Phytopath. Z., 82, 339—346 (1975)© 1975 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / ASTM-Coden: PHYZ A3

Institute of Microbiology and Virology, Ukrainian Academy of Sciences, Kiev

Red Clover Mottle Virus in Ukraine

ByL. G. LAPCHIC, L. L. KUZNETZOVA, V. S. MELNICZENKO, S. K. VOCELKO,

and A. A. SJEDIN

With 7 figures

Received Decembci 20, 1974

Red clover mottle virus (RCMV) was first isolated by SINHA in Englandin 1960 and since then has been investigated in some countries of Europe. Thereare some differences among isolates of this virus studied in the Netherlands (2),Czechoslovakia {3, 6, 7, 9,10, 11, 12, 15), GDR (13), Sweden (4) and in Ukraine(PoREMBSKAjA, unpubHshed) in symptoms on host plants and properties in vitro.

The present paper describes the results of investigations concerning theproperties of RCMV isolated from Trifolium pratense L. in Ukraine in 1973.

Materials and Methods

Virus was isolated from naturally infected red clover plants with symptoms of mosaic(dark-green areas on the green-yellow leaf tissue) which were dug up in the field, replantedin pots and placed in the greenhouse.

Uniform virus culture was obtained by several successive single-lesion transfers onPhaseolus vulgaris (Top Crop variety). Virus isolate was maintained on broad bean by me-dianical inoculation. Broad bean and pea seedlings were inoculated in the two-leaf stage; beanand cowpea — on the primary leaves stage when they have been found to be mostly susceptibleto the infection. For mechanical inoculation of test plants carborundum was used as abrasivefor dusting over the leaves before rubbing.

All plants were raised and kept in the greenhouse at a temperature of about 20 °Csupplemented with artificial (fluorescent) light in winter when the light conditions were ratherpoor. Sometimes in summer the temperature was higher for some periods. The temperature andtight conditions were found to have profound effect on the appearance of virus symptoms.

Physical properties of the virus in crude sap of broad bean plants were determinedaccording to Bos et al. methods (1).

Virus was purified by differential centrifugatioti from freshly harvested broad bean orpea plants inoculated 12—15 days previously and systemically infected.

The plant material was homogenized in 0.06 M phosphate buffer pH 5.5 containingL-cysteine-HCl and 0.01 M sodium diethyldithiocarbamate. Ratio of plant material: buffer

340 LAPCHIC, KUZNETZOVA, MELNICZENKO, VOCELKO, and SjEDrw

was 1 ; 1 w/v. The plant sap was expressed through two layers of dieesc-cloth and centrifugated20 mm at 4000 rpm for discarding off the cell debris. After isoelectric precipitation of the hostproteins at pH 4.1 virus was purified by four cycles of differential centrifugation (1.5 hoursat 30000 rpm. VAC-601 ultracentrifuge). Final virus pellets were resuspendod in 0.06 Mphosphate buffer pH 7 and stored at 4 ' 'C.

The purified virus preparations were examined in spectrophotomecer SF-4A, analyticalultracentrifuge Spinco model E, electron microscope (JEM-7), by centrifugation in sucrosedensity gradient and electrophoresis in polyacrylamide gel.

RCMV was electrophoresed in 2.7% polyacrylamide gels (the ratio of acrylamide tobisacrylamide was 30 to 0.8), 0.01 M Na-phosphaie buffer pH 1.1 during 2.5—3 hours at roomtemperature and 3—4 ma/tube. Virus bands were detected by staining gels with 0.25% Coo-massi brilliant blue R-250 in 10 % trichloracetic acid during 2 hours.

Sedimentation coefficients were determined in Bediman Model E ultracentrifuge AnD-rotor, using Schlieren optics, and calculated as described by MARKHAM (8).

Virus components were separated by centrifuging twice through sucrose density gradients.They were prepared by layering 2, 4, 2 and 1 ml respectively of 40, 30, 20 and 10% (w/v)sucrose in 0.06 M neutral phosphate buffer and allowing diffusion at 4 ^C overnight. Samples(1.5 to 2 ml) containing up to 3 mg virus were floated on the gradients. After centrifuging(MOM-317a ultracentrifuge, 28 000 rpm, 2.5 hours) viral zones were collected with the syringeneedle. Fractions containing any one virus component were pooled, diluted at least fivefoldin 0.06 M phosphate buffer pH 7 and centrifugated for 2 hours at 30 000 rpm to sediment thevirus. The virus pellets were resuspended in phosphate buffer pH 7. Approximate estimatesof the relative proportions of the virus components were made from the areas under theSdilieren peaks.

