On the basis of the investigations presented it has been established that RMV—K isolate from Roripa amphibia belongs to tobamoviruses. This virus is symptomatologically and serologically more closely related to the type strain of ribgrass mosaic virus (RMV) than to the common tobacco mosaic virus. Consequently, the virus which was isolated and investigated is a strain of RMV.Prošle godine izolirali smo virus iz divlje krucifere Roripa amphi- bia (L.) Bcss. u blizini Zagreba i dali mu oznaku RMV—K. Inficirane biljke imale su simptome u obliku blage išaranosti i nekrotičnih linija na listovima (si. 1A).
Elektronsko-mikroskopska analiza purificirane virusne suspenzije pokazala je da RMV—K ima čestice približno 300 nm duge (si. 4). Apsorpcija virusne suspenzije dosizala je maksimum u ultravioletnom dijelu spektra kod 260 nm (si. 5).
Izolirani virus prenijeli smo na 12 zeljastih biljaka (tablica 1). Virus je prouzrokovao lokalne lezije na listovima vrsta Nicotiana silves- tris, N. glutinosa, D. stramonium i vrlo jake sistemične simptome na vrsti N. tabacum, tip Samsun (si. 1). Na osnovi simptoma na tim vrstama bilo je očito da je RMV—K vrlo sličan običnom virusu mozaika trpuca.
Serološki pokusi vršeni su metodom dvostruke difuzije u agaru te unakrsnim zasićavanjem u agaru. Ti su pokusi pokazali da je istraživani virus srodniji virusu mozaika trpuca nego virusu mozaika duhana (si. 3)

Davor Miličić

Nikola Juretić

Đorđe Mamula

Prošle godine izolirali smo virus iz divlje krucifere Roripa amphi- bia (L.) Bcss. u blizini Zagreba i dali mu oznaku RMV—K. Inficirane biljke imale su simptome u obliku blage išaranosti i nekrotičnih linija na listovima (si. 1A).

Elektronsko-mikroskopska analiza purificirane virusne suspenzije pokazala je da RMV—K ima čestice približno 300 nm duge (si. 4). Apsorpcija virusne suspenzije dosizala je maksimum u ultravioletnom dijelu spektra kod 260 nm (si. 5).

Izolirani virus prenijeli smo na 12 zeljastih biljaka (tablica 1). Virus je prouzrokovao lokalne lezije na listovima vrsta Nicotiana silves- tris, N. glutinosa, D. stramonium i vrlo jake sistemične simptome na vrsti N. tabacum, tip Samsun (si. 1). Na osnovi simptoma na tim vrstama bilo je očito da je RMV—K vrlo sličan običnom virusu mozaika trpuca.

Serološki pokusi vršeni su metodom dvostruke difuzije u agaru te unakrsnim zasićavanjem u agaru. Ti su pokusi pokazali da je istraživani virus srodniji virusu mozaika trpuca nego virusu mozaika duhana (si. 3).On the basis of the investigations presented it has been established that RMV—K isolate from Roripa amphibia belongs to tobamoviruses. This virus is symptomatologically and serologically more closely related to the type strain of ribgrass mosaic virus (RMV) than to the common tobacco mosaic virus. Consequently, the virus which was isolated and investigated is a strain of RMV

