7 species)TospovirusTospovirus DistributionDistribution Host cropsHost crops

EuroEuro‐‐American type (7 species)American type (7 species)

TSWVTSWV WorldwideWorldwide Very broad (>1090 species)Very broad (>1090 species)

GRSVGRSV Brazil SouthBrazil South Peanut TomatoPeanut TomatoGRSVGRSV Brazil, South Brazil, South AfricaAfrica

Peanut, TomatoPeanut, Tomato

TCSVTCSV BrazilBrazil TomatoTomatoTCSVTCSV BrazilBrazil TomatoTomato

ZLCVZLCV BrazilBrazil Courgette, CucumberCourgette, Cucumber

CSNVCSNV BrazilBrazil Florists chrysanthemum, TomatoFlorists chrysanthemum, Tomato

MeMSVMeMSV MexicoMexico Honeydew melon, Honeydew melon, Cucurbita pepo, Cucurbita pepo, Cucumis sativusCucumis sativus

INSVINSV USA, EuropeUSA, Europe Mainly ornamentals, Common Mainly ornamentals, Common dahlia, Gloxinia, African balsamdahlia, Gloxinia, African balsam


In In GeorgiaGeorgia, tomato yields were reduced by , tomato yields were reduced by TSWVTSWV forfor $8 8 million$8 8 million (approximately NTD 3(approximately NTD 3TSWVTSWV for for $8.8 million$8.8 million (approximately NTD 3 (approximately NTD 3 hundred million) in 2000.hundred million) in 2000.

The epidemic ofThe epidemic of IYSVIYSV in in ColoradoColorado (USA) in 2003 (USA) in 2003 causedcaused $2 5$2 5‐‐5 0 million losses5 0 million losses in onionin onioncaused caused $2.5$2.5‐‐5.0 million losses5.0 million losses in onion.in onion.

WSMoVWSMoV caused melon damages as high as caused melon damages as high as NTD NTD g gg g25 million25 million in in Taiwan Taiwan in 2006.in 2006.

Sherwood et al., 2005; Schwartz et al., 2006; Cheng, 2008Sherwood et al., 2005; Schwartz et al., 2006; Cheng, 2008

G i ti f t iG i ti f t i


Genome organization of tospovirusesGenome organization of tospoviruses

F ilF il B i idB i idFamily Family BunyaviridaeBunyaviridaeGenus Genus BunyavirusBunyavirus

HantavirusHantavirusHantavirusHantavirusNairovirusNairovirusPhlebovirusPhlebovirusPhlebovirusPhlebovirusTospovirusTospovirus

3’ 5’

N29 kDa

NSs52 kDa

2916 nts3’ 5’

Gc75 kDa

Gn46 kDa NSm

37 kD

4821 ntsS RNAS RNA M RNAM RNA

3’ 5’

L

8897 nts

37 kDa

L RNAL RNAL

332 kDa


PhylogeneticPhylogenetic relationship of tospoviruses basedrelationship of tospoviruses based

WSMoV serogroupWSMoV serogroup

PhylogeneticPhylogenetic relationship of tospoviruses based relationship of tospoviruses based on NPon NP

IYSV serogroupIYSV serogroupg pg p

TSWV serogroupTSWV serogroup

EuroEuro‐‐American typeAmerican type

Asian typeAsian type


Recognized Recognized TospovirusTospovirus vector species in thevector species in the

