Characterization of Potyvirus Isolates from West African Yams (Dioscorea spp.)

J. Phytopathology 120, 166—183 (1987)© 1987 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-1785

Institut fur Pflanzenkrankheiten der Universitdt Bonn, Nussallee 9, 5300 Bonn;Biologische Bundesanstalt fur Land- und Forstwirtschaft,

Institut fur Viruskrankheiten der Pflanzen,Messeweg 11112, 3300 Braunschweig, F. R. Germany

Characterization of Potjrvirus Isolatesfrom West African Yams {Dioscorea spp.)

A. PoRTH, D.-E. LESEMANN and H. J. VETTEN

Authors' addresses: A. PORTH, Institut fiir Pflanzenkrankheiten, Nussallee 9, D-5300 Bonn (F.R.Germany). D.-E. LESEMANN and H. J. VETTEN, Biol. Bundesanstalt fiir Land- und Forstwirtschaft,Institut fiir Viruskrankheiten der Pflanzen, Messeweg 11/12, D-3300 Braunschweig (F. R. Germany).

Witb 9 figures

Received September 3, 1986; accepted February 2, 1987

Abstract

In comparative studies on potyviruses from West African yams {Dioscorea spp.) the followingisolates were used: Dioscorea greenbanding mosaic virus (DGMV) and a Nigerian yam virus (YV-N),both isolated from Dioscorea rotundata, and a beet mosaic virus isolate from D. alata (BtMV-Y)formerly designated Dioscorea alata ring mottle virus. Naturally infected D. alata containing very fewparticles of BtMV-Y, contained primarily particles of a second potyvirus {Dioscorea alata virus, DaV)which could not be transmitted but which was included in these studies wherever possible.

The normal lengths of DGMV, YV-N, DaV, and BtMV-Y were 754, 772, 805, and 812 nm,respectively. All viruses induced cytplasmic inclusions of the pinwheel type and laminated aggregates.In addition, the nucleoli of BtMV-Y infected cells contained characteristic electron dense inclusions.The buoyant density of purified DGMV and BtMV-Y in CsCl was 1.336 g/cm' and 1.321 g/cm\respectively. The sedimention velocities (Srei) of DGMV, YV-N, and BtMV-Y were 156, 158, and162 S, respectively. In SDS-polyacrylamide gel electrophoresis the coat protein of purified DGMVand YV-N all migrated as a single band with an apparent moIeciJar weight of 36 kd. Coat protein ofpurified DaV showed up to 5 bands with molecular weights of 36 to 32 kd. Polypeptides of purifiedBtMV-Y had an estimated molecular weight of 35 kd but those from infected plant extracts had amolecular weight of 36 kd. DGMV, YV-N, and BtMV-Y particles contained a single nucleic acid withan apparent molecular weight of 3.2, 3.2, and 3.1 Md, respectively. Using X-DNA digested with HindIII as a ntarker, the molecular weight of DGMV and BtMV-Y nucleic acid was calculated to be 3.6 Md± 1 0 % . The nucleic acid was determined to be single-stranded RNA by enzymatic digestion and bystaining with acridine orange.

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Characterization of Potyvirus Isolates from West African Yams 167

In serological studies using immunoelectron microscopy (IEM), electro-blot immunoassay(EBIA), and enzyme-linked immunosorbent assay (ELISA), DGMV and YV-N were closely related.Strong serological reactions were also obtained in IEM and EBIA when DGMV and YV-N weretested with antiserum to yam mosaic virus (YMV). Antisera against DGMV, YV-N, and YMV alsoreacted strongly with DaV antigen. Serological reactions between these viruses and BtMV-Y wereusually not found or were weak. A very close serological relationship could be detected betweenBtMV-Y and beet mosaic virus isolated from beet (BtMV); both isolates were also very similar in hostrange, symptomatology, and cytopathology.

Zusammenfassung

Charakterisierung von Potyvirus-Isolatenaus westafrikanischen Yam-Arten {Dioscorea spp.)

Fiir vergleichende Untersuchungen von Potyviren aus westafrikanischen Yam-Arten wurdenfolgende Isolate verwendet: Dioscorea greenbanding mosaic virus (DGMV) und ein nigerianischesYam-Vims (YV-N), die beide aus Dioscorea rotundata isoliert wurden, sowie ein Riibenmosaikvirus-Isolat aus D. aUta (BtMV-Y), das bisher als Dioscorea alata ring mottle virus bezeichnet wurde.Naturlich infizierte D. alata enthielten sehr wenige BtMV-Y-Partikel, aber zusatzlich uberwiegendPartikel eines zweiten Potyvirus {Dioscorea alata virus, DaV), das nicht ubertragen werden konnte.Dieses Virus wurde so weit wie moglich in die Untersuchungen einbezogen.