Juretić, Nikola

Miličić, Davor

Mamula, Đorđe

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Acta Bot. Croat. 32 (1973) 29—35I D E N T I F I C A T I O N  O F  A  T O B A M O V I R U S  I S O L A T E D  F R O M  R O R I P A  A M P H I B I ANIKOLA JVRETIC, DAVOR MILlClC  and DORDE MAMULA  (Botanical Institute oi the University of Zagreb)Received January 21, 1973I n t r o d u c t i o nAlthough viruses belonging to the tobacco mosaic group (tobamo- viruses; H a r r i s o n  et al. 1971) are widely spread in nature, only few of them have been found in cruciferous plants. A tobamovirus was isolated from Cardaría draba and Sisymbrium loeselii in Czecho­slovakia by P o l á k  (1964). Almost simultaneously, G o t o  and Os- h i m a (1962) found another virus in the wild crucifer Radícula sil- vestris in Japan, and P e i  (1962) in Brassica campestris in China.Last year we isolated a tobamovirus from the wild crucifer Roripa amphibia (L.) Bess, in the vicinity of Zagreb. The infected plants showed mild mottling symptoms and necrotic lines on the leaves (Fig. 1A). From these plants a virus was isolated and transmitted to a relatively large number of plants. On the basis of symptomatological, electron microscopic and serological investigations it has been esta­blished that the isolate belongs to tobamoviruses, particularly to rib- grass mosaic virus (RMV).M a t e r i a l  a n d  M e t h o d sThe virus isolated from Roripa amphibia was marked RMV—K. Several other viruses were used for comparison with RMV—K. These viruses were: common tobacco mosaic virus (TMV), type strain of RMV that was sent us by courtesy of Professor C. W e t t e r  (Sa­arbrücken) and Yugoslav strain of RMV (RMV—Y) obtained from rib- grass ( J u r e t i c  et al. 1969). For serological investigation we em­ployed the immune sera against TMV and RMV—K which were pre­pared after J u r e t i 6 et al. (1969).29The virus investigated was passed through single-lesion culture. For this purpose leaves of Nicotiana glutinosa were used. After this treatment the virus was transmitted to common herbaceous plants some of which served as test plants for differentiation of tobamoviruses.RMV—K was purified according to the method of G o o d i n g  and H e b e r t  (1967). Serological experiments were performed by means of agar-gel double diffusion test (D u d m a n 1965, v a n  R e g e n -  mo  r t e 1 1966, W e t t e r  and L u i s o n i 1969) and cross absorption test ( van R e g e n m o r t e l  1967).R e s u l t sInvestigation on herbaceous plantsThe isolate RMV—K was mechanically transmitted to 12 herbaceous test plants. Symptoms caused on these plants by RMV—K are presented in Tab. 1 (comp. Fig. IB, C, D). The reaction of some dif­ferential test species, following the inoculation with RMV—K, TMV, type strain of RMV and RMV—Y, is shown for comparison in Tab. 2. As it is visible from Tab. 2, RMV—K produced on Datura stramonium  and N. glutinosa only local lesions (Fig. IB), similar to those caused by TMV and other tobamoviruses. On the basis of these symptoms we could anticipate that RMV—K belonged to tobamoviruses.Unlike common TMV, RMV—K caused only local lesions on N. silvestris and systemic symptoms on Plantago media, i. e. the same symptoms as the common RMV and RMV—Y (comp. H o l m e s  1941, J u r e t i c  et al. 1969). Moreover, the necrotic symptoms in form of rings and spots produced on tobacco by RMV—K were similar to those provoked under the influence of RMV and RMV—Y on this plant ( H o l m e s  1941, K o v a c h e v s k . y  1963, J u r e t i c  et al. 1969).Consequently, the reactions of test plants have pointed out that RMV—K is a tobamovirus which is more closely related to RMV than to TMV.Purification, ultraviolet absorption