Thrips vector Tospovirus species

gg pp ppThysanopteraThysanoptera

p p p

Ceratothripoides claratris Shumsher CaCV

Frankliniella occidentalis (Pergande) CSNV, GRSV, INSV, TCSV, TSWV

F. schultzei (Trybom) CaCV, CSNV, GRSV, INSV, TCSV, TSWV

F. intonsa (Trybom) GRSV, INSV, TCSV, TSWV

F i B ll GRSV TSWVF. gemina Bagnall GRSV, TSWV

F. fusca (Hinds) TSWV

F. bispinosa (Morgan) TSWV

F. zucchini Nakahara & Monteiro ZLCV

Microcephalothrips abdominalis (D. L. Crawford) TYRV

S i t th i d li H d PBNV PCFV PYSVScirtothrips dorsalis Hood PBNV, PCFV, PYSV

Thrips palmi Karny CaCV, CCSV, WSMoV, PBNV, WBNV, MYSV, TSWV

T. tabaci Lindeman IYSV, TYRV, TSWVT. tabaci Lindeman IYSV, TYRV, TSWV

T. setosus Moulton TSWV

http://www.oznet.ksu.edu/tospovirus/tospo_list.htmhttp://www.oznet.ksu.edu/tospovirus/tospo_list.htm


Whitfield et al., 2005. Annu. Rev. Phytopathol. 43:459Whitfield et al., 2005. Annu. Rev. Phytopathol. 43:459––8989


Diseases caused byDiseases caused by

T t tt d ilt iT t tt d ilt i (TSWV)(TSWV)

Diseases caused byDiseases caused byEuroEuro‐‐American type tospovirusesAmerican type tospoviruses

Tomato spotted wilt virusTomato spotted wilt virus (TSWV)(TSWV)TomatoTomato

PeanutPeanut

Diseases caused byDiseases caused by


Diseases caused byDiseases caused byEuroEuro‐‐America type tospovirusesAmerica type tospoviruses

I i i iI i i i (INSV)(INSV)Impatiens necrotic spot virusImpatiens necrotic spot virus (INSV)(INSV)EpidendrumEpidendrum

ImpatiensImpatiens

GloxiniaGloxinia


Diseases caused by AsiaDiseases caused by Asia type tospovirusestype tospoviruses

Watermelon silver mottle virusWatermelon silver mottle virus (WSMoV)(WSMoV)

Diseases caused by AsiaDiseases caused by Asia‐‐type tospovirusestype tospoviruses

( )( )

WatermelonWatermelon W dW dWatermelonWatermelon Wax gourdWax gourd

MelonMelonWatermelonWatermelonC bitC bitMelon yellow spot virusMelon yellow spot virus (MYSV)(MYSV)

MelonMelonWatermelonWatermelonCucurbitCucurbit

Di d b A iDi d b A i ii


Diseases caused by AsiaDiseases caused by Asia‐‐typetype tospovirusestospoviruses

Iris yellow spot virusIris yellow spot virus (IYSV)(IYSV)Iris yellow spot virus Iris yellow spot virus (IYSV)(IYSV)

OnionOnion


Identification ofIdentification of TospovirusTospovirusIdentification of Identification of TospovirusTospovirus

TEMTEM l hN i iCCSVCCSV

TEMTEM

y

Ultrathin sectionNegative stain

CaCVCaCV

Calla lilyCaCVCaCV

amiana

N. bentha

((ZhengZheng et al., 2008. Eur. J. Plant et al., 2008. Eur. J. Plant PatholPathol. 120:199. 120:199––209)209)

Chen et al., 2005. Plant Dis. 89:440Chen et al., 2005. Plant Dis. 89:440––445445

N


Detection of tospoviruses SerologySerology‐‐basedbased Nucleic acidNucleic acid‐‐basedbased

Detection of tospoviruses

UgaUga et al., 2004 et al., 2004


The N gene‐based primers and antibodies are highly species‐specific for identification of The N gene‐based primers and antibodies are highly species‐specific for identification of tospoviruses.tospoviruses.

However, facing to the complication of viruses in fields, various primer pairs and antibodiesHowever, facing to the complication of viruses in fields, various primer pairs and antibodiesin fields, various primer pairs and antibodies should be used for virus detection. in fields, various primer pairs and antibodies should be used for virus detection.