Die Normallangen von DGMV, YV-N, DaV und BtMV-Y betrugen 754, 772, 805 und 812 nm.AUe diese Viren induzierten cytoplasmatische Einschliisse vom »pinwheer-Typ sowie ,,laminatedaggregates". Zusatzlich zeigten die Nucleoli BtMV-Y infizierter Zeller charakteristische elektronen-dichte Einschliisse. Die Schwebedichte des DGMV in CsCl konnte mit 1,336 g/cm' und die desBtMV-Y mit 1,321 g/cm' bestimmt werden. Die relative Sedimentationsgeschwindigkeit (Sr ) betrugfiir DGMV 156 S, fiir YV-N 158 S und fiir BtMV-Y 162 S. Mit HiMe der SDS-Gelelektrophoresekonnten die Hiillproteine von gereinigtem DGMV und YV-N als einzelne Banden mit einemMolekulargewicht von 36 kd ermittelt werden. Das Hiillprotein von gereinigtem DaV zeigte bis zu 5Banden mit einem Molekulargewicht von 36 bis 32 kd. Polypeptide von gereinigtem BtMV-Y hattenein Molekulargewicht von 35 kd, wogegen solche aus infizierten Pflanzenextrakten ein Molekular-gewicht von 36 kd aufwiesen. DGMV- und YV-N-Partikel enthielten eine einzelne Nukleinsaure miteinem Molekulargewicht von 3,2 Md, BtMV-Y eine mit einem Molekulargewicht von 3,1 Md. BeiVerwendung der Hind III gespaltenen X-DNA als Marker, wurde das Molekulargewicht der Nuklein-saure von DGMV und BtMV-Y mit 3,6 Md ± 10% errechnet. Als Nukleinsauretyp konnte durchenzymatischen Abbau bzw. durch Farbung mit Acridinorange einzelstrangige RNA nachgewiesenwerden.

In serologischen Untersuchungen wurden unter Anwendung immunelektronenmikfoskopi-scher Methoden (IEM), ,,electro-blot immunoassay" (EBIA) und »enzyme-linked immunosorbentassay" (ELISA) eine enge Verwandtschaft zwischen DGMV und YV-N nachgewiesen. Beide Virenzeigten starke serologische Reaktionen mit Antiserum gegen Yammosaikvirus (YMV) in ISEM,Dekoration (IEM) und EBIA. Antiseren gegen DGMV, YV-N und YMV reagierten stark mit DaV-Antigen. Die genannten Viren reagierten nicht oder nur sehr schwach mit Antiserum gegen BtMV-Y.Eine sehr enge serologische Verwandtschaft wurde zwischen BtMV-Y und einem Riibenmosaikvirus-Isolat aus Zuckerriibe (BtMV) festgestellt. Beide Viren waren sich auch in bezug auf den Wirtskreis,die Symptomatologie und die Gytopathologie ahnlich.

Yam is an economically important food crop in West Africa where morethan 90 % of the world production of yam is grown (FAO 1985). Virus diseasesare limiting factors in all yam-growing areas of the world.

Potyviruses have been isolated from Dioscorea rotundata Poir. showinggreen vein-banding, shoestring, distorted leaves, and severe stunting in Nigeria(Anonymous 1975, TERRY 1977, MOHAMED and TERRY 1979) and in the Caribbean

168 PoRTH, LBSEMAI^N and VETTEN

(MOHAMED 1976). D. cayenensis Lam. (synonymous with D. rotundata) exhibit-ing green veinbanding and mosaic symptoms in the Ivory Coast, contained apotyvirus which was described as yam mosaic virus (YMV) by THOUVENEL andFAUQUET (1979). From D. rotundata with greenbanding in Togo, a potyvirusdesignated Dioscorea greenbanding mosaic virus (DGMV) was isolated (RECK-HAUS 1980, RECKHAUS and NIENHAUS 1981). DGMV had a very restricted hostrange, caused the formation of pinwheeis and laminated aggregates, and wasfound to be serologically related to YMV from the Ivory Coast.

Symptoms of infected D. alata L. in Nigeria (Anonymous 1980) and theCaribbean (MOHAMED and MANTELL 1976) were described as severe chlorosis,veinal necrosis, and green veinbanding or also in the Caribbean as mosaic onleaves and internal brown spots in tubers (HARRISON and ROBERTS 1973). In TogoD. alata showed mottle symptoms (REGKHAUS 1980) and chlorotic ringspots,necrotic flecks, and malformation of leaves (PORTH and NIENHAUS 1983). Whereassap transmission of viruses from D. rotundata was relatively easy, transmission ofviruses from D. alata was not successful in most cases (HARRISON and ROBERTS1973, MOHAMED and MANTELL 1976, REGKHAUS 1980, Anonymous 1982). Only inNigeria and Togo potyvirus isolates have been obtained by mechanical (Anonym-ous 1980) or aphid transmission (PORTH and NIENHAUS 1983), respectively. Thepotyvirus isolated from Togolese yam was described as Dioscorea alata ringmottle virus (DaRMV) by PORTH and NIENHAUS (1983) although it was not shownto be the cause of the disease. In the present study DaRMV was found to beclosely related or identical to beet mosaic virus (BtMV) and hence it is referred tohere as the yam isolate BtMV (BtMV-Y). In addition to BtMV-Y, D. alata fromTogo also contained a second potyvirus {Dioscorea alata virus, DaV) which waspartially characterized.