and electron microscopyRMV—K was purified after the method of G o o d i n g  and H e- b e r t (1967). The purified virus suspension had and absorption ma­ximum in ultraviolet at 260 nm (Fig. 5). The shape of the curve indi­cated that the virus was well purified.The analysis of infected sap and purified virus suspension in the electron microscope showed that the cause of disease was a rod-shaped virus about 300 nm long (Fig. 2, 4). This length is a characteristic of tobamoviruses.Serological investigationsThe purpose of these investigations was to establish whether RMV—K was more closely related to the common TMV or to the RMV. With this aim in view some experiments were made by agar-gel double diffusion and by intragel cross absorption method.30T a b l e  1. Reactions of herbaceous plants infected with the isolate RM V-K  of ribgrass mosaic virusExplanation of signs: I symptoms in inoculated leaves; II symptoms in non- -inoculated top leaves; O local infection; [ |. systemic infection; A unsusceptible.AmaranthaceaeGomphrena globosa L. O  local necrotic lesions surrounded with a red ring.ChenopodiaceaeChenopodium amaranticolor Coste & Reyn. O  necrotic lesions.Ch. murale L. O  necrotic lesions.Ch. quinoa Willd. O  necrotic lesions.CruciferaeBrassica rap a L. var. rapa □  I necrotic lesions; II vein clearing, mottling.CucurbitaceaeCucumis sativus L. cv. Delicatesse A- LeguminosaePhaseolus vulgaris L. cv. Pinto A.PlantaginaceaePlantago media L. □  II mottling.SolanaceaeDatura stramonium L. O  necrotic lesions.Lycopersicum esculentum L. A-Nicotiana glutinosa L. O  necrotic lesions (Fig. IB).N. megalosiphon Heurck & Muell. Arg. O  necrotic lesions.N. tabacum L. cv. Samsun □  I sporadic necrotic lesions; II necro­tic rings, line pattern and oak leaf pattern, mottling, mosaic (Fig. 1C, D).N. silvestris Speg. and Comes O  necrotic lesions Petunia hybrida hort. ex Vilm. O  necrotic lesions.T a b l e  2. Symptoms in some test plants infected by RM V-K  isolate and three other tobamovirusesTest plants TMV RMV RM V-Y RM V-KPhaseolus vulgaris L O O OPlantago media L s S sDatura stramonium L Li L LNicotiana glutinosa L L L LN. silvestris S L L LN. tabacum cv. Samsun S S(necroticrings)S(necroticspots)S(necrotic rings and spots)L local symptoms, S systemic symptoms, O not infected31First, experiments in agar-gel were performed to examine the sero­logical relationship between RMV—K and common TMV. As Fig. 3A shows, the precipitin bands of TMV and RMV—K made a spur indi­cating that RMV—K differs from TMV, i. e. that these two viruses are not closely related.Afterwards, we carried out an experiment which was to answer the question of whether RMV—K is more closely related to the com­mon TMV or to the RMV. As can be seen from Fig. 3B the immune serum against TMV was absorbed by the type strain of RMV. The absorbed serum could react neither with RMV nor with RMV— K but it reacted with the homologous virus. On the basis of this experi­ment we could conclude that RMV—K is more closely related to the type strain of RMV than to the common TMV, i. e. that RMV—K sha­res more common determinants with the type strain of RMV than with the common TMV.D i s c u s s i o nSo far, RMV has been isolated from a large number of wild and cultivated plants belonging to various families ( H o l m e s  1941; G o l ­d i n  1953; H a r r i s o n  1956; S c h u m a n n  1963a, b; S i l v a  and P o p  1965; K o v a c h e v s k y  1963; J u r e  t i c  et al. 1969). It is in­teresting that the tobamoviruses isolated so far from crucifers were more closely related to RMV than to common TMV (P o 1 a k 1964, G o t o  and O s h i m  a 1962. P e i  1962). It seems that the crucifers are suitable natural hosts to RMV. It should be noted that the