UgaUga et al., 2004 et al., 2004


S l i l l iS l i l l iImmunodiffusionImmunodiffusion ELISAELISA

Serological analysis Serological analysis –– Antisera against NPAntisera against NPELISAELISA

Western blotWestern blot

Chu et al., 2001. Chu et al., 2001. PhytopathologyPhytopathology 91:85691:856‐‐863863


Serological response ofSerological response of MAbMAb WNSs against the NSsWNSs against the NSsSerological response of Serological response of MAbMAb‐‐WNSs against the NSs WNSs against the NSs protein of WSMoVprotein of WSMoV

3’ 5’

NNSsNSs

Chen et al., 2006. Chen et al., 2006. PhytopathologyPhytopathology 96:129696:1296‐‐13041304


The commonThe common epitopeepitope presents among the NSs proteins ofpresents among the NSs proteins ofThe common The common epitopeepitope presents among the NSs proteins of presents among the NSs proteins of WSMoVWSMoV‐‐serogroupserogroup tospovirusestospoviruses

Chen et al., 2006. Chen et al., 2006. PhytopathologyPhytopathology 96:129696:1296‐‐13041304


MAbMAb‐‐WNSs reacts with all Asian tospovirusesWNSs reacts with all Asian tospoviruses

kDakDa727255554343

MAbMAb--WNSsWNSs

2 5

3

3.5AA

405

405

1

1.5

2

2.5

No backgroud, highly specific

0

0.5

1

1

highly specific to Asian type tospovirusestospoviruses


The commonThe common epitopeepitope presents among the NSs proteins ofpresents among the NSs proteins ofThe common The common epitopeepitope presents among the NSs proteins of presents among the NSs proteins of WSMoVWSMoV‐‐ and IYSVand IYSV‐‐serogroupserogroup tospovirusestospoviruses

WNSscon98 120

WSMoVWSMoV serogroup

IYSV serogroup

E i f INSV NS t i b ETE i f INSV NS t i b ET 28b( ) f28b( ) f


Expression of INSV NSs protein by pETExpression of INSV NSs protein by pET‐‐28b(+) for 28b(+) for production of antiserum RAsproduction of antiserum RAs‐‐INSsINSs

kDakDaElution from gelElution from gelElution from NiElution from Ni2+2+‐‐NTANTA

kDakDa

5555 NSsNSs7272

9595

NSsNSs5555

7272

NSsNSs4343

3434

4343

3434

26262626

NSsNSs

RAsRAs--HisHisRARA HiHi

NSsNSs

RAsRAs HisHisRAsRAs--HisHis

Serological response of RAsSerological response of RAs‐‐INSVINSV‐‐NSs reacted withNSs reacted with


Serological response of RAsSerological response of RAs‐‐INSVINSV‐‐NSs reacted with NSs reacted with tospovirusestospoviruses

727255554343

kDakDa

1.4

1.6

RAsRAs-- INSsINSs

0 8

1

1.2

AA40

540

5

0.4

0.6

0.8AA

0

0.2


MAbMAb‐‐WNSsWNSs

RAsRAs‐‐INSsINSs

?? MAb recognizes the?? MAb recognizes the common eiptope of Euro-American NSs proteins is

thon the way

P ifi i f PYSV NP d bP ifi i f PYSV NP d b ETET ff


Purification of PYSV NP expressed by Purification of PYSV NP expressed by pETpET vector for vector for production of RAsproduction of RAs‐‐PYSV NPPYSV NP

Elution from gelElution from gelkDakDa

Elution from NiElution from Ni2+2+‐‐NTANTAkDakDa

4343

3434

kDakDa

4343

72725555

NPNP2626NPNP2626

3434

17171717

NPNPRAsRAs--HisHis

AA ii

NPNPRAsRAs--HisHis


Serological response of RAsSerological response of RAs PYSV NP reacted withPYSV NP reacted withSerological response of RAsSerological response of RAs‐‐PYSV NP reacted with PYSV NP reacted with tospovirusestospoviruses

