red clover mottle virus and red clover necrotic mosaic virus in sweden

Phytopath. Z., 76, 67—79 (1973)© 1973 Verlag Paul Parey, Berlin und Hamburg

Department of Plant Pathology and Entomology.,Agricultural College of Sweden, S--7SQ07 Uppsala 7

Red clover mottle virusand Red clover necrotic mosaic virus in Sweden

ByB. GERHARDSON and K. LINDSTEN

With 8 figures

Received January 14, J972

Introduction

In an investigation concerning virus diseases on ley plants in Sweden cloverplants were collected from different fields during the summers of 1968—1971and examined for virus diseases. Among the viruses isolated from these plantswe often found two which we consider to be identical to red clover mottle virus(RCMV) and red clover necrotic mosaic virus (RCNMV).

Neither of these viruses has been earlier reported to occur in Sweden andred clover necrotic mosaic virus has previously only been reported from Cze-choslovakia (MUSIL 1969a).

Investigations concerning test plant reaction, physical properties, serologyand electron microscopy of the Swedish isolates of these viruses were made andthe results are briefly reported in this paper.

Materials and Methods

The viruses were isolated frotn naturally infected red clover plants which were dugup in tbe field, replanted in clay pots and placed in an ordinary greenhouse.

Three isolates of RCNMV (49/69, 63/70 and 68/70) collected from different parts ofSweden were studied in more detail. Only one isolate of RCMV, originating from aninfected red clover plant grown near Uppsala, was used in these studies.

Transmission in the laboratory took place by mechanical inoculation. Phosphate bufferpH 7 was used when macerating the tissue of infected leaves, and carborundum powder(size 600 mesh) was dusted over the leaves of the test plants before inoculation. Inoculationwas done with a piece of foam-plastic held by a pair of pincers.

The test plants were grown from seeds planted in steam sterilized soil and wereusually Inoculated at a very young stage. The plants were kept in an insect proof greenhouse

6g GERHARDSON and LINDSTEN

at 18—24 °C. Additional light from fluorescent lamps was used to compensate for the shortperiod of daylight daring tbe darker seasons.

Physical properties of the viruses in crude plant sap were tested according to themethods proposed by Bos, HAGEDORN and QUANTZ (1960).

The viruses were purified by differential centrifugation using a 1 : 1 mixture of n-butanol and chloroform for clarifying tbe sap and 0.05 M citrate-phosphate buffer pH 7 asa suspending medium after high speed centrifugation. The procedures used for purificationand also for electron microscopy and particle measurement were mainly the same as thosereported for alsike clover vein mosaic virus (GERHARDSON and LINDSTEN 1971). RGNMVwas purified from systemically infected plants of Nicotiana clevclandii Gray and RCMV fromplants of Pisum sativum L.

Antisera were obtained from rabbits subjected lo a combination of intravenous andintramuscular injections. As a rule two cycles of intravenous injections about one weekapart were followed by three or more cycles of intramuscular injections. For Intramuscularinjections the virus was emulsified in Freund's adjuvant (Bacto). Serological tests werecarried out as Ouditerlony double diffusion tests in agar gels using crude sap of infectedplants, clarified by low-speed centrifugation, as antigens.

Field Observations of the Diseases

Of the viruses found on red clover in Sweden RCMV and RCNMV seemto be among those most prevalent in clover fields.

RCMV was identified for the first time in our country in 1968 and sincethen it has been isolated from red clover plants on several occasions. In theeastern part of central Sweden the virus seems to occur in most red clover fieldsand often in high frequencies.

RCNMV seems to be more widespread than RCMV and occurs both in thecentral and southern parts of the country. However, RCNMV mostly occurs inlower frequencies.

A more detailed report on the occurrence and distribution of viruses foundto infect legumes in Sweden is in preparation and will be published elsewhere.

Both viruses induce easily observed symptoms on naturally infected redclover plants (see under Host range and symptoms) and are readily found in thefields. However, a certain masking of the symptoms of RCNMV in the summerhas been observed and this seems to be true especially for newly infected plants,

At present we know very Httle about the damage caused by these twoviruses under field conditions. However, infected plants in the fields often showa rather marked growth reduction and are probably more sensitive to unfavour-able winter conditions.