agent isolated by P o 1 a k (1964) from crucifers was a mixture of two toba­moviruses; only one component was a strain of RMV, the second com­ponent being another virus (J u r e t i c and W e t t e r  1971).The investigations of RMV have shown that this virus is widespread in nature. It would be interesting to know whether this virus has in nature a larger number of hosts than the common TMV (comp. K o- v a c h e v s k y  1963).The detailed serological investigations of RMV—K will be reported elsewhere.S u m m a r yOn the basis of the investigations presented it has been established that RMV—K isolate from Roripa amphibia belongs to tobamoviruses. This virus is symptomatologically and serologically more closely rela­ted to the type strain of ribgrass mosaic virus (RMV) than to the common tobacco mosaic virus. Consequently, the virus which was iso­lated and investigated is a strain of RMV.*W e wish to thank Dr N. L j u b e s i c  (Ruder Boskovic Institute, and Institute of Biology, University of Zagreb, Zagreb) for the electron micro­scopic examination and the micrograph presented.32Fig. 1. — SL 1.Fig. 2. — SI. 2.Fig. 3. —  SI. 3.Length of ParticlesFig. 4. —  si. 4.Wavelength (nm)Fig. 5. —  si. 5.<Fig. 1. Symptoms caused by the isolate RM V-K  of ribgrass mosaic virus. A  —  Leaf of Roripa amphibia with vein banding and necrotic spots. B —  Leaf of Nicotiana glutinosa with necrotic local lesions. Leaves of N. tabacum ’Samsun’ with systemic symptoms in the form of ne­crotic rings, lines (C) and of necrotic oak leaf pattern (D).SI. 1. Simptomi oboljenja koje uzrokuje izolat RM V-K virusa mozaika trpuca. A  —  list vrste Roripa amphibia s vrpcama uz nerve i nekrotičnim pjegama. B —  list vrste Nicotiana glutinosa s nekrotičnim lokalnim lezijama. Listovi vrste N. tabacum, tip Samsun, sa sistemičnim simp­tomima u obliku nekrotičnih prstenova i linija (C) i simptoma poput hrastova lista (D).Fig. 2. Particles oí ribgrass mosaic virus (RMV-K) in a purified prepara­tion treated with phosphotungstic acid. Magn. 50 000 X. Photogr. in Ruder Bošković Institute in Zagreb.SI. 2. Elementarne čestice virusa mozaika trpuca (RMV-K) u purificiranom preparatu obrađenom fosforno-volframskom kiselinom. Snimljeno u Institutu Ruder Bošković u Zagrebu.Fig. 3. Precipitin reactions in immuno-diffusion tests in agar. A  —  Reaction of RM V-K (K) and TMV (T) with the serum against RM V-K (sK). B —  Result of the absorption test in agar: The middle well was first filled with the suspension of the type strain of ribgrass mosaic virus (H) and two hours later with the serum against TMV (sT). The ab­sorbed sT still reacted with TMV (T) but not any more with RM V-K  (K) or the type strain of ribgrass mosaic virus (H).SI. 3. Precipitacijske reakcije u imunodifuzijskim pokusima u agaru: A  —  reakcija RM V-K (K) i virusa mozaika duhana (T) s antiserumom od RM V-K (sK). B —  rezultat pokusa zasićavanja u agaru: središnji bazen bio je početno napunjen suspenzijom tipičnog virusa mozaika trpuca (H), a 2 sata kasnije antiserumom od virusa mozaika duhana (sT). Zasićeni sT još je reagirao s virusom mozaika duhana (T), ali ne i s RM V-K (K) i tipičnim sojem virusa mozaika trpuca (H).Fig. 4. Size distribution of rods of ribgrass mosaic virus (RMV-K).SI. 4. Raspodjela štapićastih čestica virusa mozaika trpuca (RMV-K).Fig. 5. Ultraviolet absorption of purified isolate RM V-K of ribgrass mosaic virus.SI. 5. Ultravioletna apsorpcija purificiranog izolata RM V-K virusa mozaika trpuca.3 33R e f e r e n c e sDudtnan, W. F., 1965: Differentiation of strains of tobacco mosaic virus by immune diffusion in agar plates. Phytopathology 55, 635— 639.Goldin, M. I., 1953: A  mosaic of ribgrass. (In Russian.) Dokl. Akad. Nauk SSSR 88, 933— 935.Gooding, G. V., and T. T. Hebert, 1967: A  simple technique for purification of tobaco mosaic virus in large quantities. Phytopathology 57, 1285.Goto, T. T., and N. Oshima, 1962: A  strain of tobacco mosaic virus isolated from a wild crucifer plant, Radicula sylvestris Druce. Ann. Phytopath. Soc. Japan 27, 109— 114.Harrison, B. D„ 1956: A  strain of tobacco mosaic virus infecting Plantago spp. in Scotland. Plant Pathol. 