This paper describes comparative studies on the cytology, physico-chemicalproperties, and serology of DGMV, BtMV-Y, DaV, and a potyvirus isolate fromNigerian D. rotundata (yam virus-Nigeria; YV-N).

Material and Methods

Virus isolates and their multiplication

Tubers of virus-infected D. rotundata and D. alata were collected in Togo and planted in Bonn.Dioscorea greenbanding mosaic virus (DGMV) (RECKHAUS 1980, RECKHAUS and NlE>fHAUS 1981)was isolated from D. rotundata and a beet mosaic virus (BtMV-Y), formerly designated Dioscoreaalata ring mottle virus (DaRMV, PoRTH and NIENHAUS 1983), was isolated from D. alata. Anotherpotyvirus (YV-N) was isolated from D. rotundata collected in Nigeria. All three viruses werepropagated in N. benthamiana Domin. An isolate of beet mosaic virus (BtMV) from sugar beet wasmaintained in N. benthamiana and sugar beet. DaV, which we failed to transmit, was maintained inD. alata and propagated by cuttings. All plants were cultivated in a greenhouse (20—25 °C, 70—80%rel. humidity, 16 h artificial light).

Electron microscopy

Particles were visualized by floating pioloform-carbon coated copper grids for 5 min on a dropof plMit %^ or of purified preparations, washing the grids with 40 drops of distilled water and stainingwith a few drops of 2% aqueous uranyl aceute (UA). Plant sap was generally homogenized in 0.1 M

Characterization of Potyvims Isolates from West African Yams 169

phosphate buffer (pH 7.0) (PB). Extracts from yam and beet were prepared using the same buffercomplemented widi 2% polyvinylpyrrolidone (PVP) and 0.5% Na2SO3.

ISEM was done as described by LESEMA fN et al. (1980). Filmed nickel grids were coated for5 min with antiserum diluted 1 : 1000 or widi IgG's (5 jug/ml), washed with 20 drops of PB, drainedwith filter paper and floated for 30 min or 1 h (short-time ISEM) or overnight (long-time ISEM) oncrude leaf extracts. After washing with 40 drops of distilled water the grids were stained with UA.ISEM with long incubation time was also made by floating nickel grids for 5 min on a drop of proteinA (diluted 1 :100). After washing and draining the grids were incubated for 5 min on a drop ofantiserum (diluted 1 : 50) or IgG's (100 |Mg/ml), washed and drained again and incubated overnight onplant extract.

For decoration tests (MILNE and LUISONI 1977) grids were first floated for 5 min on plantextracts or purified virus preparations, washed with PB and incubated for 15 min on a drop ofantiserum (1 : 50) or of IgG's (100 jUg/ml) in PB, washed with distilled water and stained with a fewdrops of UA. When particle concentration was very low, decoration was done following trapping ofparticles on antibody-coated grids. The normal length of particles was determined at an electronmicroscope magnification of 50,000 times using the Morphomat 30 image analysing system (Zeiss).For calibration a carbon grating replica was used.

Cytological observations were performed on ultrathin sections which were cut from leaf tissue,fixed and embedded in Epon as described by KOENIG and LESEMANN (1985).

For all studies a Zeiss EM 10 C electron microscope was used.

Purification

Virus was purified from leaves of N. benthamiana collected 2—3 weeks after inoculation andfrom infected cuttings of D. alata. All purifications were done following the method of LISA et al.(1981) with slight modifications. After the first high-speed centrifugation, pellets were resuspended inan appropriate volume of 0.05 M sodium citrate buffer (pH 7.5) to which cesium chloride (400 mgCsCl/ml) was added. Following centrifugation in a Beckman SW 55 Ti rotor at 35,000 rpm for15—17 h at 10°C, the virus band was collected, diluted with 0.05 M sodium citrate buffer and thevirus was sedimented at 30,000 rpm for 4 h in a Beckman 35 rotor. If virus preparations were not freeof host components, as judged by electron microscopical examinations, they were subjected to one ortwo further CsCl gradient centrifugations. When virus was purified from D. alata, filtration throughcelite pretreated with 1 % PVP and 0.1 % ovalbumin was used to remove mucilage from plant extractsafter low-speed centrifugation.

Determination of physico-chemical properties

Ultraviolet absorption spectra of virus preparations were measured in a Leitz Unicam SP 800 Bspectrophotometer. The percentage of nucleic acid in the virus particles was determined by themethods of LAYNE (1957) and PAUL (1959) after correction of the spectrophotometrical data for lightscattering (ENGLANDER and EPSTEIN 1957).

Buoyant densities of viruses were determined in preformed CsCl gradients (4 ml with anaverage concentration of 400 mg CsCl/ml) on which 1 ml of purified virus (0.1 ± 0.02 mg virus/ml)was layered, and the tubes were centrifuged (35,000 rpm, 10°C, 18 h) in a Beckman SW 55 Ti rotor.The gradients were fractionated into 0.25 ml fractions with an ISCO density-gradient fractionator andan UA-5 ultraviolet absorption monitor. The densities of selected fractions were determined bymeasuring the refractive index with a Zeiss Opton Abbe refractrometer at 25 °C and using Interna-tional Critical Tables.