According to our observations both RCMV and RCNMV are easily spreadin the field but nothing is known about any vector or the way of spread undernatural conditions.

Host Range and Symptoms of RCMV

Trifolium pratense L. In the greenhouse the first symptoms appeared abouttwo weeks after inoculation when the young leaves showed a diffuse mosaic,often beginning with vein clearing. Later a distinct mosaic developed with

Red clover mottle virus and Red clover necrotic mosaic virus in Sweden 69

. 1 (left). A plant of Trifolium pratense infected with red clover mottle virus (RCMV)Fig. 2 (right). Local lesions on Pbaseolus vulgaris ten days after inoculation

with red dover mottle virus (RCMV)

irregular dark green areas sharply contrasting to the light green tissue surround-ing them (fig. 1). The leaves were often more or less distorted.

Naturally infected plants in the field showed similar symptoms and accord-ing to our experience this type of mosaic was clearly different from field symp-toms of other viruses infecting clover in Sweden.

Gomphrena globosa L. About two weeks after inoculation the inoculatedleaves developed red spots and reddening of the veins. The virus could berecovered from these leaves by bade inoculation but not from unmoculatedleaves, and no systemic symptoms were seen.

Phaseolus vulgaris L. The varieties tested, 'Konserva II' and 'Cita', bothdeveloped small necrotic local lesions four to five days after inoculation (fig. 2).As a rule no systemic symptoms appeared and recovery tests from uninoculatedleaves gave negative results. Occasionally test plants of the variety 'Cita'developed necrosis of segments of the veins and slight malformation of unino-culated trifoliate leaves. In these cases the virus could be recovered from suchleaves.

Pisum sativum L. The symptoms became apparent as a vein clearing fourto six days after inoculation, which was later followed by a conspicuous andcharacteristic mosaic. The plants became stunted and malformed to a certainextent. The leaves were smaller than corresponding ones on healthy plants. Al!the pea varieties tested — 'Torsdags', 'Witor', 'Hero', 'Parvus', 'Engelsk sabel','Dvargsabel' and 'Goncordia' — became systemically infected and all gavesimilar symptoms.

Trifolium hybridum L. Vein clearing started to develop about two weeksafter inoculation. Subsequently mosaic, crinkling of the leaflets and depressionso{ plant growth were observed. The mosaic symptoms were not as distinct as onT. pratense.

70 GERHARDSON and LINDSTEN

T. incarnatum L. The symptoms were as for T. hybridum, but a morepronounced mosaic, leaf malformation and inhibition of plant growth wereobserved.

Vicia faba L. Necrotic local lesions developed on inoculated leaves afterabout a week. Systemic infection soon followed with necrosis of the stem apexand the upper leaves, which often became crinkled and malformed. The plantsusually wilted and died after two weeks (fig. 3).

Fig. 3. TbrcL' diUcrcnt stages ol symptoms on Vicia faha after infection with red clovermottle virus (RCMV). To the right typical symptoms two weeks after inoculation

Vigna sinensis L. Numerous small necrotic local lesions developed on in-oculated leaves about 6—7 days after inoculation. No systemic infection wasdetected.

The following plant species were found not to be susceptible to the isolateinvestigated. Chenopodium amaranticolor Coste et Reyn., C. quinoa Willd.,Cucumis sativHs L., Nicotiana clevelandii Gray and N. glutinosa 1.

Minor differences in symptoms were observed between different isolates ofRCMV. Thus some isolates collected in 1970 gave local lesions on C. amaran-ticolor and C. quinoa similar to those reported by Bos and MAAT (1965) whileother isolates had no sudi effects.

Host Range and Symptoms of RCNMV

Trifolium pratense. Naturally infected plants found in the field oftenshowed distmct symptoms with mosaic, malformation, necrosis on parts of theveins and frequently a pronounced growth reduction (fig. 4). In some cases,however, the virus was also isolated from field plants showing only a slightmosaic and chlorosis on parts of the veins.

