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Tubular structures containing virus-like particles were found by Walkey & Webb (1968) in squashes of meristems of Nicotiana rustica L. systemically infected with cherry leaf roll virus (CLRV), a nepovirus. These tubules occurred in the cytoplasm of undifferentiated meristematic cells of N. rustica, but were not found in differentiated leaf or stem cells (Walkey & Webb, 1970). We have examined differentiated leaf cells of Nicotiana clevelandii Gray infected with four strains of CLRV and here report that tubules occur often in the cell walls of these cells, but rarely in their cytoplasm. We also describe ultrastructural changes induced by CLRV that resemble those produced by bean pod mottle and cowpea mosaic viruses. N. clevelandii leaves systemically infected with CLRV were used.

The forms of raspberry ringspot (RRV) and tomato black ring (TBRV) viruses known from southern England are serologically related to the forms known from Scotland, but only distantly. They also seem to have different vectors. Longidorus elongatus (de Man), which transmits the forms of both viruses occurring in Scotland, was not found at outbreaks of the viruses in England. Instead, L. macrosoma Hooper occurred where RRV was spreading, and L. attenuatus Hooper where TBRV was spreading. Adults and juveniles of L. macrosoma readily transmitted the English but not the Scottish form of RRV, or either form of TBRV. Adult females and juveniles of L. attenuatus transmitted the English form of TBRV, but in the one test failed to transmit the Scottish form. Ability to be transmitted by a given species of nematode appears to be correlated with the antigenic constitution of the virus.

Sixteen groups of plant viruses are described. The type members and main characteristics of each group are listed. The extension of this system to other plant viruses is discussed, and it is recommended that only well studied viruses should be grouped. Names are proposed for twelve of the groups: bromovirus, carlavirus, caulimovirus, comovirus, cucumovirus, nepovirus, potexvirus, potyvirus, tobamovirus, tobravirus, tombusvirus, and tymovirus. We suggest that the value of these names should be tested by using them for an experimental period.

Our recent studies have shown that raspberry ringspot virus (R/1:2.4/43 + 1.4/30 (or 2 × 1.4/46):S/S:S/Ne, nepovirus group) has two functional RNA species, of mol. wt. 2.4 × 106 (RNA-1) and 1.4 × 106 (RNA-2) (Harrison, Murant & Mayo, 1972a, b; Murant et al. 1972). There is also evidence that tomato top necrosis (Bancroft, 1968), tobacco ringspot (Harrison et al. 1972a) and cherry leaf roll (Jones & Mayo, 1972) viruses have divided genomes and we suggested (Harrison et al. 1972a) that this property is characteristic of nepoviruses. In this paper we present results with another nepovirus, tomato black ring virus (TBRV), in which we have found three RNA components. Two of these seem essential for infection and presumably comprise the genome of the virus. The third seems to be a ‘satellite’ RNA, analogous to that found in some cultures of tobacco ringspot virus (Schneider, 1969, 1971).

SUMMARY: Serological and plant-protection experiments provided evidence that potato bouquet and beet ringspot viruses are strains of tomato black ring virus. Tomato black ring and potato bouquet viruses are more closely related to each other than either is to beet ringspot virus. The three produce similar symptoms and have a similar host range. There was no evidence that they are related to tobacco ring spot or peach yellow bud mosaic viruses. In the plant-protection tests, protection between serologically related virus strains often was not reciprocal; when protection was incomplete, the symptoms produced by the second virus were milder than those in plants infected by it alone. Strains of tomato black ring virus could be arranged in order of their ability to cause symptoms in plants already infected with other strains. Although results of the plant-protection and serological tests agreed broadly, the correlation was not complete, suggesting that the tests measure different properties of the virus particles. The degree of difference between strains of tomato black ring virus seems to reflect their geographical separation, something that is perhaps to be more expected with soil-borne than with other viruses.