Sedimentation coefficients (velocities) were determined as described by PLESE et al. (1979) bycentrifugation (35,000 rpm, 8°C, 2 h) through 5 ml of 10—40% linar sucrose gradients in a BeckmanSW 55 Ti rotor. Gradients were analyzed in an ISCO density gradient fractionator. Sedimentationvelocity (Srei) was estimated by co-centrifugation with bottom component of tobacco ringspot virus(TRSV, 126 S) (STACE-SMTTH 1970) and with tobacco mosaic virus (TMV, 194 S) (ZAITLIN and ISRAEL1975) or by parallel centrifugation with potato virus Y (PVY, 145 S) (HUTTINGA1975).

170 PORTH, LESEMANN and VETTEN

SDS-polyacrylamid gel electrophoresis (SDS-PAGE)

SDS-PAGE in 1 mm thick polyacrylamide slab gels (4% stacking gel and 12% separatmg gel)was done by the method of LAEMMLI and FAVRE (1973). SDS-degraded proteins were obtained byboiling virus preparations in LAEMMLI and FAVRE'S sample buffer for 3 min. Crude plant extraas wereused for electrophoresis after the following treatment: 1 g of fresh leaves was pulverized in liquidnitrogen. 4 ml of sample buffer were added and an aliquot of 1 ml was boiled for 5 min, clarified bylow speed centrifugation and stored frozen until use. Electrophoresis was carried out in a vertical slabgel apparatus (POOMA-PHOR, Labor-MuUer, D-3510 Hann.-Munden) at 10°C. Gels were eitherimmediately used for electro-blotting or stained and destained according to STEGEMANN et al. (1985).Low molecular weight marker proteins ranging from 14.4 to 94.0 kd (Pharmacia Fine Chemicals,Uppsala, Sweden) were co-electrophoresed.

Antisera

Antisera to DGMV, YV-N, and BtMV-Y were produced in rabbits using intramuscularinjections of 1 ml of purified virus emulsified in the same volume of Freund's complete adjuvant. Asecond injection was given one week later using incomplete adjuvant. The rabbits were bled weekly,starting 10 days after the last injection.

Antisera to YMV and BtMV were kindly provided by Dr. THOUVENEL ( O R S T O M , Abidjan,Ivory Coast) and Dr. PAUL (BBA, Braunschweig, FRG), respectively.

Electro-blot immunoassay (EBIA)

EBIA was done as described by BURGERMEISTER and KOENIG (1984) at 30 V/hxcm. The blotswere reacted with antisera or IgG's to DGMV, YV-N, YMV, BtMV-Y, BtMV, pre-immune serum,and antiserum to proteins of healthy plants. Serological reactions were visualized using a conjugate ofgoat anti-rabbit IgG's and alkaline phosphatase prepared by the method of CLARK and ADAMS (1977)and a mixture of equal volumes of naphthol AS-MX phosphate (Sigma; 0.4 mg/ml distilled water) andFast Red TR salt (Sigma; 6 mg/ml in 0.2 M Tris-HCl, pH 8.0 containing 2 mM MgCla) (BODE et al.1984) as substrate. Marker proteins were electrophoresed in the same gel as the samples. Afterblotting, the part of the nitrocellulose paper containing the markers was cut off, washed for 15 minwith phosphate buffer saline (PBS) containing 0.3% Tween 20 and stained for 3—18 h in the samebuffer containing 0.3 % Tween 20 and 1 % India ink. For destaining the sheets were washed withwater (EHLERS and PAUL 1986).

ELISA

Serological relationships, virus concentrations, and possible host plants were determined usingthe ELISA method described by CLARK and ADAMS (1977).

Characterization of nucleic acids

Two different methods were used. In the first method, virions were disrupted as described byHULL and LANE (1973) and the nucleic acid was electrophoresed for molecular weight determinationin vertical 1 % agarose slab gels under non-denaturating conditions using the buffer system ofPEACOCK and DINGMAN (1968). Nucleic acids of tobacco mosaic virus (TMV, 2.1 Md) (HUTH et al.1984), turnip yellow mosaic virus (TYMV, 2.2 Md) (per. comm. Dr. HuTH, BBA, Braunschweig),potato virus Y (PVY strain GO 16; 3.1 Md) (MAKKOUK and GuMPF 1974) and brome mosaic virus(BMV, 1.09, 0.99, 0.75, and 0.28 Md) (LANE and KAESBERG 1971) were used as markers. In thesecond method, nucleic acid was isolated using two cycles of phenol extraction, denaturated withglyoxal, electrophoresed in vertical 0.8% agarose slab gels (MCMASTER and CARMICHAEL 1977,MANIATIS et td. 1982) and stained with acridine orange (30 jug/ml in 0.01 M phosphate buffer, pH 7.0).The gel was destained by several changes of 0.01 M phosphate buffer. The following markers wereused: Hind III digested X-DNA (15.27, 6.21, 4.33, 2.88, 1.53, 1.34, and 0.37 Md) and ssRNA ofpltim pox virus (PPV, 3.6 Md) (pers. comm. Dr. MAISS, BBA, Braunschweig) and potato vims Y.