On the other hand, red clover plants inoculated mechanically and placedin the greenhouse gave very mdistinct and unreliable symptoms. Sometimes


Fig. 4. A field plant of Trifoliurrj pratensenaturally infected with red clover necro-

tic mosaic virus (RCNMV)

necrotic local lesions developed oninoculated leaves about ten daysafter the inoculation. This develop-ment of local lesions was observedmostly during the autumn andwinter seasons and was possiblydependent on environmental con-ditions. As a rule no local lesionswere observed and systemic symp-toms were weak and showed onlysome leaf crinkling and a veryslight mosaic or mottle. Not all theinoculated red clover plants becameinfected and according to our experiences it is not easy to transmit the virusmechanically to red clover in the laboratory.

Chenopodium amaranticolor. Sometimes a few dilorotic local lesionsdeveloped. These lesions later spread and coalesced into irregular chlorotic areas.No systemic infection was obtained. The local lesions did not show up on allinoculated plants and in such cases it was not possible to recover the virus frominoculated leaves.

C. quinoa. A few chlorotic, and later, necrotic local lesions sometimesdeveloped on inoculated leaves. Like the symptoms on C. amaranticolor theselocal lesions were not seen on all inoculated plants. We also found a certaindifference in susceptibility of C. quinoa to different isolates of RCNMV. Nosystemic infection could be detected.

Cucumis sativus. Small necrotic dots developed on inoculated cotyledonsfour to five days after inoculation. No systemic infection could be detected.

Gomphrena globosa. A few necrotic local lesions with red margins devel-oped on inoculated leaves. No systemic spread of the virus was detected.

Lathyrus odoratus L. The inoculated leaves wilted and died about a weekafter inoculation. Systemic infection induced growth reduction, necrosis of theveins of the lower leaves and wilting symptoms beginning in the top leaves.

Nicotiana clevelandii. A few white chlorotic local lesions developed oninoculated leaves after four to five days. The infection rapidly spread system-ically inducing mosaic, slight crinkling of the leaves and a marked growthreduction (fig. 5).

Petunia hybrida (Hook) Wilm. Only a few plants of those inoculatedbecame infected. No symptoms were observed on infected plants but the viruswas readily recovered by back inoculation to plants of Phaseolus vulgaris.


Fig. 5. Symptoms on Nicotiana clevelandii infected with red clover necrotic mos,iic virus(RCNMV). To the right a healthy control plant

Phaseolus vulgaris. (Varieties 'Konserva II' and 'Cita'). Necrotic locallesions and vein necrosis developed on inoculated leaves four to five days afterinoculation. These leaves usually bent down and later wilted and usually drop-ped. The systemic infection showed up as a slight mosaic, necrotic streaks on thestem and necrotic veins on the upper leaves or top necrosis. Systemically infectedplants often showed a rather sudden inhibition of plant growth (fig. 6).

Pisum sativum. A few necrotic local lesions developed on inoculated leaveswhidi later wilted. Usually no systemic infection followed but in a few plantsthe infection spread systemically inducing necrosis on parts of the stem andnecrotic areas and necrotic veins on the upper leaves. Such plants usually gotvery stunted and soon died. In those plants not showing systemic symptoms thevirus could not be demonstrated in the upper leaves by back inoculation.

Tetragonia expansa Murr.Small whitish local lesions develop-ed on inoculated leaves. No sys-temic infection could be detected.

Trifolium hybridum. Infectedplants showed a systemic infectionwith reduction of plant growth,crinkling of the leaflets and a dif-fuse mosaic. Like T. pratense alsikeclover was not easily infected inthe laboratory.

T. incarnatum. Infection wasmanifested as a marked stunting ofplant growth, malformation of the

1-ig. 6. Symptoms of red clover necroticmosaic virus {RCNMV) on Phaseolus

vulgaris 12 days after inoculation

Red clover mottle virus .ind Red clover necrotic mosaic virus in Sweden 73

leaves and a pronounced mosaic. Often the infected plants died prematurely.In contrast to the other clover species tested, T. incarnatum was easily infectedby RCNMV.

Vicia faba. Diffuse necrotic local lesions developed on inoculated leaveswhich sometimes wilted and dropped. No systemic infection could be foundwith back inoculation from uninoculated leaves.

Vigna sinensis. Necrotic local lesions developed on inoculated leaves about4—5 days after inoculation. A systemic necrosis then followed causing dwarfingand premature death of the infected plants.