For electron micro.scopy study purified virus preparations were stained in uranyl acetate(2 %) or phosphotungstic acid pH 7 (2 %). Photographs were taken at fixed magnification ofX 52 000.

ResultsHost range and symptomatology

Trifolium pratense L. In the field red clover plants from which the viruswas isolated showed dark green spotting pattern. Plants inoculated medianicailyand maintained in the greenhouse developed similar symptoms.

Vicia faba L. Usuallyinoculated leaves showed nosymptoms, but some timesreddish-brown rings ap-peared on them after five toseven days (fig. 1). Typicalsymptoms of RCMV inVicia faba are necroticstreaks on the stem, brown-ing and death of growingpoint then wilting and pre-mature death of plant. AU

hig. 1. Symptoms of red clovermottle virus (RCMV) on Vicia faba

Red Clover Mottle Virus in Ukraine 341

Fig.2. Chlorotic ring spots onthe young trifoliate leaves of

Vigna iinensis var. Gibridnaj

most all infected plantsdied about two weeksafter inoculation. Thesesymptoms were observedon all varieties tested:Piculovichsky, Russkiecherny and Aushra.

Pisum sativum L.Inoculated leaves devel-oped chlorotic ring-spotsabout a week after inocu- /lation. The infection —gspread systemically caus-ing vein-clearing of the youngest leaves. Later mosaic symptoms followed byvein necrosis and leaf malformation developed.

Vigna sinensis L. Chlorotic ring spots were observed on primary inoculatedleaves of cowpea var. Gibridnaj 7 about 7—8 days after inoculation. Similarspots developed on the young trifoliate leaves (fig. 2). High concentration ofvirus in these leaves was demonstrated with back inoculation to Phaseolus vul-

garis Top Crop variety. Cow-pea var. Black Eye showed thesame symptoms.

Phaseolus vulgaris L.Only necrotic symptoms weredetected on all varieties tested,no systemic infection was ob-served. Two types of lesionsdeveloped on inoculated pri-mary leaves: reddish-brownand pale chlorotic, often sur-rounded by concentric rings asfor example on var. Saxa(fig. 3). Such varieties as

Fig. 3. Chlorotic local lesions on in-oculated leaves of Phaseolus vulga-

ris var. Saxa

342 LAPCHIC, KUZNETZOVA, MELNICZENKO, VOCELKO, and SJEDIN

Fig. 4. Chlorotic spots surroundedby concentric rings of Lupinns albus

Midielit, Pinto, Top Crop,Wisconsin Refugee, Bountifulshowed numerous browtilesions.

Lupinus albus and L. lit-teus L. Inoculated leavesshowed chlorotic spots sur-rounded by concentric rings(fig. 4) Similar symptomsdeveloped on the youngleaves. The virus could berecovered from these leavesabout two months afterinoculation of seedlings, but

virus concentration was very low according to a number of necroses on P/>«ieo/«5vulgaris var. Michelit. The infected plants severely stunted.

Chenopodium amaranticolor Coste et Reynier and Nicotiana glutinosashowed no symptoms and virus could not be detected by back inoculation.

Properties of RCMV in sapTests of properties of RCMV in vitro were done using sap from infected

I.Toad bean leaves and assayed on Phaseolus vulgaris var. Michelit. The dilutionend point was between 10~^ and 10"''. The thermal inactivation point was63 " C. Sap retained infectivity after 27 days of storage (chloroform was usedas anticeptic) at room temperature.

Intracellular inclusions

For revealing intracellular inclusions in cells of infected plants epidermiswas stripped from the lower surface of broad bean and pea leaves, fixed andstained with pironin-methyl green or by Giemsa. Amorphous inclusions likeX-bodies were detected in epidermal cells of broad bean and pea leaves.

The effect of pH on virus infectivity

The effect of pH on infectivity of RCMV was investigated within therange from 2.3 to 10.5. Suspension of purified virus in 0.06 M phosphate bufferat defmtte pH allowed to stand at room temperature within a matter of an hourand assayed on primary leaves of bean var. Michelit. Virus concentration inthese experiments was 0.5 mg/ml. Concentration was estimated using assumedextinction coefficient (E° 'J''") of 8 by analogy with cowpea mosaic virus (13).Results of experiment showed high virus stability within the pH range from3.5 to 10.5 (fig.5).

