J[ Phytopathology 036\ 154Ð158 "0888#Þ 0888 Blackwell Wissenschafts!Verlag\ BerlinISSN 9820!0674

Horticulture Research International\ Wellesbourne\ and John Innes Centre\ Norwich\ UK

The Partial Characterization of a Badnavirus Infecting the Greater Asiatic or

Water Yam "Dioscorea alata#

S[ PHILLIPS0\ R[ W[ BRIDDON1\ A[ A[ BRUNT0 and R[ HULL1

Authors| addresses] 0Horticulture Research International\ Wellesbourne\ Warwickshire\ CV24 8EF\ UK^ 1John InnesCentre\ Norwich Research Park\ Colney\ Norwich NR3 6UH\ UK "correspondence to R[ Hull[ E!mail]HULLÝBBSRC[AC[UK#

With 2 _gures

Received July 29\ 0887^ accepted September 12\ 0887

Abstract

A bacilliform virus from Dioscorea alata\ designatedDioscorea alata bacilliform virus "DaBV#\ from Bar!bados and West Africa and from other Dioscorea spp[from West African\ Carribean\ Asian and South Amer!ican countries\ has been characterized[ The virus wastransmitted by the mealybug\ Planococcus citri and bymechanical transmission of partially puri_ed prep!arations to several Dioscorea spp[ DaBV was sero!logically related to a distinct bacilliform virus fromDioscorea bulbifera\ to one isolate of sugarcane bacilli!form badnavirus and two isolates of banana streak bad!navirus "BSV# but was not related to another isolate ofBSV or to Kalanchoe top spotting or cacao swollen shootbadnaviruses[ The coat protein of DaBV was about45 kDa and the nucleic acid was double!stranded DNAof about 6[4 kbp\ part of which showed distant homologywith other badnaviruses[ Thus\ DaBV is a distinct hith!erto uncharacterized badnavirus[

Zusammenfassung

Die Teilcharakterisierung des Badnavirus in Wasseryams "Dios!corea alata#

Charakterisiert wurde ein Bacilliformvirus aus Dioscoreaalata bezeichnet als das Discorea alata bacilliform Virus"DaBV#\ von Barbados und Westafrika sowie aus ande!ren Dioscorea spp[ von La�ndern in Westafrika\ der Ka!ribik\ Asien und Su�damerika[ Das Virus wurde durchdie Mehllaus\ Planococcus citri\ und durch mechanischeUÝbertragungswege von teilgereinigten Proben zu ver!schiedenen Dioscorea spp[ u�bertragen[ Serologisch warDaBV mit einem speci_zschen Bacilliformvirus aus Dios!corea bulbifera\ mit einem Isolat des sugarcane bacill!iform Badnavirus sowie mit zwei Isolaten des bananastreak Badnavirus "BSV# verwandt\ jedoch nicht miteinem anderen Isolat des BSV oder mit dem Kalanchoetop spotting oder dem cacao swollen shoot Badnavirus[

U[ S[ Copyright Clearance Center Code Statement] 9820Ð0674:88:3694Ð9154 , 03[99:9

Das Hu�llprotein des DaBV war circa 45 kDa und dieNukleinsa�ure eine doppelstra�ngige DNA von circa 6\4kp\ einen Teil dessen eine entfernte Homologie mit ande!ren Badnaviren zeigte[ Von daher ist DaBV ein spe!zi_sches\ bisher unbeschriebenes Badnavirus[

Keywords] badnavirus\ Dioscorea alata badnavirus\ mea!lybug transmission

Introduction

Yams "Dioscorea spp[# are important sources of food inmany tropical and semitropical countries\ especially inthose of West Africa\ South and Central America "par!ticularly so in Caribbean countries# and Oceania[ In 0885\22[00 million tonnes of yam tubers were harvested froma world production area of 2[06 million hectares^ of these\20[56 million tonnes were harvested from 2[92 millionhectares in Africa "Anon\ 0886#[ Dioscorea alata "thegreater Asiatic or water yam# is the most widely grownspecies\ although _ve others "Dioscorea bulbifera\ Dios!corea cayenensis\ Dioscorea esculenta\ Dioscorearotundata and Dioscorea tri_da# are also important insome countries[ Some species "especially Dioscorea ~o!ribunda and Dioscorea composita# are grown for theextraction of diosgenin\ the precursor of some steroidaldrugs "Martin et al[\ 0855#[