The following plant species did not show symptoms after inoculation withthe Swedish isolates of RCNMV and the virus could not be recovered with backinoculation to Phaseolus vulgaris:

Callistephus chinensis Nees., Glycine soya L., Lycopersicum esculentumMill., Medicago sativa L., Physalis floridana L., Trifolium repens L., and Zinniaelegans Jacq.

During the work so far done on RCNMV in Sweden we have found somedifferences in symptom expression between different isolates of the virus. Thussome of the isolates gave very weak symptoms, especially on P. vulgaris andN. clevelandii, but others more or less killed plants of P. vulgaris, once infectionwas established.

Transmission Experiments')

As RCMV-infected red clover plants are often found in the clover fieldsalready the first year after sowing the leys, we thought it possible that sometransmission of the virus can occur with the seeds.

An experiment was set up where seeds from infected red clover plants werecollected in the autumn and sown in the laboratory. The seedlings were placedin the greenhouse and observed for the development of symptoms for abouteight weeks.

About 800 seedlings were grown in this way but none of them showed anysymptoms.

Attempts were made to transmit RCNMV with the aphid species Acyr-thosiphon pisum (Harries), Myzus persicae (Sulzer) and Aphis faba Scop. Theaphids were first starved for four to six hours and then placed on infected Tri~folium incarnatum plants for aquisition feeding during 20 sec, 1 min., 1 hourand 48 hours. The aphids were then placed on healthy seedlings of T. incarnatumfor 48 hours after whidi they were killed. About 400 aphids of each species weretested but no transmission of the virus occurred.

Soil transmission of RCNMV was tried by planting seedlings of T. pra-tense, T. incarnatum and N. clevelandii in soil collected around the roots of

') While this paper was in press additional experiments have shown that clover shootweevils, Apian spp., arc able to transmit RCMV. These experiments are in progress and willbe published later on.


diseased plants in the field. After eight weeks macerated tissue of both the rootsand the leaves of the Trifolium species were inoculated to test plants of P. vul-garis and N. clevelandii. None of these plants developed any symptoms andneither did N. clevelandii planted in soil from the field.

Properties in vitro of RCMV

D i l u t i o n e n d p o i n t : When sap of infected pea plants was seriallydiluted m distilled water and inoculated on pea plants infectivity of the sap wasobtained at a dilution to I0~^ but not at a dilution to 10~^. Using P. vulgaris asassay plants gave the same results.

L o n g e v i t y in v i t r o : Sap of infected pea plants stored at room tem-perature was infective after three weeks of storage but not after four weeks.Infectivity of the sap was tested by inoculation on test plants of Pisum sativum.

T h e r m a l i n a c t i v a t i o n p o i n t : Sap of infected pea plants heated at63 ' C for ten minutes and inoculated on pea plants was still infective althoughmfectivity was low. In sap heated at 65 ' C for ten minutes all infectivity waslost.

Properties in vitro of RCNMV

D i l u t i o n e n d p o i n t : Sap of infected P. vulgaris plants serially dilutedin distilled water and inoculated on test plants of P. vulgaris was infective afterdilutions of 10"" to 10~^. Sap diluted to 10~^ had a low infectivity and abouthalf of the test plants used became infected.

L o n g e v i t y in v i t r o : Sap from infected P. vulgaris stored at roomtemperature for four weeks was still infective. This was the longest time tested.

T h e r m a l i n a c t i v a t i o n p o i n t : Sap of all isolates was infective afterheating at 86 C for ten minutes. Heating at 88 'C destroyed most infectivitybut in some cases a few test plants became infected after heating the sap at thistemperature. No infectivity was obtained after heating the sap to 90 C forten minutes.

Electron Microscopy

Partially purified preparations for electron microscopy, immunization andanalytical ultracentrifugation were obtained according to the proceduresdescribed above (see Materials and methods).

Electron microscopy of partially purified preparations, negatively con-trasted with phosphotungstic acid (PTA) pH 7, revealed isometric particles ofboth viruses (fig. 7).

About 300 particles of each virus were measured, giving an average size forRCMV of about 29 nm 0 and for RCNMV of about 30 nm 0 .


Fig. 7. Electron micrographs of negatively cuntrascfd particles ol partially purified red clovermottle virus (RCMV) to the left and red clover necrotic mosaic virus (RCNMV) to the right.

