Phytopath. Z., 103, 361—368 (1982)© 1982 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / InterCode: PHYZA3

Biologische Bundesanstalt fiir Land- und ForstwirtschaftjInstitut fiir Viruskrankheiten der Pflanzenund Institut fiir Pflanzenschutz im Obstbau

Identification of Tombusvirus Isolatesfrom Cherry in Southern Germany

as Petunia Asteroid Mosaic Virus

By

R. KoENiG and L. KUNZE

With 4 figures

Received February 9, 1982

Tombusviruses have been found in sweet cherries showing necrotic areason fruits, leaves, and shoots in Ganada (ALLEN and DAVIDSON 1967), Gzecho-slovakia (ALBRECHTOVA, GHOD and ZIMANDL 1975), Switzerland (STOUFFER1973, pers. comm.) and the German Democratic Republic (KEGLER and KEG-LER 1980). Often the appearance of these necrotic symptoms is the first stageof a harmful disease named cherry detrimental canker in Gzechoslovakia(BLATTNY 1962) and Switzerland (SGHMID 1968). The most striking symptomsof this disease are bark splitting, stunting of shoots and sideways bending oftheir tips, and a tight clustering of distorted leaves (Figs. 1 and 2). During thelast years these symptoms were also observed in Southern Germany in sweetcherry orchards north of Niirnberg (KUNZE, KRAUSE and KOENIG 1982). Fromdiseased trees at two different locations in this area a tombusvirus wasisolated which serologically could not be distinguished from petunia asteroidmosaic virus (LOVISOLO, BODE and VOLK 1965), although it differed from thisvirus in its electrophoretic properties and its pathogenicity for Petuniahybrida.

Materials and Methods

Transmission and host rangeThe investigated material originated from diseased sweet cherries at Walkersbrunn near

Grafenberg (isolate 121/81) and Sdilaifhausen near Fordiheim (isolate 122/81), respectively.For mechanical virus transmission flower buds were ground m a mortar in three volumes of asolution containing 0.01 M DIECA, 0.02 M sodium thioglycolate and 0.4% (w/v) charcoal in

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362 KOENIG and KUNZE

Fig. 1. Leaves of sweet cherry with necrosis on the main vein

Fig. 2. Stunted shoots of sweet cherry with bark splits and sideways bended tip

Identification of Tombusvirus Isolates as Petunia Asteroid Mosaic Virus 363

a 0.03 M phosphate buffer at pH 7.0. The same buffer without charcoal was used whenherbaceous test plants served as source of inoculum in further studies. The plant sap wasgently rubbed on carborundum dusted leaves of herbaceous test plants using a brush. Cucum-bers were inoculated at the cotyledon stage, French beans after unfolding of the primaryleaves and the other plants at the four to six leaf stages. After inoculation the test plants wereobserved during a period of about four weeks.

Purification

The cherry isolate 122/81 was purified by homogenizing 100 ;5 leaves from systemicallyinfected Nicotiana clcvelandii in 100 ml 0.1 M phosphate buffer pH 7.2 containing sodiumsulfitc at 0.05 M. The liquid obtained after passage through cheesecloth was stirred for 20 minwith 1/4 volume each of diloroform and butanol. The virus was sedimented by high speedcentrifugation from the aqueous phase obtained after low centrifugation for 15 min at7000 X g.

Purified preparations of Lovisolo's petunia asteroid mosaic virus (PAMV) and the BS3strain of tomato bushy stunt virus (TBSV-BS3) and their respective antisera were from thestod^ of the Plant Virus Institute in Braunschweig.

Serology

A rabbit was immunized with the purified cherry isolate 122/81 by two intramuscularinjections spaced one week apart of virus suspensions emulsified in an equal volume of Freund'sadjuvant complete and incomplete, respectively. Bleedings were taken at two weeks intervals.Agar gel double diffusion tests were done with 0.85 9v Difco Noble agar containing 0.85 %sodium chloride, 0.25 % sodium azide, and O.OI M Tris-HCl buffer, pH 8.0. The reactantwells, 4 mm in diameter, were spaced 2 mm apart. For immunoelectrophoresis, 1 % agarose in0.025 M phosphate buffer, pH 7.0, was used. Enzyme-linked immunosorbent assay (ELISA)was done as described by CLARK and ADAMS (1977).