Viruses are reported to be widespread and often dam!aging in Dioscorea spp[ Of these\ yam mosaic potyvirus"Thouvenel and Fauquet\ 0868^ Duterme et al[\ 0885^Aleman!Verdaguer et al[\ 0886# and Dioscorea latent pot!exvirus "Hearon et al[\ 0867^ Phillips et al[\ 0875# havebeen well characterized^ another potyvirus\ designatedDioscorea alata potyvirus "DaPV#\ has yet to be fullydescribed "Hughes\ 0875^ Brunt et al[\ 0878#[ An unde!scribed virus with bacilliform particles in complex with apotyvirus has been associated with the internal brownspot disease of D[ alata cv[ White Lisbon in Barbados"Harrison and Roberts\ 0862#^ a similar virus was later

155 PHILLIPS et al[

detected in D[ cayenensis in the eastern Caribbean"Mohamed and Mantell\ 0865#[

During preliminary investigations of viruses and virusdiseases of yams\ a virus with bacilliform particles wasdetected in plants of D[ alata from Barbados and othercountries which was subsequently shown to be those ofa badnavirus[ Badnaviruses are para!retroviruses whichhave a double!stranded DNA genome encapsidated in abacilliform particle "Lockhart\ 0889#[ This article reportsthe results of further studies on the virus which\ as it hasnot been previously described\ it is proposed to designateDioscorea alata badnavirus "DaBV#[

Materials and Methods

Plant material

Tubers of D[ alata\ D[ bulbifera\ D[ cayenensis\ Dioscoreadumetorum\ Dioscorea nummularia\ Dioscorea opposita\Dioscorea pentophylla and D[ rotundata were importedunder a licence from Bangladesh\ Barbados\ Benin\Ghana\ Japan\ Nigeria\ Papua New Guinea\ Puerto Rico\Sri Lanka\ Togo and Vanuatu and grown in greenhousesmaintained at 19Ð14>C[ Leaf samples of several specieswere received also from Ghana\ Sri Lanka\ Togo andReading University\ UK[

Small plants of healthy or virus!infected yam specieswere cultured in vitro as described by Forsyth and vanStaden "0871#[ The infected plants so obtained were usedas virus sources in mealybug transmission tests\ and heal!thy cultures and seedlings were used as test plants[

Mealybug transmission

Mealybugs "Planococcus citri# were allowed to colonizesmall infected plants\ kept in isolation\ and then trans!ferred to uninfected plantlets and seedlings at the rate of3 to 14 per plant[ The mealybugs were removed afterapproximately 4 days or\ more often\ left to colonize\ andthe test plants grown on and checked for symptoms atregular intervals[ Virus tests for symptomatic and latentinfection were made by immunosorbent electronmicroscopy "ISEM#[

Virus puri_cation

Yam leaves\ usually with conspicuous virus!like symp!toms were stored at Ð19>C for up to 5months prior tobeing blended "0 g:09ml# in an extractant at pH6[3 con!taining 9[14 M borate bu}er\ 9[94 M disodium ethy!lenediaminetetraacetate "Na1!EDTA#\ 9[90 M

diethyldithiocarbamate "DIECA# and 9[0) "v:v# mer!captoacetic acid[ The homogenate was stirred for 0 h at19>C and then overnight at 1>C[ After stirring for a fur!ther hour at 19>C\ Triton X!099 "4) v:v# was addedslowly with stirring[ After 29min\ the ~uid was separatedfrom the gross plant debris by squeezing the extractthrough muslin^ the virus was then separated by di}er!ential centrifugation "19min at 01 999× g^ 099min at71 999× g# and resuspended in approximately one!thirdvolume of 9[1 M borate bu}er containing 9[94 M Na1!EDTA and 9[1) "v:v# mercaptoacetic acid "pH6[9#[After stirring overnight at 1>C and 29min at 19>C\ 1)"v:v# crude Celluclast suspension "Novo Industri A:S\

Copenhagen\ Denmark# was added\ and the mixture stir!red for 29min before di}erential centrifugation "04minat 03 499× g^ 89min at 71 999× g#[ The sedimentedvirus was resuspended in 9[92 M phosphate bu}er "pH6[2#and centrifuged at 049 999× g for 1[4 h through 9Ð39)caesium chloride step gradients in 9[994 M borate con!taining 9[90 M Na1!EDTA\ 19) "w:w# sucrose and9[991) "w:v# sodium azide "pH4[7#[ The virus!con!taining zones were removed\ dialysed overnight against9[90 M phosphate bu}er "pH6[2#\ clari_ed to remove thecoagulated plant contaminants\ and sedimented for 0 hat 74 999× g[ The sedimented virus was _nally resus!pended in 9[92 M phosphate bu}er "pH6[2# "approxi!mately 0ml:14 g leaf material#[