Magnification X 120 000

Analytical Ultracentrifugation

Analytical ultracentrifugation (Spinco model E) of partially purified pre-parations of the viruses revealed three components of RCMV with the sedimen-tation coefficients of about 56, 95 and 118 Soo.w (fig-8, left). RCNMV showeda single component with a sedimentation coefficient of about 126 S n.w (fig-8,right).

Fig. 8. Sdilieren diagrams of red clover mottle virus (RCMV), left, and red clover necroticmosaic virus (RCNMV), right, sedimenting in tbe analytical ultracentrifuge. Both pictures

taken after running the centrifuge for 15 min. at 31 410 rev./min. SchlJeren angle 60°

Serology

An antiserum prepared against the Swedish isolate of RCMV gave a homo-logous titre of 1 : 512. Antisera prepared against two different isolates of


RCNMV (49/69 and 63/70) both gave a positive reaction up to a dilution of1 :1024 when tested against homologous antigens. Crude sap of infected plantswas used as antigen when testing the titre and all tests were carried out asOuchterlony double diffusion tests. In no case any precipitation lines wereobtamed with healthy plant sap.

Antigens of alfalfa mosaic virus, alsike clover vein mosaic virus, arabismosaic virus and RCNMV were combined with Swedish RCMV-antiserum fortesting possible serological relationships with the Swedish isolate of RCMV. Nc)positive reaction was obtained with any of these viruses.

However, an antiserum against the Dutdi isolate of RCMV, kindly suppliedby Dr. L. Bos, Wageningen, gave a strong positive reaction when tested againstthe Swedish isolate of RCMV showing that these two viruses are serologicall)related. I

The two RCNMV-antisera were also tested against alfalfa mosaic virus,alsike clover vein mosaic virus, arabis mosaic virus and red clover mottle virus.No positive reaction was obtained whidi indicates that there are no serologicalrelationships between RCNMV and any of these viruses.

Antisera of two Czechoslovakian isolates of RCNMV, kindly supplied byDr. M. MUSII., Bratislava, were tested against Swedish isolates of RCNMV.A positive reaction was obtained with these antisera against some of the Swedishisolates showing that there is a serological relationship between the Swedish andthe Czechoslovakian isolates of RCNMV. Antisera of two Swedish isolates ofRCNMV (49/69 and 63/70) sent to Dr. MUSIL and tested in Bratislava againstantigens of his isolates of RCNMV also reacted positively (pcrs. comm.).

It was, however, also found that the antiserum prepared against one isolateof RCNMV (49/69) did not react with antigens of some of the other isolates.The same phenomenon was found by MUSIL with differetit isolates found itiCzedioslovakia (MUSIL 1969 b). This indicates, as reported by MUSIL, the possi-bility of different serological groups of the virus in different isolates or that thedifferent isolates might represent different serological strains of the virus orperhaps even different viruses.

Discussion

The RCMV-isolates studied caused local lesions on Phaseolus vulgaris.systemic mosaic on Pisum sativum and local lesions and later a lethal systemic-necrosis on Vicia faba. These symptoms, including the characteristic severemosaic on pea varieties were always typical and very similar to the symptomsdescribed for RCMV by SINHA (I960), Bos and MAAT (1965), MUSIL andLESKOVA (1969a) and SCHUMANN and UMLAND (1970). Even if the reaction ofRCMV on P. vulgaris is similar to symptoms caused by alfalfa mosaic virus amiconfusions may be made with broad bean mottle virus on V. faba, we consider,however, as unlikely that confusions can be tnade with hitherto described virusesattadting clover when all these three species mentioned give typical sytnptoms.

According to our experience the symptoms of RCMV on our red clovervarieties are usually not a mottle but rather a mosaic of dark-green patches


unevenly surrounded by a lighter leaf area. Well developed symptoms of thistype frequently make it also possible to distinguish a RCMV-infection fromother types of mosaic on red clover plants (cf. Bos and MAAT 1965).

The host range for the Swedish isolate of RCMV is in agreement with thefindings of other workers on this virus. Some discrepancy seems to occur con-cerning the reaction of RCMV on Chenopodium spp. indicating differences alsobetween different strains as shown by Bos and MAAT (1965). With our typeisolate we got no reaction on C. amaranticolor and C. quinoa. However, someisolates of RCMV collected in 1970 gave local lesions on both these species. Thismay indicate that different strains of the virus occur in Sweden but so far noreal effort has been made to distinguish between strains of this virus.