When turnip, sugar beet, or spinach seedlings were grown for varying periods in soils containing tomato black ring (TBRV) and raspberry ringspot viruses, and then transplanted to virus-free soil, some seedlings became infected in 3 days and many in 9 days. The ease with which TBRV infected and multiplied in roots decreased with increasing age of seedling. The presenee of the viruses in soils was shown by growing bait seedlings in them, but not by inoculating leaves of test plants with soil extracts. In highly infective soils, one bait seedling became infected per 100–200 g soil. Infectivity of soils was abolished by air-drying for a week at 20°C and was decreased by freezing at −10°. It was also abolished by treating soils with pentachloronitrobenzene, tetramethylthiuram disulfide, ethylene dibromide, and Nemagon, but not by griseofulvin or by mixing the soil with lime or ammonium sulfate. Infectivity of soils containing TBRV was associated with particles that sedimented through 7 cm water in between 30 seconds and 5 minutes, and to a lesser extent with particles sedimenting in 30 seconds. Pot experiments showed that seedlings grown in noninfective field soil or autoclaved soil did not become infected when watered with highly infective sap. TBRV became established in autoclaved sand when watered with crude eelworm preparations from infective soils, but not when the sand was mixed with washed roots of naturally infected plants. TBRV also became established in noninfective field soil when it was cropped with artificially infected sugar beet. The receptivity of this soil to virus seemed specific to the beet ringspot strain of tomato black ring virus and was abolished by autoclaving. Evidence is adduced that an agent occurring in soil plays a part in transmission of TBRV, and its possible identity among soil-inhabiting organisms is discussed.

Raspberry ringspot virus (R/1:2.4/43 + 1.4/30 (or 2 × 1.4/46):S/S:S/Ne, nepovirus group) sediments as three major components (T, M and B) with sedimentation coefficients of 52, 92 and 130 S, respectively (Mayo, Murant & Harrison, 1971). We reported (Murant et al. 1972) that particles of M component yielded one type of RNA molecule, of mol. wt. 1.4 × 106 (RNA-2), whereas B component yielded both RNA-2 and RNA of mol. wt. 2.4 × 106 (RNA-1). We suggested that some particles of B component contain one RNA-1 molecule and others contain two RNA-2 molecules. Confirmation of this suggestion comes from the finding, which we now report, that dimers of RNA-2 are formed when purified virus is irradiated with u.v. light. The culture of raspberry ringspot virus, and the methods by which it was purified and assayed for infectivity, were as described by Murant et al. (1972).

Raspberry ringspot virus (R/1:2.4/43 + 1.4/30 (or 2 × 1.4/46):S/S:S/Ne, nepovirus group) is a multicomponent plant virus with two sizes of RNA having mol. wt. of 2.4 × 106 (RNA-1) and 1.4 × 106 (RNA-2) (Murant et al. 1972). When obtained by centrifuging in sucrose density gradients or by electrophoresis in polyacrylamide gels, preparations of RNA-2 mostly did not infect the local lesion assay host, Chenopodium amaranticolor Coste & Reyn., and those of RNA-1 were only moderately infective, whereas mixtures of the two kinds of preparation were very infective (Harrison, Murant & Mayo, 1972). No such increase in infectivity occurred when one RNA species from raspberry ringspot virus was mixed with the other from tobacco ringspot virus (another nepovirus), or when either of the RNA species from raspberry ringspot virus was u.v. irradiated before use. A possible explanation of these results is that each RNA species carries genetic information not carried by the other.

Summary Purified preparations of the golden elderberry strain of cherry leaf roll virus contain two spherical nucleoprotein components with sedimentation coefficients of 115s and 128s. Purification of the components by centrifuging several times through sucrose density gradients decreased their specific infectivity. The original specific infectivity of unfractionated virus was restored by mixing the separated components, indicating that both components are necessary for maximum infectivity. The components were indistinguishable serologically, in their extinction spectra and in their appearance in the electron microscope. Polyacrylamide gel electrophoresis of preparations from each component indicated that both contained a single protein species of 54,000 mol. wt, but that whereas the 115s component contained a RNA molecule of 2.1 × 106 mol. wt the 128s component contained one of 2.4 × 106. These and other properties suggest that cherry leaf roll virus can be considered a member of the nepovirus group. The present cryptogram of the golden elderberry strain is R/1 : 2.1/40 + 2.4/43 : S/S : S/*.

Longidorus elongatus (de Man) was always found in soil from patches in raspberry and strawberry plantations where the plants were infected with the beet ringspot (Scottish) strain of tomato black ring virus but rarely elsewhere in the same plantations. L. elongatus hand-picked from virus-containing field soil transmitted the virus to sugar beet, turnip, and spinach seedlings, but did so less often than might have been expected from the infectivity of the whole soil from which the nematodes came. Noninfective L. elongatus acquired and subsequently transmitted the virus from infected cucumber and potato plants. When hand-picked adult and larval L. elongatus were used separately, only larvae transmitted. Also, when infective soil was fractionated by sieving through meshes of successively smaller size, the infectivity of the fraction retained on each sieve, relative to the number of L. elongatus it contained, was greatest for samples containing the smallest larvae. A previously undescribed species of Longidorus , closely related to L. elongatus , was associated with an outbreak of the lettuce ringspot (English) strain of tomato black ring virus.