Results

Host range

Viruses were transmitted from the diseased sweet cherries to Nicotianaclevelandii Gray, Chenopodium quinoa Willd. and Cucumis sativus L. cv.Delikatess. From these herbaceous hosts further transmissions were made toCapsicum annuum L., Chenopodium murale L., Datura stramonium L., Pha-seolus vulgaris L. cv. Saxa and Petunia hybrida hort. cv. Himmelsroschen. OnPetunia hybrida a few necrotic lesions were produced only during the summermonths. Systemic infections did not occur in this host as ascertained by sero-logical tests. LOVISOLO'S PAMV which was studied in comparative tests pro-duced a systemic mottle with chlorotic asteroid spots on Petunia all yearround. On the other plant species the cherry isolates produced chlorotic ornecrotic local lesions. The only host which developed systemic symptoms wasN. clevelandii. In this species the young leaves showed a systemic yellow greenmottle and later turned necrotic. Finally the plants died.

Agar gel double diffusion tests

In the agar gel double diffusion test the cherry isolates reacted stronglywith antisera to LOVISOLO'S PAMV and to TBSV-BS3. A strong spur was

364 KOENIG and KUNZE

Fig. 3. Agar gel double diffusion tests with Lovisolo's petunia asteroid mosaic virus (P), thecherry isolate 122/81 (C) and tomato bushy stunt virus (T). The central wells contained anti-serum to petunia asteroid mosaic virus (Fig. 3a), the cherry isolate 122/81 (Fig. 3b) or tomato

bushy stunt virus (Fig. 3c), respectively

Identification of Tombusvirus Isolates as Petunia Asteroid Mosaic Virus 365

observed when the cherry isolates were tested side by side with TBSV-BS3.With PAMV, however, a continuous line was formed regardless of whetherantisera to PAMV, the cherry isolate 122/81 or to TBSV-BS3 were used(Fig. 3). No serological differences were found between the two cherryisolates.

Immunoelectrophoresis

In spite of their apparent serological identity LOVISOLO'S PAMV and thecherry isolate 122/81 could be differentiated in immunoelectrophoresis. AtpH 7.0 the latter migrated faster towards the anode than LOVISOLO'S PAMV(Fig. 4).

Fig. 4. Immunoelectrophoresis of Lovisolo's petunia asteroid mosaic virus (upper row)and the cherry isolate 122/81 (lower row) in 0.025 M phosphate buffer pH 7.0

Direct double-antibody sandwich ELISA

Antisera to TBSV-BS3 and LOVISOLO'S PAMV yielded a strong yellowcolor with their homologous antigens, but the cross-reactions with the cor-responding heterologous antigens were very weak or missing. The cherryIsolates were readily detected by the antiserum to PAMV, but no or extremelyweak reactions were observed with antiserum to TBSV-BS3.

Discussion

The serologically related petunia asteroid mosaic (PAMV), pelargoniumleaf curl, carnation Italian ringspot (GIRV) and artichoke mottled crinkleviruses have sometimes been regarded as strains or major variants of tomatobushy stunt virus (TBSV) (e.g. MARTELLI, QUAGQUARELLI and Russo 1971).In more recent reviews, however, they are listed as separate members of thetombusvirus group (e.g. MATTHEWS 1979, MARTELLI 1981).

The first report of a tombusvirus from a woody plant was by LOVISOLO,BODE and VOLK (1965) who isolated PAMV from roots of Ligustrum vulgareL. in Italy. In 1967, ALLEN and DAVIDSON isolated a tombusvirus from cherrytrees in Ganada that was more closely related to the type strain of TBSV than

Phytopath. Z., Bd. 103, Heft 4 24

366 KOENIG and KUNZE

to GIRV. In a detailed study of tombusvirus interrelationships HOLLINGS andSTONE (1975) found that ALLEN'S cherry isolate and LOVISOLO'S PAMV wereindistinguishable in the serological agar gel double diffusion test. The twoviruses were clearly differentiated, however, from the type strain of TBSVand other tombusviruses.

BERCKS (1967) isolated a tombusvirus from grapevines in Germany thatwas likewise indistinguishable serologically from PAMV. PAMV seems alsowidespread in grapevines in Italy (GONTI and PROTA, cited by MARTELLI

1981). Tombusvirus isolates from grapevines and hop (NOVAK and LANZOVA

1976) and from cherries (ALBREGHTOVA, GHOD and ZIMANDL 1975) in Gzecho-slovakia may also represent PAMV, but exact comparisons with this virus inthe agar gel double diffusion test were not made. The antiserum produced byALBREGHTOVA et al. strongly reacted with serologically identical viruses fromapple, pear and cherry in Fast Germany (KLEINHEMPEL et al. 1971, RIGHTER

etal. 1977).Our cherry isolates resemble the Ganadian cherry and the German grape-

vine isolates by being serologically indistinguishable from LOVISOLO'S PAMV.Different PAMV isolates differ, however, in their pathogenicity for Petuniahybrida the host after which the virus was named originally. Only LOVISOLO'S