11.5

2

A40

5

00.5

IYSVTYRVMoVBNVBNVCaCVCCSVMYSVPYSVPCFVSWVTCSV

RSVINSV H

A

IY TYWSMo

WBNPB CaCCC MY PY PC TSW TC GR IN

3434

2626

kDakDa

RAsRAs--PYSV NPPYSV NP

PYSV serogroupPYSV serogroup

SummarySummarySu a ySu a y

Plant PathologyPlant PathologyNCHUNCHUWSMoV serogroupWSMoV serogroup

Serological relationship of Serological relationship of Asian typeAsian type tospoviruses tospoviruses ypyp pp

based on based on PolyclonalPolyclonalPolyclonal Polyclonal

antibodies to NPsantibodies to NPs

kDakDa

2626

3434

RAsRAs--WSMoV NPWSMoV NPkDakDa

3434

RAsRAs--MYSV NPMYSV NP2626

3434


Serological relationships of Serological relationships of AsianAsian‐‐type type tospoviruses tospoviruses ypyp pp

based on based on Polycolandal antibodiesPolycolandal antibodies

IYSV serogroupIYSV serogroupPolycolandal antibodies Polycolandal antibodies toNPstoNPs

kDakDa

3434

2626

MAsMAs--TYRV NPTYRV NP

3434

kDakDa

MAsMAs--IYSV NPIYSV NP

2626

Plant PathologyPlant PathologyNCHUNCHUWSMoV serogroupWSMoV serogroup

Serological relationship of Serological relationship of AsianAsian‐‐typetype tospoviruses tospoviruses ypyp pp

based on based on

IYSV IYSV serogroupserogroup MAb to the MAb to the common eiptope ofcommon eiptope ofcommon eiptope of common eiptope of NSs proteinsNSs proteins

kDakDa

72727272

55554343

MAbMAb--WNSsWNSs


Serological relationships of PYSVSerological relationships of PYSVSerological relationships of PYSV Serological relationships of PYSV serogroup based on serogroup based on

NPNP‐‐polycolonal Abpolycolonal Ab

kDakDa

MAbMAb--PCFV NPPCFV NP

3434

2626

kDakDa

3434

2626

kDakDa


3434

2626

kDakDa RAsRAs--PYSV NPPYSV NP

RAsRAs--PCFV NPPCFV NP

2626


Serological relationship ofSerological relationship ofSerological relationship of Serological relationship of EuroEuro‐‐American typeAmerican type

i b di b dtospoviruses based on tospoviruses based on

NPNP‐‐polyclonal Abpolyclonal AbNPNP polyclonal Abpolyclonal Ab

3434

2626

kDakDa

RAsRAs--TSWV NPTSWV NP3434

kDakDaTSWV serogroupTSWV serogroup

2626

RAsRAs--INSV NPINSV NP


Serological relationship ofSerological relationship ofSerological relationship of Serological relationship of EuroEuro‐‐American typeAmerican type

i b di b dtospoviruses based on tospoviruses based on

NSsNSs Polyclonal AbPolyclonal AbNSsNSs‐‐Polyclonal AbPolyclonal Ab

7272

kDakDa

TSWV TSWV serogroupserogroup5555

4343

RAsRAs-- INSsINSs


Serological relationships of tospoviruses based onSerological relationships of tospoviruses based onWSMoV serogroupWSMoV serogroup

Serological relationships of tospoviruses based on Serological relationships of tospoviruses based on NPsNPs

IYSV serogroupIYSV serogroupg pg p

TSWV serogroupTSWV serogroup



D t ti f t i b t bl ttiD t ti f t i b t bl ttiDetection of tospoviruses by western blottingDetection of tospoviruses by western blotting

EE A i iA i iNSs protiens EuroEuro‐‐American type tospovirusesAmerican type tospovirusesNSs protiens

72725555

4343

kDakDa

RAsRAs-- INSsINSs4343

72725555

kDakDa

7272

kDakDa RAsRAs-- PNSsPNSs4343

55554343

MAbMAb--WNSsWNSs

Asian type tospovirusesAsian type tospoviruses


ConclusionsConclusions Based on the serological relationships of NPs,Based on the serological relationships of NPs,

TospovirusTospovirus is suggested to be reclassified into four is suggested to be reclassified into four serogroups, including as WSMoV, TSWV, IYSV and PYSV serogroups, including as WSMoV, TSWV, IYSV and PYSV serogroups.serogroups.