Gharacterization of Potyvirus Isolates from West African Yams 171

For determining the type of nucleic acid in DGMV and BtMV-Y particles, aliquots of nucleicacid extracted with phenol were incubated with 10 or 50 ng pancreatic RNase/ml for 10 or 30 min,respectively. These samples and untreated nucleic acid of DGMV and BtMV-Y were co-electrophor-esed in 1 % agarose slab gels using the buffer system of PEACOCK and DiNGMAN (1968).

Results

Particle morphology

Filamentous particles were present in crude sap of D. aUta, D. rotundata,and various inoculated host plants (RECKHAUS 1980, RECKHAUS and NIENHAUS1981, PoRTH and NIENHAUS 1983) and in purified virus preparations. The normallength of particles of DGMV, YV-N, DaV, and BtMV-Y was 754, 772, 805, and812 nm, respectively (Fig. 1) in extracts of virus-infected plants. ISEM did notinfluence the length values. The length of purified particles decreased and wasmore variable because of particle fragility. BtMV-Y particles seemed to be morefragile than the others. The particles had a diameter of 13—14 nm after stainingwith uranyl acetate (UA).

Cytological observations

In all tissues infected with DGMV, YV-N, and BtMV-Y cylindrical inclu-sions consisting of pinwheels and laminated aggregates, but not of scrolls wereobserved (Figs. 2 and 4). In addition to these inclusions the cells infected withDGMV and YV-N consistently contained bundles of filamentous structureswhich apparently did not consist of virions and which were up to severalmicrometers long and often closely associated with elements of the endoplasmic

4 0 -

30-

20-

10

Fig. 1. Length distribution ofparticles of

a) DGMV in extracts of infectedN. benthamiana,

h) YV-N in extracts of infectedN. benthamiana after trappingwith homologous antiserum,

c) DaV in extracts of infectedD, alata after trapping withantiserum to DGMV.

Measurement of all samples weredone after negative staining with2% uranyl acetate

•2 30

01

i 10

s 40

30

20

10

i

to PI••

1 f 1111 rP"'! n •

c

r-n n n n fTi 1

DGMV

1 n 1 t 1 n

I YV-N

1 1 1 1 11 r-1

OaV

rlTw-rfWi700 BOO 900 {nml

172 PoRTH, LESEMANN and VETTEN

Figs. 2 and 3. Ultrathin sections from N. benthamiana infected with DGMV.2. Cross sections of pinwheel inclusions which are modified into laminated aggregates.3. Bundles of filamentous structures associated with elements of the endoplasmic reticulum in

cytoplasm of an infected cell.

characterization of Potyvirus Isolates from West African Yams 173

reticulum (Fig. 3). Cells infected with BtMV-Y often contained cytoplasmiccomplexes of structures resembling membranes which appeared modified byapposition of dark staining material (Fig. 5). Also, accumulation of vesicles with

"• ' i

Figs. 4 and 5. Ultrathin sections of N. benthamiana infected with BtMV-Y.4. Cross sections of pinwheel inclusions most of which are modified into laminated aggregates.5. Electron-dense bands of altered membraneous material.

174 PORTH, L E S E M A N N and V E T T E N

Figs. 6 and 7. Ultrathin sections of M benthamiana infected by BtMY-Y.6. Cytopiasmic clusters of vesicles characteristic of BtMV-Y infected cells.7. Electron-dense inclusion in the nucleolus of an infected cell. Note electron-transparent areas in the

inclusion containing small aggregates of filamentous material (inset).


fibrillar content was found (Fig. 6). BtMV-Y infected cells sometimes showedaltered nucleoli which contained rounded, strongly osmophilic inclusions with adiameter of up to 3—4 /im. These inclusions contained electron transparent areassometimes filled with filamentous material (Fig. 7).

Purification and particle properties

The average yield of purified DGMV, YV-N, and BtMV-Y was 1.5, 0.7, and1.0 mg/100 g of fresh leaves of N. benthamiana, respectively. Virus purificationfrom D. alata was difficult and yielded only a partially purified preparationcontaining about 0.4 mg DaV/100 g of tissue. This preparation contained noparticles which could be decorated with antiserum to BtMV-Y. Purified viruspreparations had ultraviolet absorption spectra with a maximum at 260—265 nm,a minimum at 242—247 nm, and a shoulder at 293 nm. After correction for lightscattering the ratios of A280/A260 for DGMV, YV-N, and BtMV-Y were 0.850,0.860, and 0.866, indicating a nucleic acid content of c. 5.4, 5.3, and 5.1%,respectively.

Buoyant densities of DGMV and BtMV-Y in GsCl were 1.336 and 1.321 g/cm' , respectively.

The sedimentation velocities (Srei) of DGMV, YV-N, and BtMV-Y were156, 158, and 162 ± 1 S, respectively, when compared with TRSV (bottomcomponent, 126 S), PVY (145 S), and TMV (194 S).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

In SDS-PAGE the coat protein of purified DGMV and YV-N had anapparent molecular weight of 36 ± 1 kd and that of purified BtMV-Y had amolecular weight of 35 ± 1 kd. With YV-N a minor band with a molecularweight of 31 ± 1 kd was sometimes observed. SDS-PAGE of purified DaVpreparations yielded up to five bands with molecular weights of 36, 35, 34, 33,and 32 ± 1 kd. The molecular weight of the major bands ranged from 34 to32 ± 1 kd (compare Figs. 8 and 9).