Electron microscopy

Samples of puri_ed virus were negatively stained in 1)phosphotungstic acid adjusted to pH6[9 with KOH andexamined in a JEOL "JEOL Ltd[\ Tokyo\ Japan# 099Selectron microscope[

Serology

An antiserum to DaBV was produced by injecting a rab!bit intramuscularly on three occasions\ at 17 day inter!vals\ with virus puri_ed from D[ alata cv[ Sagbe emulsi_edwith Freund|s incomplete adjuvant[ Antiserum was col!lected 10 days after the third injection\ and stored at 1>Cafter the addition of an equal volume of glycerol[

Immune!trapping of particles from sap extracts wasessentially as described by Lockhart "0875#[ ISEM anddecoration tests were performed using antisera diluted0:499 to 0:0999 with 9[92 M phosphate bu}er "pH6[2#[Grids were ~oated carbon!side down on drops of diluteantisera for 29min\ washed dropwise with phosphate!bu}ered saline containing 9[94) "v:v# Tween 19 "PBS!T#\ ~oated on samples for 29min\ then washed\ blotteddry and stained with 1) neutral potassium phos!photungstate "KPT#[ For decoration tests\ the washedgrids were ~oated again for 0 h on dilute antisera priorto staining[ Puri_ed virus preparations were used todetermine the possible serological relationships of DaBVby ISEM[ Additional antisera for such tests were kindlyprovided by Drs B[ E[ L[ Lockhart and J[ d|A[ Hughes[

For enzyme!linked immunosorbent assay "ELISA#\ thegamma!globulin fraction of the antiserum was extractedand conjugated with alkaline phosphatase as described byHughes and Ollennu[ "0882#[ Double antibody sandwichELISA "DAS!ELISA# was performed essentially asdescribed by Clark and Adams "0866#[ Absorbance wasmeasured at 394 nm using a Titertek Multiscan MCC:239absorptiometer "Labsystems\ Helsinki\ Finland#[ Di}er!ent extractants were used in attempts to minimize thenon!speci_c coloration induced by leaf constituents "pos!sibly polyphenols# of some Dioscorea species\ and di}er!ent sap dilutions were compared in attempts to overcomedi.culties of manipulating mucilaginous extracts[

Micro!double di}usion agar gel tests "Mansi\ 0847#were also used to investigate the serological relationshipsof DaBV[

156Partial Characterization of a Badnavirus Infecting Greater Asiatic or Water Yam

)

)CMYK Page 156

Coat protein

The size of the coat protein was estimated by sodiumdodecylsulfate "SDS#!gel electrophoresis in a 09) acry!lamide gel according to Laemmli "0869#[ The proteins inthe gel were blotted onto nitrocellulose "Schleicher andSchuell^ from Amersham:Pharmacia\ Little Chalfont\Bucks\ UK# "Towbin et al[\ 0868# and were probed withDaBV antiserum[ DaBV!speci_c bands were visualizedusing antirabbit immunoglobulin!G alkaline phos!phatase conjugate\ the presence of the enzyme beingrevealed by the colour reaction with Sigma FASTNBT:BCIP tablets "Sigma Chemical Co[\ St[ Louis\ MO\USA#[

Nucleic acid

Virion nucleic acid was puri_ed by digestion of puri_edvirus particles with Proteinase K at 0mg:ml in 099mM

TrisHCl pH7[9\ 1mM CaCl1\ 1) SDS for 1 h at 54>C[Following phenol extraction\ the nucleic acid was etha!nol!precipitated\ washed in 69) ethanol and resus!pended in 49ml TE "09mM TrisHCl\ 0mM EDTA pH7[9#[RNase\ DNase and restriction endonuclease digestionswere performed according to the manufacturers| instruc!tions and DNA fragments were separated by elec!trophoresis in 0) agarose gels in TBE bu}er "89mM Tris!borate\ 0mM EDTA\ pH7[9# and detected by ethidiumbromide staining[ Cloning was by the methods describedby Sambrook et al[ "0878# and sequencing was carriedout manually using Sequenase version 1[9 "USB\ UnitedStates Biochemicals\ London\ UK#[ Sequences were ana!lysed using the GCG sequence package "Devereux et al[\0873#[