Based on the serological relationships of NSs proteins, Based on the serological relationships of NSs proteins, tospoviruses are clustered into three major serogroups.tospoviruses are clustered into three major serogroups.tospoviruses are clustered into three major serogroups.tospoviruses are clustered into three major serogroups.

GroupGroup‐‐specific antibodies and antisera can be used to specific antibodies and antisera can be used to develop commercial detective kits for detectingdevelop commercial detective kits for detectingdevelop commercial detective kits for detecting develop commercial detective kits for detecting tospoviruses effectively.tospoviruses effectively.

Plant PathologyPlant PathologyNCHUNCHUDetection by Nucleic Acid tools

RTRT--PCR PCR S ifi i d i d f NS ifi i d i d f NSpecific primers designed from N geneSpecific primers designed from N gene

Degenerate primersDegenerate primersDegenerate primersDegenerate primers

ZhengZheng et al., 2008. Eur. J. Plant et al., 2008. Eur. J. Plant PatholPathol. 120:199. 120:199––209209


Determined sequences Determined sequences –– S RNAS RNASpecies S-RNA

length (nt)5’-UTR

(nt)NSs IGR

(nt)N 3’-UTR

(nt)nt aa nt aa

CCSV 3172 66 1383 460 825 834 277 64

CaCV 3477 66 1320 439 1196 828 275 67

GRSV 3049 87 1404 467 630 777 258 151

INSV 2992 62 1350 449 642 789 262 149

IYSV 3105 70 1332 443 811 822 273 70

MeSMV 3283 80 1368 455 887 789 262 159

MYSV 3232 68 1410 469 847 840 279 67

PBNV 3057 66 1320 439 773 831 276 67

PCFV 2833 67 1419 472 455 813 270 79

PYSV 2970 57 1443 480 653 741 246 76

PolRSV 2484 72 1332 443 183 825 274 72

TSWV 2837 46 1404 467 585 777 258 25

TYRV 3061 71 1332 443 762 825 274 71

TZSV 3279 64 1380 459 934 837 278 64TZSV 3279 64 1380 459 934 837 278 64

WSMoV 3534 66 1320 439 1255 828 275 65

WBNV 3402 66 1320 439 1421 828 275 67

TCSV 1410 469 780 777 258TCSV - - 1410 469 780 777 258 -

ZLCV - - - - - 783 260 -

CSNV - - - - - 783 260 -


Determined sequences Determined sequences –– M RNAM RNASpecies M-RNA

length (nt)5’-UTR

(nt)NSm IGR

(nt)GP 3’-UTR

(nt)nt aa nt aa

CCSV 4704 54 930 309 303 3372 1123 45

CaCV 4823 56 927 308 427 3366 1121 47

INSV 4972 85 912 303 473 3333 1110 169

IYSV 4838 63 936 311 379 3411 1136 49

MYSV 4815 58 927 308 398 3384 1127 48

PBNV 4801 56 924 307 408 3366 1121 47

PolRSV 4689 62 924 307 267 3408 1135 28

TSWV 4829 100 909 302 328 3408 1135 84

TZSV 4945 54 930 309 546 3369 1122 46

WSMoV 4880 55 939 312 473 3366 1121 47

WBNV 4794 55 924 307 402 3366 1121 47WBNV 4794 55 924 307 402 3366 1121 47

TYRV 4787 62 927 308 355 3393 1130 50

PCFV - - 921 306 - - - -

ZLCV 108 912 303 3408 1135 85ZLCV - 108 912 303 - 3408 1135 85

CSNV - 101 912 303 - 3408 1135 83

TCSV - 100 912 303 - 3405 1134 84

GRSV - - 912 303 - 3402 1133 85GRSV - - 912 303 - 3402 1133 85

MeSMV - - - - - - - -

PYSV - - - - - - - -

Plant PathologyPlant PathologyNCHUNCHUDetermined sequences Determined sequences –– L RNA L RNA

Species L-RNA length (nt)

5’-UTR (nt)