Red Clover Mottle Virus in Ukraine 343

300--

200 -

100 -

0 2 4 6 8 10 pHFig. 5. The effect of pH on the infectivity of purified red clover mottle virus

Virus purification

Method of differential centrifugation gave a diance to obtain the purifiedvirus preparations. Purified preparations of virus had spectra typical of nucleo-protein with maximum UV absorption at 260 nm and minimum at 240 nm.H uii/Eo o was 1.7 and E..,,,,/E24(, ratio was 1.4. The yields generally were about100 mg and 150 mg of nucleoprotein for 1 kg plant tissue of broad bean and pearespectively.

Polyacrylamide gel electrophoresis of purified virus showed two electro-phoretic forms. Centrifugation in sucrose density gradients and analyticalcentrifugation revealed three components assigned as top (T), middle (M) andbottom (B) respectively (fig.6). Sedimentation coefficients (S:.'o.u) at infinitedilution were found to be 55 S, 95 S and 125 S for T, M and B-componentsrespectively. Unfractionated preparations of the virus contained usually 4 to9% of T, 50—60% of M and 31—46% of B. Top component is unstable;in unfractionated virus preparations stored at 4 C its proportion decreasedgradually and after two weeks it was completely lost. In the analytical ultra-centrifuge such preparations separated only into the M and B components.Purified preparations of the virus retained their infectivity during six monthsat 4 C in 0.06 M phosphate buffer pH 7.

I'ig. 6. Siiiliercn diagrams of a sedimentation analysis of a purified virus preparation showing(left to right) T, M and B components. Photographs were taken at 5, 10, 15 min after centri-

fuging at 30 000 rev/min. SdiHeren angle 60'-

344 LAPCHIC, KUZNETZOVA, MELNICZENKO. VOCELKO, and

Fig. 7. Virus particles of apurified red clover mottlevirus preparation showingsome particles completely,some partially and some notpenetrated by phosphotungsticacid. Magnification ^ 92 000

Isometric particlespenetrated and not-pe-netrated by the stainwere revealed by elec-tron microscopy of puri-fied preparations. Themean diameter of par-ticles was 28—29 nm

{fig- 7).Investigations of in-

fectivity of separatedcomponents and their mixtures are in progress. The data obtained showed thatboth middle and bottom components are necessary for the infectivity of RCMV,thereby supporting the assumption that RCMV has divided genome.

The resuits of these experiments will be discussed in the next paper.

Discussion

The properties of the virus isolate described herein establish its identity asRCMV-isolate. Its resembles the Czechoslovakian RCMV-isoIate described byMusiL et al. (9) and the isolate reported from Sweden by GERHARDSON andLiNDSTEN (4). These isolates have similar host range and symptomatology.Common typical symptoms for all these RCMV-i.solates are premature death ofinfected broad bean plants and severe reaction on pea cultivars.

The properties of these virus isolates in crude sap are in good agreement:the thermal inactivation point 63 "C, dilution end point - \Q~\ longevity invitro 27 days.

As was shown by M. MUSIL for Czechoslovakian isolate (11) we also foundthat appearing of symptoms on bean var. Saxa are affected by temperature.At high temperature (above 30 C) no symptoms can be detected during oneweek after inoculation but if the temperature lowers small chlorotic spotsdeveloped on leaves.

Our attempts to transmit the virus with the aphid species Acyrthosiphonpisum (Harries) were unsuccessful. The aphids were placed on infected Viciafaba plants after starving for six hours and then after aquisition feeding duringI min, 30 min, 1, 24, 48 hours they were placed on healthy seedling of broadbean.

Red Clover Mottle Virus in Ukraine 345

Double-diffusion tests in 1 % agar gel with an antiserum against theCzechoslovakian isolate of RCMV (kindly supplied by Dr. MUSIL) showedstrong serological relationship between described isolate and Czedioslovakianisolate of RCMV.

The presence of three components with the sedimentation coefficients atinfinite dilution of 58 S, 95 S and 125 S give supporting evidence that theinvestigated virus is an isolate of red clover mosaic virus.

Our isolate of RCMV and the isolate previously recovered in Ukraine(POREMBSKAJA, Unpublished) differ in longevity in vitro (27 days against3 days) and in severity of symptoms on broad bean and other host plants.