Infectious nucleic acid has been isolated from purified tobacco ringspot virus by a modification of the heat-denaturation method. The infectivity of these preparations ranged from 0.1 – 1.0% as compared with the nucleic acid in the virus. Tests conducted with this nucleic acid have given evidence that the associated infectivity cannot be due to contamination with residual whole virus: (1) Infectivity of the nucleic acid was destroyed by incubation with ribonuclease whereas whole-virus infectivity was not. (2) Infectivity of the nucleic acid remained in the supernatant after 2 hours ultracentrifugation, while that of whole virus was pelleted under identical conditions. (3) Infectivity of the nucleic acid was influenced by the concentration of phosphate buffer present in the medium used for inoculation, whereas that of whole virus was not. (4) Infectivity of the nucleic acid was usually destroyed during storage for several days at 4°, whereas whole virus activity remained constant for weeks at 4°. No characteristic virus particles could be found in electron micrographs of nucleic acid preparations which had ten times the activity of whole virus preparations that were shown to contain many characteristic particles. Phosphorus, nitrogen, and protein analyses and nucleotide composition have been obtained for lyophilized nucleic acid preparations. From these data it has been determined that there was 0.7% or less protein contaminant. There was, however, 20% less phosphorus and nitrogen than should be expected on the basis of the nucleotide composition. This discrepancy suggests that a nonnitrogenous contaminant was present in this material.

Among plant viruses that are moved from host to host by living organisms, those transmitted by nematodes constitute, as a whole, a heterogeneous collection of infectious entities, which can grossly be differentiated by virtue of particle morphology and relationship with vectors. All these viruses comprise two of the sixteen groups presently included in the virus classification scheme proposed by Harrison et al. (47), i.e. nepovirus and tobravirus groups.

Publisher Summary If a viral genome consists of two or more nucleic acid molecules of different length, which are separately encapsidated, the resulting particles are likely to differ in sedimentation rate but will not necessarily differ in size and buoyant density. The different classes of particles, which are called the components of the virus, can be separated by physical means. This chapter summarizes the different modes of particle heterogeneity that have been described and the methods suited for the detection and separation. If the size of the capsid is adapted to the size of the genome parts, the particles can be separated by electrophoresis in sieving media provided that an adequate pore size can be found and by sedimentation in density gradients. Since the RNA:protein ratio, and thus the buoyant density, does not differ appreciably, the particles cannot be separated by banding in equilibrium gradients. Tubular (tobraviruses) and bacilliform viruses (alfalfa mosaic virus) as well as spherical viruses (tobacco streak virus, pea enation mosaic virus) belong to this class. The particles of como- and nepoviruses do not differ in size, and thus cannot be separated by electrophoresis in sieving media, but they sediment and band differently. Separation of the parts of the genome can only be accomplished after the extraction of the RNA's. It is conceivable that viruses exist that have genome parts of the same length. The nepovirus tomato ring-spot virus approaches this situation. In the animal DNA virus, adeno-associated virus the complementary strands are present in different particles. In preparations of bromo- and cucumoviruses, a similar RNA is found encapsidated together with the smallest part of the genome.

After dagger nematodes Xiphinema diversicaudatum (Micol.), were shown to transmit arabis mosaic virus to strawberry plants, the mode of transmission was studied using Petunia hybrida Vilm. seedlings as test plants. Approximately 30% of the nematodes reared on roots of infected strawberry plants transmitted the virus, and batches of ten nematodes caused infection in 90% of the test plants. Three days was the minimum test-feeding period in which transmission occurred, but longer periods resulted in a higher proportion of plants becoming infected. The vector retained the virus for long periods; some nematodes transmitted the virus to test plants in a second series without infecting those in the first on which presumably they fed for periods of 5, 10, and 20 days. Nematodes were still infective after fasting periods of up to 30 days in autoclaved sand. Noninfective nematodes acquired the virus in 24 hours, the minimum acquisition period tested, but a higher proportion were infective after 3 days on the source plants.