Leaf extracts from DGMV- and YV-N-infected plants showed only onevirus-specific band which migrated to the same position as the major band of thecoat protein of purified virus. Extracts from BtMV-Y-infected leaves, however,contained a virus-specific band with a slightly higher molecular weight than thatof capsid protein of purified BtMV-Y, indicating that its coat protein is degradedduring purification. The molecular weight of undegraded BtMV-Y was36 ± 1 kd and thus similar to that of DGMV and YV-N.

Serology

Antisera to DGMV, YV-N, and BtMV-Y had homologous titers in slideprecipitin tests of 1 : 8,192, 1 : 256, and 1 : 1,024, respectively.

In EBIA the major capsid proteins of DGMV, YV-N, and DaV reactedequally strongly with antisera to DGMV, YV-N, and YMV, but only weaklywith antiserum to BtMV-Y and not at all with antiserum to BtMV (Figs. 8 and 9).BtMV-Y antigen only reacted with BtMV and homologous antisera and not with


DQMV Ab

1 2 3 4

43 K -

30 K

YV-N Ab

1 2 3 4YMV Ab

1 2 3 4

Pi Ab

2 3 4

Ab3 4

BtMV Ab1 2 3 4

I i

43 K

30K-

Figs. 8 and 9. Electro-blot immunoassays of antigens of YV-N, DGMV, DaV, and BtMV-Y (lanes 1to 4) using antibodies to DGMV, YV-N, YMV, and pre-immune (PI) serum (Fig. 8), and to BtMV-Y

and BtMV (Fig. 9).

the other antisera used (Figs. 8 and 9). Beside the major capsid proteins, someminor bands of lower molecular weight were revealed by EBIA but not by SDS-PAGE.

Following co-electrophoresis and blotting of coat proteins of purifiedDGMV, YV-Y, and BtMV-Y and of leaf extracts from plants infected with theseviruses, leaf extracts of DGMV- and YV-N-infected plants yielded one bandwhich was identical to that of coat protein of the purified viruses but also revealedprotein degradation in purified BtMV-Y preparations, as shown before in SDS-PAGE.


Table 1Serological relations of potyviruses from Dioscorea rotundata. D. alata, and of beet mosaic virus

assessed by immunosorbent electron microscopy (ISEM)'

Antigens

1. DGMV (N. bentbamianaf2. DGMV (D. rotundata)3. YV-N (7V. bentbamiana)4. DaV and BtMV-Y (D. alata)5. BtMV-Y {N. benthamiana)6. BtMV {Beta vulgaris)

DGMV

9,000^*1,6852,785

126428495

YV-N

10,0001,5904,460

44199203

Antisera

YMVigG

10,0001,8552,775

4115122

BtMV-Y

1009

1057

3,1251,525

BtMV

331207612

1,5101,030

PI^

3252556

1

430

' Grids were coated with 10 ^ug/ml protein A, incubated with antisera diluted 1 : 50 and incubatedwith virus samples for 16 h. Antiserum to YMV was used as IgG fraction at 100 /xg/ml.

• PI = pre-immune serum.' Plant species used as virus source.•* Figures represent means of counts per 500 ixvr^ on duplicate grids.

In ISEM experiments high trapping rates were obtained with DGMV andYV-N and their antisera in homologous and heterologous combinations(Table 1). Antibodies to YMV trapped particles of DGMV and YV-N to levelssimilar to those of the respective homologous antisera (Table 1). DGMV and YV-N did not react with BtMV-Y or BtMV antisera. From virus-infected plants ofD. alata moderate numbers of particles were trapped with antisera to DGMV,YV-N, and YMV (Table 1). These particles could be decorated with DGMVantiserum (Table 2). From the same plant of D. alata, a few particles weretrapped which could be decorated with BtMV-Y antiserum. This result indicatedinfection of D. alata by two distinct potyviruses. BtMV-Y particles in extractsfrom N. benthamiana could be efficiently trapped by homologous and BtMVantisera. From these extracts low but significant trapping of BtMV-Y particleswas observed with DGMV, YV-N, and YMV antisera.

DGMV and YV-N particles but not those of BtMV-Y were decorated withDGMV, YV-N, and YMV antisera. BtMV-Y and BtMV particles but not those ofDGMV and YV-N showed antibody coating when treated with homologous andBtMV antisera (Table 2). When comparing the decoration titers of antisera toDGMV and YV-N with homologous and heterologous antigens, differences ofone dilution step were obtained. Decoration titers of DGMV and YV-N withYMV antiserum also showed a difference of one dilution step. BtMV-Y andBtMV also differed by maximally one dilution step when tested in homologousand heterologous combinations (Table 2).