The shape and size of the particles, the way of transmission and the pro-perties in vitro of the Swedish RCMV-isolate are in good agreement, too, withthe data found in the literature for this virus. As shown by MUSIL and LKSKOVA(1969 b) the thermal inactivation point and dilution endpoint vary considerablyfor RCMV owing to the original virus content of the plants. However, differencesbetween isolates may also occur as our RCMV-isolates never showed infectivityafter heating ten minutes at 65 ' C.

The strong serological reaction between antigens of the Swedish RCMV.lnd antiserum of the Dutch isolate as well as the behaviour in the analyticalultracentrifuge (56, 95 and 118 So,i,\v) give further evidence that the virusdescribed in this paper is to be considered as an isolate of red clover mottle virus.

The symptoms caused by RCNMV, the second virus studied, were especiallyconspicuous and reliable on P. vulgaris even if the severity may vary fordifferent isolates. Characteristic for RCNMV, in contrast to most other legumeviruses, is that it causes a systemic infection on Nicotiana clevelandii and someother non-leguminous plants.

These symptoms, as well as symptoms caused on various other test plantsand the host range, strongly suggest a close relationship to the red clover necroticmosaic virus reported from Czechoslovakia by Musii. (1969 a).

However, some differences in symptoms seem to occur between the Swedishand Czechoslovakian isolates. Thus, no systemic infection in V. faha could bedetected when this plant was infected with the Swedish isolates although severalplants were tested in all seasons of the year. The symptoms caused on C. amaran-ticolor and C. quinoa were mostly very weak and unreliable. These discrepanciesmay be due to differences in the genetic constitution of the test plants or theenvironmental conditions but it may also indicate that different strains of thevirus occur.

We found that our isolates of RCNMV had isometric particles about 30 nmin diameter which are somewhat smaller than the size (35 nm) reported by MUSIL(1969 a). However, in all other respects the physical properties of the Swedishisolates of RCNMV show a close resemblance to the Czedioslovakian virus.

A mutual exchange of antisera with Dr. M. MUSIL, Bratislava, has displayeda serological relationship between Swedish and Czechoslovakian isolates.


Although there seem to be some differences between our isolates ofRCNMV and those described from Czechoslovakia we do not consider theSwedish isolates as a special strain of RCNMV, at least not until more is knownabout the differences between various isolates which are isolated in each country.

Thus, the two viruses dealt with in this paper are considered to be Swedishisolates of red clover mottle virus (RCMV) and red clover necrotic mosaic virui(RCNMV) and neither of these have previously been reported from the Scandi-navian countries.

Red clover mottle virus (RCMV) was first described from England bySiNHA (1960) and since then has been reported from The Netherlands (Bos andMAAT 1965), Czechoslovakia (MUSIL and LE§KOVA 1966, MUSIL and LE§KOVA1969 a) and Germany (SCHUMANN and UMLAND 1970). However, as far as weknow RCMV has only been reported to occur in lower frequencies and is con-sidered to be of only minor importance. In Sweden on the other hand we con-sider RCMV to be one of the most common viruses on red clover plants and incertain areas it seems to occur in high frequencies.

Red clover necrotic mosaic virus (RCNMV), hitherto reported only fromCzechoslovakia (MUSIL 1969 a) also seems to be common in Sweden. However,many of the legume viruses common in other countries, e. g. bean yellow mosaicvirus (including pea mosaic virus) and alfalfa mosaic virus, seem to be less com-mon on clover in Sweden, suggesting that the importance of different legumeviruses may vary considerably among various countries.

We are indebted to the Swedish Council for Forestry and Agricultural Research forfinancial support. Thanks are also due to Mrs. ELSA ANUERSSON, Mrs. BARBRO LINDSTEN andMrs. MAJKEN DBERG for tedinical assistance.

Summary

This paper deals with two viruses on red clover, earlier unknown inSweden.

The symptoms caused on Phaseolus vulgaris (local lesions), Pisum sativum(systemic) and Vicia faba (lethal systemic necrosis) as well as the host range, thephysical properties and the serological relationship led to the conclusion thatone of these viruses is red clover mottle virus (RCMV).