5, 147— 148.Harrison, B. D., J. T. Fineh, A. J. Gibbs, M. Hollings, R. J. Shepherd, V. Valenta and C. Wetter, 1971: Sixteen groups of plant viruses. Viro­logy 45, 356— 363.Holmes, F. O., 1941: A  distinctive strain of tobacco mosaic virus from Plan­tago. Phytopathology 31, 1089— 1098.Juretic, N., and C. Wetter, 1973: Serological relationship among some nec­rotic strains of the Holmes’ ribgrass mosaic virus. Plant Virology, Proc. of the 7th Conf. Czechosl. Plant Virologists.Juretic, N., M. Wrischer and Z. Poldk, 1969: A  strain of Holmes’ ribgrass virus occurring in Yugoslavia. Biol. Plant. 11, 284— 290.Kovachevsky, I. C„ 1963: Untersuchungen über das Wegerichmosaik in Bul­garien. Phytopath. Z. 49, 127— 146.Pei, M. Y., 1962: Preliminary studies on several isolates of TMV from dif­ferent plants. Acta Microbiol. Sinn. 8, 420— 427.Poldk, Z., 1964: In nature occurring distinctive necrotic strain of tobacco mosaic virus. Plant Virology. Proc. of the 5th Conf. Czechosl. Plant Virologists, pp. 168— 169. Academia -  Prague.Schumann, K.,  1963: Das »Digitalis-Mosaik« —  eine Viruskrankheit an Digi­talis lanata Ehrh. Pharmazie 18, 497— 501.Schumann, K.,  1963: Über ein Mosaik an Plantago lanceolata L. und seine Beziehung zum Digitalis-Mosaik. Pharmazie 18, 573— 575.Silva, F., und I. Pop, 1965: Mosaikvirus an Digitalis lanata Ehrh. und sein Einfluß auf den Gehalt an Wirkstoffen. Pharmazie 20, 110— 112.van Regenmortel, M. H. V., 1966: Plant virus serology. Advan. Virus Res. 12, 207— 271.van Regenmortel, M. H. V., 1967: Serological studies on naturally occurring strains and chemically induced mutants of tobacco mosaic virus. Vi­rology 31, 467— 480.Wetter, C., and E. Luisoni, 1969: Precipitin, agar gel diffusion, and intragel absorption tests with three strains of tomato bushy stunt virus. Phy- topathol. Z. 65, 231— 242.34S A D R Ž A JID E N T IF IK A C IJ A  T O B A M O V IR U S A  IZ O L IR A N O G  IZ  V R S T E  R O R I P A  A M P H 1B IANikola Juretić, Davor Miličić i Đorde Mamula (In stitu t za b o ta n ik u  S veu č iliš ta  u Z a grebu )Prošle godine izolirali smo virus iz divlje krucifere Roripa amphi- bia (L.) Bcss. u blizini Zagreba i dali mu oznaku RMV—K. Inficirane biljke imale su simptome u obliku blage išaranosti i nekrotičnih linija na listovima (si. 1A).Elektronsko-mikroskopska analiza purificirane virusne suspenzije pokazala je da RMV—K ima čestice približno 300 nm duge (si. 4). Ap­sorpcija virusne suspenzije dosizala je maksimum u ultravioletnom di­jelu spektra kod 260 nm (si. 5).Izolirani virus prenijeli smo na 12 zeljastih biljaka (tablica 1). Vi­rus je prouzrokovao lokalne lezije na listovima vrsta Nicotiana silves- tris, N. glutinosa, D. stramonium i vrlo jake sistemične simptome na vrsti N. tabacum, tip Samsun (si. 1). Na osnovi simptoma na tim vrstama bilo je očito da je RMV—K vrlo sličan običnom virusu mozaika trpuca.Serološki pokusi vršeni su metodom dvostruke difuzije u agaru te unakrsnim zasićavanjem u agaru. Ti su pokusi pokazali da je is­traživani virus srodniji virusu mozaika trpuca nego virusu mozaika duhana (si. 3).Dr Nikola Juretić, prof. đr Davor Miličić 1 Đorđe Mamula, mr biol.,Institut za botaniku SveučilištaMarulićev trg 20/1141000 Zagreb (Jugoslavija)35

Identifikacija tobamovirusa izoliranog iz vrste Roripa amphibia

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