RdRp 3’-UTR (nt)nt aa

CCSV 8911 29 8652 2883 230

CaCV 8912 31 8634 2877 247

INSV 8776 33 8604 2867 139

IYSV 8880 33 8622 2873 225

MYSV 8918 32 8613 2870 273

PBNV 8911 32 8634 2877 245

TSWV 8897 33 8628 2875 236

TZSV 8919 28 8658 2885 233

WSMoV 8917 32 8637 2878 248

WBNV 8916 32 8637 2878 247

TYRV - - - - -

CSNV - - - - -

GRSV - - - - -

TCSVTCSV - - - - -

ZLCV - - - - -

PCFV - - - - -

PYSVPYSV - - - - -

MeSMV - - - - -

PolRSV - - - - -

D t iD t iDegenerate primer Degenerate primer pairs designed forpairs designed forpairs designed for pairs designed for quick identificationquick identificationquick identification quick identification of tospovirusesof tospovirusespp


The degenerate primers designed from theThe degenerate primers designed from theThe degenerate primers designed from the The degenerate primers designed from the consensus sequences of L RNAsconsensus sequences of L RNAs Degenerate primers: gL3637, gL4435c or gL4510c (Chu et al., 2001)


The degenerate primers designed from theThe degenerate primers designed from theThe degenerate primers designed from the The degenerate primers designed from the consensus sequences of L RNAsconsensus sequences of L RNAs Degenerate primers: t2740 and t3920c (Lin, 2007)


L3637/ L4510L3637/ L4510gL3637/gL4510cgL3637/gL4510c

t2740/t3920ct2740/t3920c


The degenerate primers designed from theThe degenerate primers designed from theThe degenerate primers designed from the The degenerate primers designed from the consensus sequences of M RNAsconsensus sequences of M RNAs

Degenerate primers: tNSm410 and tNSm870c (Lin, 2007)


The degenerate primers designed from theThe degenerate primers designed from theThe degenerate primers designed from the The degenerate primers designed from the consensus sequences of M RNAsconsensus sequences of M RNAs Degenerate primers: WG1 and WG960c (Lin, 2007)


Tospoviruses can be detected by the degenerate Tospoviruses can be detected by the degenerate

NSm GP L

primer pairsprimer pairs

Virus tNSm410/tNSm870

G1/G960

gL3637/gL4510c

t2740/t3920

TSWV + - + +TSWV + - + +

TCSV + - + +

INSV + - + +

GRSV + - + +

IYSV + + + +

TYRV + + + +

WSMoV + + + +

MYSV + + + +

C CVCaCV + + + +

CCSV + + + +

WBNV + + + +

PBNV + + + +

PCFV - - + -


Multiplex RTMultiplex RT‐‐PCR for detection of tospovirusesPCR for detection of tospoviruses

Application of degenerate primers for field surveyApplication of degenerate primers for field survey


Application of degenerate primers for field surveyApplication of degenerate primers for field survey


Concl sionsConcl sionsConclusionsConclusions All tested tospoviruses can be detected by the primer pair All tested tospoviruses can be detected by the primer pair

gL3637/gL4510c.gL3637/gL4510c.

Twelve of them except PCFV can be detected by theTwelve of them except PCFV can be detected by the Twelve of them, except PCFV, can be detected by the Twelve of them, except PCFV, can be detected by the primer pairs tNSm410/tNSm870c and t2740/t3920c.primer pairs tNSm410/tNSm870c and t2740/t3920c.

Eight tospoviruses, such as CaCV, CCSV, IYSV, MYSV, TYRV, Eight tospoviruses, such as CaCV, CCSV, IYSV, MYSV, TYRV, WBNV, PBNV and WSMoV, can be detected by the primer WBNV, PBNV and WSMoV, can be detected by the primer pairs G1/G960cpairs G1/G960c..

The degenerate primer pairs can be used in inspection forThe degenerate primer pairs can be used in inspection forThe degenerate primer pairs can be used in inspection for The degenerate primer pairs can be used in inspection for preventing import of foreign tospoviruses.preventing import of foreign tospoviruses.


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