In direct ELISA using different dilutions of conjugate and plant sap thehomologous reactions of DGMV and YV-N yielded shghtly higher extinctionvalues (E405) than the heterologous reactions. Almost identical results wereobtained when different concentrations of purified DGMV and YV-N weretested with homologous and heterologous antisera in direct ELISA (Table 3).

J. Phytopathology, Bd. 120, Heft 2 12


Table 2Relationship among potyviruses studied by antibody decoration tests

Antigens AntiseraDGMV YV-N YMV BtMV-Y BtMV

1. DGMV {N. benthamianaf 4-4- 4-to 4-4- 4-to 4-4-decoration titre > 1 :12,800 1 :6,400 1 :400

2. DGMV {D. rotundata) 4-4- + to 4-4- 4-y. YV-N {N. benthamiana) 4-4- 4-4- 4-

decoration titre > 1 : 12,800 > 1 : 12,800 1 : 2004. DaV and BtMV-Y {D. alata)

DaV 4- to 4--1- 4- (4-) to 4-BtMV-Y - n.t.^ n.t. 4-4- n.t.

5. BtMV-Y {N. benthamiana) - - - -1-4- 4-to-l--l-decoration titre 1 :6,400 1 : 1,600

6. BtMV {Beta vulgaris) - - - 4 - to - l -4 - - l - to4 -4 -decoration titre 1 : 6,400" 1 : 3,200"

' Plant species used as virus source.^ Symbols indicate intensity of decoration at antiserum dilution of 1 : 50.

4-4- = strong4- = medium

(4-) = weak— = no decoration

' n.t. = not tested" Determined with BtMV in extract of infected N. benthamiana.

This result suggested that the isolates are serologically very closely related, butdistinct. BtMV-Y and the BtMV isolate from beet reacted also better with theirhomologous antisera than in respective heterologous combinations indicating thatthey are two slightly distinct isolates of BtMV.

Characterization of nucleic acid

When using the method of HULL and LANE (1973) and RNA of potato virusY (PVY) as marker for which a molecular weight of 3.1 Md was assumed(MAKKOUK and GUMPF 1974), particles of DGMV, YV-N, and BtMV-Y containeda single nucleic acid species with a molecular weight of 3.2, 3.2, and3.1 ± 0.2 Md, respectively. When phenol extracted nucleic acids of PVY,DGMV, and BtMV-Y were co-electrophoresed under denaturating conditionsusing Hind Ill-digested X-DNA as a marker, a single nucleic acid species wasobserved with an apparent molecular weight of 3.5, 3.6, and 3.6 Md (± 10%),respectively.

Phenolized nucleic acids of DGMV and BtMV-Y were stained orange withacridine orange and digested by pancreatic RNAse, indicating that they aresingle-stranded RNA.

Because of the low YV-N and DaV yields their nucleic acids could not becharacterized.


Table 3Reaction of different concentrations of purified DGMV and YV-N with homologous

and heterologous antisera in direct ELISA

Antigens Concentration Antisera toDGMV YV-N

YV-N^

20010050

25012562.5

ng

ng

ngngng

0.991 ± .039.641 ± .033.389 ± .013

0.743 ± .012.510 ± .013.289 ± .005

0.609 ± .007.437 ± .012.278 ± .003

0.790 ± .005.570 ± .015.316 ± .008

IgG's to DGMV and YV-N were both used at a coating concentration of 1 jug/ml. The conjugate toDGMV and YV-N were diluted 1 : 4,000 and 1 : 1,000, respectively.Average A405 values of three replicate wells. Substrate reaction time was 30 minutes.

Discussion

Particle morphology and cytological alterations induced by DGMV, YV-N,DaV, and BtMV-Y are typical for potyviruses (HOLLINGS and BRUNT 1981).DGMV, YV-N, and BtMV-Y induce the formation of cylindrical inclusions ofthe pinwheel type and laminated aggregates, thus all viruses studied here are in thesubdivision II of the potyvirus group as described by EDWARDSON (1974). In thisrespect our viruses are similar to potyviruses of Dioscorea rotundata andD. trifida in Nigeria (MOHAMED and TERRY 1979) and Guadeloupe (MIGLIORI andCADILHAC 1976), respectively. A potyvirus of D. alata from Togo induce pin-wheels and scrolls (RECKHAUS 1980), Dioscorea greenbanding virus, a potyvirus ofD. floribunda from Puerto Rico, induce pinwheels, scrolls, and laminatedaggregates (HEARON et al. 1978) and are thus clearly differentiated from virusesdiscussed here.

The shape of the inclusions and other cytological alterations are very similarin tissues infected with DGMV and YV-N. Minor differences in pinwheelstructure do not also allow a clear distinction of DGMV and YV-N from BtMV-Y. However, the nucleolar inclusions of BtMV-Y, which appear to be identical tothose described for BtMV (Bos 1969, MARTELLI and Russo 1969), clearly differ-entiate BtMV-Y and BtMV from DGMV and YV-N. In addition, complexes ofdark staining structures obviously derived from cytoplasmic membranes wereobserved in the cytoplasm of BtMV-Y-infected cells but not in those infectedwith DGMV or YV-N. These structures may be similar to those described forsweet potato russet crack virus (LAWSON et al. 1971). On the other hand, bundlesof filamentous structures were found in DGMV and YV-N infected cells but notin BtMV-Y infected cells.