PAMV causes a systemic asteroid mosaic in this host which is not infected bythe grapevine isolate (BERGKS 1967) and only occasionally by our cherryisolates which may cause a few necrotic lesions. The Ganadian cherry isolateswere reported to cause chlorotic or necrotic spots on this host (ALLEN andDAVIDSON 1967). Differences in the pathogenicity of serologically indistinguish-able isolates have also been found with other viruses (e.g. KOENIG, FRIBOURG

and JONES 1979).Inspite of their serological identity, PAMV isolates may also differ in

their electrophoretic mobility. HOLLINGS and STONE (1975) found that theGanadian cherry isolate migrated slightly faster towards the anode thanLOVISOLO'S PAMV. The same observation was made with the German cherryisolate 122/81 (Fig. 4).

Although PAMV seems to attack a number of woody plants, not all tom-busvirus isolates from woody plants are apparently PAMV. ALLEN (1969)described an apple isolate which serologically was very different from hischerry isolate, i.e. from PAMV, and from the type strain of TBSV. NOVAK

and LANZOVA (1977) described a lilac isolate which — in contrast to their hopand grapevine isolates that seem to resemble PAMV — reacted well with anantiserum to artichoke mottled crinkle virus, but failed to react with an anti-serum to PAMV. The isolates from cherry and plum obtained by these authorsreacted well with both antisera and it was therefore suggested that they repre-sent a mixture of different tombusviruses.

An exact typing of isolates is important in the routine diagnosis of tombus-viruses by the direct double antibody sandwich method of FLISA, because inthis test antisera to PAMV, TBSV-BS3 and other tombusviruses gave a strongyellow color only with their homologous viruses. Heterologous reactions were

Identification of Tombusvirus Isolates as Petunia Asteroid Mosaic Virus 367

very weak or not detectable (this paper and MAKKOUK, KOENIG and LESEMANN1981). This specificity may explain an observation made by FUGHS, MERKERand KEGLER (1979) who succeeded to detect unclassified tombusviruses withELISA in mazzard {Prunus avium L.), but not in infected apple, pear, plumand sweet cherry.

Summary

Sweet cherry trees in orchards located north of Nurnberg showed typicalsymptoms of detrimental canker. From these trees tombusviruses were isolatedwhich were indistinguishable in the serological agar gel double diffusion testfrom LOVISOLO'S Petunia asteroid mosaic virus (PAMV) and German grape-vine isolates. With tomato bushy stunt virus (TBSV), however, strong spurswere observed indicating non-identity. In the direct double antibody sandwichform of ELISA the cherry isolates reacted well with antiserum to PAMV, butnot with antiserum to TBSV. PAMV isolates from Petunia, cherry and grape-vine differ in their pathogenicity for Petunia hybrida and their electrophoreticproperties.

Zusammenfassung

Identifizierung von Tombusviren aus Kirschbiiumen in Siiddeutschlandals Petunia asteroid mosaic virus

Von Kirschbaumen mit Zweignekrose (detrimental canker) aus Obst-anlagen nordlich von Nurnberg wurden Tombusviren isolierr, die im sero-logischen Agargeldiffuslonstest nicht von LOVISOLOS Petunia asteroid mosaicvirus (PAMV) und von deutschen Rebenisolaten zu unterscheiden waren. MitTomatenzwergbuschvirus (TBSV) wurden dagegen kraftige Sporne gebildet,die auf Nichtidentitat mit diesem Virus schliefien lassen. In der direkten,,double-antibody-sandwich"-Form von ELISA reagierten die Kirschenisolategut mit Antiserum gegen PAMV, nicht aber mit Antiserum gegen TBSV.PAMV-Isolate von Petunien, Kirschen und Reben unterscheiden sich in ihrerPathogenitat fiir Petunia hybrida und ihren elektrophoretischen Eigenschaften.

The authors are greatly indebted to Mrs. WALTRAUD PICHL, Miss ANGELIKA SIEG andMiss PETRA RAHSE for reliable tedinical assistance, to the Deutsche Forschungsgemeinschaftfor financial support of the work in Braunschweig and to Dr. CH. KRAUSE, Amt fur Landwirt-schaft und Bodenkultur, Bayreuth, for kindly sending us infected sweet dierry scions withflower buds.

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Author's addresses: Dr. R. KOENIG. Institut fiir Viruskrankheiten der Pflanzen, Bio-logische Bundesanstalt, Messeweg 11/12, D-3300 Braunschweig. Dr. L. KUNZE, Institut fiirPflanzenschutz im Obstbau, Biologische Bundesanstalt, Sdiwabenheimer Strafte 101, D-6901Dossenheim iiber Heidelberg (Federal Republic of Germany).