According to classification of HARRISON et al. (5) RCMV belongs to theComovirus group. Such members of this group as cowpea mosaic virus, squashmosaic virus and bean pod mottle virus are known to have genome dividedbetween two nucleoprotein components.

It appeared of considerable interest therefore to continue the investigationof the component properties of the described RCMV-isolate. The evidence forthe divided genome of RCMV will be discussed in the next paper.

Summary

This paper deals with the virus on red clover isolated in Ukraine. Hostrange studies and physical properties of the virus in crude sap, serological andelectron microscopical examinations as well as analytical centrifugation andseparation in sucrose density gradients into three components give evidencethat the investigated virus is red clover mottle virus (RCMV).

Zusammenfassung

Das Sdieckungs-Virus des Rotklees (Red clover mottle virus, RCMV)in der Ukraine

Es wird uber ein in der Ukraine von Rotklee isoliertes Virus beriditet.Prufung des Wirts pfl an zenk reises, physikalische Eigenschaften des Virus imRohsaft, serologische und elektronenmikroskopisdie Untersuchungen sowie dieErgebnisse der Ultrazentrifugierung zeigten, dafi es sidi um das Sdiedtungs-Virus des Rotklees Kandelt.

We thank Dr. M. MUSIL for kindly supplied antiserum against RCMV.

Literature

1. Bos, L., D. J. HAGEDORN, and L. QUANTZ, 1960: Suggested procedures for internationalidentification of legume viruses. T. Plantenziekten 66, 328—343.

2. , and D. Z. MAAT, 1965: A distinctive strain of the red clover mottle virus in theNetherlands. Netherl. Plant Path. 71, 8—13.

Phyiopath. Z., Bd. 82, Heft 4 23

346 LAPCHIC, Red Clover Mottle Virus in Ukraine

3. CALLO, J., 1973: Some knowledge on purification of viruses infesting leguminous plants.Plant Virology. Proc. 7th Conf. Czechosl. Plant Virologists.

4. GERHARDSON, B., and K. LINDSTEN, 1973: Red clover mottle virus and red clover necroticmosaic virus in Sweden. Phytopath. Z. 76, 67—79.

5. HARRISON, B. D., J. FINCH, A. J. GIBBS, M. HOLLINGS, R. J. SHEPHERD, V. VALENTA, andC. WETTER, 1971: Sixteen groups of plant viruses. Virology 45, 356—363.

6. MARCINKA, K., 1970: Changes in peroxidase isozymes in bean leaves following infectionwith red clover mottle virus. Bjologia, Bratislava, 25 (9), 599—602.

7- , and V. VALENTA, 1969: Effect of bentonite on the infection of bean leaves withred clover mottle virus. Plant Virology. Proc. 6th Conf. Czediosl. Plant Virologists,Olomouc 1967, 85—88.

8. MARKHAM, R., I960: A graphical method for the rapid determination of sedimentationcoefficients. Biochem. J. 77, 516.

9. MUSIL, M., 1966: O vyskytu viru strakatosti jetele lucniho na Slovensku. Biol6gia, Bratis-lava, 21 (9), 663—669.

10- , and O. LESKOVA, 1969a: Course of red clover mottle virus infection in some hostplants. Biol6gia, Bratislava, 24 (1), 23—31.

11- T and , 1969b; Studies on the effect of temperature on the red clover mottlevirus in vivo and in vitro. Biol6gia, Bratislava, 24, 535—544.

12. NiBLETT, C. L., and J. S. SEMANCHIK, 1970: The significance of the coat protein in in-fection by the electrophoretic forms of cowpea mosaic virus. Virology 41, 201—207.

13. SCHUMANN, K., und T. UMLAND, 1970: Zum Vorkommen des Schedtung-Virus des Rotklees(red clover mottle virus) in der DDR. Phytopath. 2. 67, 73—77.

14. SINHA, R. S., 1960: Red clover mottle virus. Ann. Appl. Biol. 48, 4, 742—748.15. VALENTA, V., and K. MARCINKA, 1968: Enhanced infectivity of combined bottom and

middle components of red clover mottle virus. Acta Virol. 12 (3), 288.

Author's address: Institute of Microbiology and Virology, Ukrainian Academy ofSciences, Zabolotnogo street 59, Kiev-143 (USSR).