The other virus gave quite different symptoms on P. vulgaris (systemic),Pisum sativum (usually not systemic) and V. faba (necrotic local lesions). Non-legumes were infected systemically, too, e.g. Nicotiana clevelandii, and locallesions were caused on others, sudi as Cucumis sativus, Gomphrena globosa andTetragonia expansa. The particles of this virus were isometric and about 30 nmin diameter. The sedimentation coefficient of the particles was 126 So,, w. Thedilution endpoint was high (lO"" ) and the thermal inactivation point 88—90'' C.Together, these data and additional serological evidence suggest that the secondvirus is probably identical to red clover necrotic mosaic virus (RCNMV) whichpreviously has been reported only from Czechoslovakia.

RCMV seems to occur in high frequencies in certain areas of Sweden andRCNMV is also likely to be common.


Zusammenfassung

Das Scheckungs-Virus des Rotklees (Red clover mottle virus)und »Red clover necrotic mosaic virus" in Schweden

Es werden zwei in Schweden bisher unbekannte Rotkleeviren beschrieben.Auf Grund der Symptome auf Phaseolus vulgaris (Lokalla.sionen), Pistim

sativum (systemisch) und Vicia faba (systemische Letalnekrose), dem Wirts-p flan 7, en bereidi, der physikalischen Eigenschaften und der serologischen Ver-wandtschaft wird gefolgert, das eines dieser Viren das Sdieckungsvirus des Rot-klees (red clover mottle virus, RCMV) ist.

Das andere Virus zeigte sehr abweichende Symptome auf P. vulgaris (syste-misch), P. sativum (gewohnlich nidit systemisdi) und V. faba (nekrotische Lokal-la.sionen). Es verursadite auch systemische Infektionen auf Nicht-Leguminosen,z.B. Ntcotiana clevelandti und Lokallasionen auf Cucumis sativus, Gomphrenaglobosa und Tetragonia expansa. Seine Partikein sind lsometrisch und ungefahr30nm 0 . Ihr Sedimentationskoeffizientist 126 Sodw, ihr Verdiinnungsendpunktliegt hoch {10-") und ihr thermischer Inaktivierungspunkt liegt bei 88 bis 90 "C.Diese Daten und zusatzliche serologische Untersuchungen weisen darauf hin, dafidieses zweite Virus wahrscheinlich mit dem »red clover necrotic mosaic virus"(RCNMV) identisdi ist. Dieses Virus ist bisher nur in der Tsdiedioslowakeigefunden worden.

RCMV sdieint in bestimmten Gebieten Schwedens sehr haufig vorzukom-men. RCNMV sdieint ebenfalls allgemein zu sein.

Literature

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

_ _^ and D. Z. MAAT, 1965: A distinctive strain of the red clover mottle virus in theNetherlands. Nether!. J. Plant Path. 71, 8—13.

GERHAKDSON, B., and K. LINDSTEN, 1971: Alsike clover vein mosaic virus, a new virus in-fecting alsike clover {Trifolium hybridum L.) in Sweden. Phytopath. Z. 72, 76—85.

MusiL, M., 1969a: Red clover necrotic mosaic virus, a new virus infecting red clover {Tri-folium pratense) in Czedioslovakia. Biologia, Bratislava, 24, 33—45.

^ 1969b: Serological properties of certain isolates of red clover necrotic mosaic virus.Acta virol. U. 226—234.

• , and O. LESKOVA. 1969a: Course of red clover mottle virus infection in some hostplants. Biologia, Bratislava, 24, 23—32.

, and , 1969b: Studies on the effect of temperature on the red clover mottle virusin vivo and in vitro. Biologia, Bratislava, 24, 535—544.

SCHUMANN, K., and T. UMLAND, 1970t Zum Vorkommen des Schediungs-Virus des Rotklees(red clover mottle virus) in der Deutschen Demokratisdien Republik. Phytopath. Z.67, 73—77.

SINHA, R . C , 1960: Red clover mottle virus. Ann. appl. Biol. 48, 742—748.

Authors' address: BERNDT GERHARDSON and KLAS LINDSTEN, Department of PlantPathology and Entomology, Agricultural College of Sweden, S-750 07 Uppsala 7 (Sweden).

red clover mottle virus and red clover necrotic mosaic virus in sweden

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