Results

Natural occurrence

Badnavirus particles that were serologically indis!tinguishable from those found in Nigerian D[ alata cv[Sagbe\ were detected alone or in complex with DaPV\yam mosaic potyvirus or an unidenti_ed _lamentousvirus in D[ alata from Barbados\ Ghana and Togo\ D[dumetorum and D[ nummularia from Papua New Guinea\D[ opposita from Japan\ D[ pentophylla from Vanuatu\D[ rotundata from Benin\ Brazil\ Ghana and Nigeria andD[ tri_da from Guyana and Barbados[

Plants of the Nigerian D[ alata cv[ Sagbe\ in whichDaBV was detected alone and in high concentrations\had distorted leaves with interveinal chlorotic patchingand\ later\ veinal necrosis "Fig[ 0#[ This isolate of DaBVwas used for subsequent studies[

Transmission

Mealybug transmission

DaBV was readily transmitted by P[ citri from infectedD[ alata to seedlings of Dioscorea abyssinica\ D[ bulbifera\Dioscorea praehensilis\ Dioscorea preussii and D[rotundata[ Infected seedlings usually showed conspicuousinterveinal chlorosis and occasionally veinal necrosisafter 4Ð01weeks and were shown to contain virus byISEM[

Fig[ 0 Symptoms of Dioscorea alata badnavirus in D[ alata cv Sagbeshowing leaf distortion and vein necrosis

Mechanical transmission

DaBV was transmitted by mechanical inoculation of par!tially puri_ed preparations from D[ alata cv Sagbe to D[alata\ D[ bulbifera\ D[ abyssinica\ Dioscorea hirti~ora\ D[preussii\ Dioscorea similacifolia\ D[ rotundata\ and Dios!corea to`oensis^ symptoms of chlorotic spotting\ mosaicand vein!banding were especially conspicuous in D[ abys!sinica and D[ preussii[ The virus was not so transmittedto D[ praehensilis or D[ dumetorum\ nor to hosts of otherbadnaviruses including Colocasia esculenta "taro#\ Musasapientum "banana# and Piper ni`rum "black pepper#[

The virus also failed to infect mechanically inoculatedseedlings of Capsicum annuum\ Chenopodium quinoa\Cucumis sativus\ Lycopersicon esculentum\ Nicotianabenthamiana\ Nicotiana clevelandii\ Nicotiana `lutinosa\Nicotiana me`alosiphon\ Nicotiana rustica\ Nicotiana tab!acum or Tetra`onia expansa[

Properties of DaBV

Virus puri_cation

Many problems were encountered in extracting DaBVfrom infected yam leaves and especially in obtainingnucleic acid which could be used for enzyme digestion[The procedure described in Materials and Methods gavevirus preparations with relatively little normal plant pro!teins but yields were low and somewhat variable[

Electron microscopy

Puri_ed DaBV preparations contained numerous intactbacilliform particles mostly measuring approxi!mately 29 nm×029 nm "Fig[ 1#[ Particles were detectedin negatively stained sap from infected yams only whenthe grids had _rst been coated with dilute homologousantiserum to trap particles[

Serology

The antiserum to DaBV reacted well with the hom!ologous virus in ISEM\ ELISA and gel immuno!di}usion

157 PHILLIPS et al[

Fig[ 1 Electron micrograph of puri_ed preparation of Dioscorea alatabadnavirus negatively stained in 1) neutralized phosphotungstate acid[The bar indicates 099 nm

tests[ In ISEM\ the diluted serum trapped and decoratedequally well particles of DaBV and those of a closelyrelated virus from D[ bulbifera designated Dioscorea bul!bifera badnavirus "DbBV#[ In reciprocal tests\ DaBV wastrapped and decorated to a lesser extent by two antiserato DbBV "DbBV!L and DbBV!EM# but almost equallyby one antiserum to sugarcane bacilliform virus "ScBV!3MX#[ Some trapping and decoration was also notedwith a second antiserum to ScBV "ScBV!SB# and twoantisera to banana streak virus "BSV!RW and BSV!MO#but not a third "BSV!MYS#\ nor with one antiserum eachof kalanchoe top!spotting virus "KTSV# or cocoa swollenshoot virus "CSSV#[