The spectrophotometric properties of DGMV, YV-N, and BtMV-Y, thebuoyant densities of DGMV and BtMV-Y, the relative S values of DGMV and

12*


YV-N as well as the number and size of the coat proteins and the number, size,and type of nucleic acids agree with those reported for other potyviruses(MoGHAL and FRANCKI 1976, HUTTINGA 1975, DAMIRDAGH and SHEPHERD 1970,PuRCiFULL 1966, HoLLiNGS and BRUNT 1981). Since no differences in the polypep-tide size of virus from purified preparations and from quickly prepared leafextracts were found, the coat proteins of DGMV and YV-N seem to be fairlystable. The molecular weight of undegraded BtMV-Y protein is very similar tothat of DGMV and YV-N but it is slightly degraded in purified preparations.Although only one major capsid protein was usually visible in SDS-PAGE, minorbands assumed to be degradation products of the major band were revealed inEBIA.

DGMV and YV-N both isolated from D. rotundata are very similar in hostrange, serology, and other properties determined. Therefore, YV-N should beregarded as an isolate of DGMV, RECKHAUS and NIENHAUS (1981) have recentlydescribed a serological reaction of DGMV antigen with YMV antiserum usingagar gel double diffusion tests. We could provide further evidence that antiserumto YMV gives strong reactions with DGMV isolates in some serological tests.Although the degree of relationships between YMV and DGMV could not beassessed accurately as YMV antigen was not available, YMV is apparently clearlydistinct from the DGMV isolates. Whereas the reaction of DGMV, YV-N, andDaV polypeptides was equally strong in EBIA when DGMV, YV-N, and YMVantisera were used, an effective trapping of DGMV and YV-N particles withYMV antiserum was only obtained after long-time ISEM. However in short-timeISEM (data not shown) YMV antibodies trapped much lower numbers of theseviruses than DGMV and YV-N antisera, and in decoration tests a slightly weakerantibody coating was observed on DGMV and YV-N particles with YMVantiserum than with the homologous antisera. This suggests that the serologicalrelationship between the DGMV isolates and YMV is either not as close asbetween DGMV and YV-N or that the available antibodies to YMV were not asgood as those of the DGMV and YV-N antisera.

Since particles of the BtMV isolates were weakly trapped by antisera toDGMV, YV-N, and YMV in ISEM tests but not in reciprocal tests, a very distantserological relationship seems to exist between the BtMV isolates on the one handand DGMV, YV-N, and YMV on the other hand. However, a weak reaction ofBtMV-Y antiserum was obtained with DGMV, YV-N, and DaV coat-protein inEBIA, but, again, not in reciprocal combinations. These weak one-way reactionswere not detected in short-time ISEM (data not shown), decoration tests, and inELISA.

In addition to BtMV-Y which occurred only at a very low concentration inD. alata, naturally infected D. alata predominantly contained particles of DaVwhich could be trapped and decorated with antisera to DGMV, YV-N, andYMV. We could only transmit BtMV-Y but not DaV from D. alata to test plants.Sap transmission of potyviruses from D. alata has been notoriously difficult(HARRI«>N and ROBERTS 1973, MOHAMED and MARTELL 1976, RjECKHAm 1980,Anooymoiis 1982). TTiis does not seem to be caused by inhibitory constituents in

Charaaerization of Potyvirus Isolates from West African Yams 181

D. alata extracts as suggested in previous studies (RECKHAUS 1980, PORTH andNiENHAUS 1983), because it has been reported (Anonymous 1982) that a potyvirusisolated from D. rotundata in Nigeria and identified as an isolate of YMV wasmechanically transmitted to D. alata and also transmitted back to N. hen-thamiana. Our data suggest that DaV is serologically related to DGMV and YMVto an unknown extent and is clearly distinct from both potyviruses in terms ofhost range and transmissibility. We detected BtMV-Y in D. alata and identified itas an isolate of BtMV on the basis of host range, serology, and cytopathology.We do not know whether DaV or BtMV-Y is responsible for symptom expres-sion in D. alata, because our attempts to transmit DaV and BtMV-Y to healthyD. alata plants were unsuccessful. It is also not known whether BtMV-Y is anindigenous West African yam virus or it infected D. alata by accidental transmis-sion at the onset of our experiments.

Since several other potyvirus isolates from various Dioscorea spp. (for reviewsee PoRTH and NIENHAUS 1983) have been insufficiently described, it is difficult todraw any conclusions concerning their identity.

We are grateful to SABINE BONSE, ANGELIKA SIEG-MOLLER, and SABINE SCHUHMANN forexcellent technical assistance, to Dr. J. C. THOUVENEL (Ivory Coast) and Prof. H. L. PAUL(Braunschweig) for gifts of antisera, and to Drs. W. HuTH and E. MAISS for assistance in nucleic acidcharacterization. Financial support was given to A. P. by the Deutsche Forsehungsgemeinschaft andthe Graduiertenforderung of the University of Bonn.

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Characterization of Potyvirus Isolates from West African Yams (Dioscorea spp.)