In gel immuno!di}usion tests\ reactions were notedonly when highly concentrated puri_ed virus prep!arations were used[ Clear sharp reaction lines thenoccurred with the homologous antiserum\ with one oftwo antisera to DbBV "DbBV!EM#\ and with ScBV!3MXand BSV!RW antisera[ Weaker reactions were noted withantiserum to ScBV!SB but not DbBV!L\ BSV!MO\ BSV!MYS\ CSSV or KTSV[

ELISA tests using the homologous antiserum detectedvirus in dilute puri_ed preparations and in some Dios!corea species\ although spurious coloration was notedwith some other species\ particularly D[ dumetorum[However\ whilst tests using extracted g!globulin fromDbBV!L and DbBV!EM antisera detected both DbBVand DaBV\ that from DaBV antiserum detected only thehomologous virus\ except when DbBV was present in ahigh concentration[

Viral protein

Western blotting of puri_ed DaBV "Fig[ 2# gave a majorband of 45 kDa and minor bands of 33 kDa and less[

Nucleic acid

The nucleic acid of DaBV ran as a major band and someminor bands on electrophoresis in agarose gels[ Digestionof the nucleic acid extracted from puri_ed preparations

Fig[ 2 Western blot of Dioscorea alata badnavirus "DaBV# coat pro!tein separated on a 09) polyacrylamide gel[ Track A is molecularweight markers with their weights indicated\ Track B\ DaBV coatprotein

yielded four bands of approximately 2[4\ 1[9\ 0[2 and 0[9kbp upon digestion with restriction endonuclease EcoRI"data not shown#[ This indicated that the viral genomewas double!stranded DNA of approximately 6[7 kbp[

A 0905!bp EcoRI fragment was cloned from DaBVDNA and was sequenced "EMBL accession numberX84315#[ Comparison of this sequence with those ofother badnaviruses showed that it spanned the reversetranscriptase motif of gene III and that it was distinctfrom other badnaviruses[ The DaBV fragment showedthe highest levels of nucleotide similarity to banana streakbadnavirus "56)^ Harper and Hull\ 0887# and to cacaoswollen shoot badnavirus "51[6)^ Hagen et al[\ 0882#[The nucleotide similarities to this region of other bad!naviruses were 59[5) to sugarcane bacilliform virus"Bouhida et al[\ 0882#\ 48[1) to Commelina yellow mot!tle virus "Medberry et al[\ 0889# and 32[8) to rice tungrobacilliform virus "Hay et al[\ 0880] Qu et al[\ 0880#[

Discussion

DaBV has properties characteristic of badnaviruses"Lockhart\ 0889#[ Thus\ like other species now included inthe genus "Lockhart et al[\ 0884#\ the virus has bacilliformparticles mostly measuring 029 nm×29nm which aretransmitted from infected to healthy plants by mealybugsand contain ds!DNA of approximately 6[4 kbp[ Themolecular weight of the major capsid protein "45 kDa# issomewhat larger than those of other badnaviruses\ those

158Partial Characterization of a Badnavirus Infecting Greater Asiatic or Water Yam

of rice tungro bacilliform virus "RTBV# being 26 kDa"Hay et al[\ 0883# and of cocao swollen shoot virus being32 kDa "Uhde et al[\ 0882#[ As with RTBV\ the antiserumdetected several protein bands which are considered tobe processing or degradation products from a poly!protein "Hay et al[\ 0883#[ The serological tests carriedout in the present study have shown that DaBV is relatedto\ but distinct from\ some strains of ScBV and BSV[Thus\ from these data\ from its host range and fromlimited nucleic acid sequencing it appears that DaBV isa distinct badnavirus species[

The authors| surveys indicate that DaBV occurs com!monly in several species of Dioscorea in Africa\ Asia\South and Central America and Oceania[ Until recently\the virus has not been recognized in yams in any of thesegeographical locations[ The apparent wide geographicaldistribution of the virus is thus possibly due to past inter!national exchange of infected germplasm[ Improved sero!logical and molecular diagnostic procedures can now beused for the rapid and unequivocal detection of the virus[Their use should prevent further inadvertent inter!national distribution of infected plants^ moreover\ theuse of such procedures should facilitate the testing and\perhaps\ international dissemination of improved germ!plasm and virus!tested stocks produced by axenic culture[

Acknowledgements

This research was supported by grants from the Overseas DevelopmentAdministration Crop Protection Programme and the Gatsby CharitableFoundation[ The virus was imported and held under MAFF licenceNo[ PHF0040B:0996:33

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