Tomato black ring virus

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A Microbial Biorealm page on the genus Tomato black ring virus

Classification

Higher order taxa

Virus; ssRNA viruses; ssRNA positive-strand viruses, no DNA stage; Picornavirales; Comoviridae; Nepovirus; Subgroup B

Description and Significance

Tomato Black Ring Virus (TBRV) is a RNA-containing virus that occurs in Europe. It infects a wide range of herbaceous and wood monocotyledonous and dicotyledonous species including many that are important crop plants. It appears on these plants as necrotic rings, spots and flecks, systemic chlorotic ringspots, mottle, stunting, leaf malformation and vein yellowing.1 In addition to black ring of tomato, the various strains of this virus cause ringspot diseases of bean, sugarbeet, lettuce, raspberry and strawberry, yellow vein of celery, shoot-stunting of peach and unnamed diseases of leek and onion. It occurs in many other plants, including cabbage, grapevine and lucerne. Plants infected with TBRV are patchily distributed in crops because of slow migration of the soil-inhabiting vectors, Longidorus spp. Some strains tend to occur in soils together with strains of raspberry ringspot virus because they share the vector L. elongatus.2

Genome structure

The TBRV has isometric particles between 26 to 30 nm in diameter with hexagonal outlines. In purified preparations, particles exist as three sedimenting components with sedimentation coefficients (S20,w) of 55S, 97S and 121S, termed T, M and B, respectively. All particles consist of 60 protein subunits each of molecular weight 57 000 but, whereas T particles are nucleic acid-free protein shells, M and B particles contain linear ssRNA with molecular weight of 1.7 x 10^6 and 2.7 x 10^6, respectively.2 Some virus isolates contain in addition a satellite RNA of molecular weight 0.5 x 10^6. Several different satellites have been described for different TBRV isolates.7

The complete sequence genome for TBRV RNA1 and RNA 2 has been sequenced. The sequence of TBRV RNA 1 is 7356 nucleotides long. A putative initiation codon at nucleotide 261 was considered to be the start of an open reading frame which terminates at a UAG codon at position 7053.3 The sequence of TBRV RNA 2 is 4618 nucleotides long and contains an open reading frame endcoding a polypeptide of 1344 amino acids.8

Cell structure and metabolism

Virions consist of a capsid. The virus capsid is not enveloped and is round with icosahedral symmetry. These capsids appear hexagonal in outline and the capsomer arrangement is not ovbious.2,3

In 2 distantly serologically related isolates (A and G12) of TBRV that each produce 2 RNA species, RNA-1 is found only in bottom component (B) particles and RNA-2 is found only in middle component (M) particles. Preparations of separated M and B particles are barely infective, but produce 8 to 30 times more lesions when mixed. This indicates that both kinds of particle are needed for infection because they contain different parts of the genome. Infectivity is not enhanced when M particles of isolate G12 are mixed with B particles of isolate A, but it increases when M particles of isolate A are mixed with B particles of isolate G12. The lesions produced are abnormally small; isolates cultured from some of them are slower than the parental isolates to produce systemic symptoms in Chenopodium quinoa and have serological properties indicating that their coat protein cistron is in RNA-2.4

Ecology

The virus can best be detected in all parts of the host plant and particularly in the mesophyll. Virions are found in the cytoplasm, cell vacuole (tubules extending through plasmodesmata, which is possibly a sign of degenerating nuclei).5

The two types of RNA present in TBRV are both genomic RNA species and are necessary for infectivity.4 Natural transmission between plants is by vector nematodes of the dorylaimid genus Longidorus: L. attenuatus and L. elongatus.5, Larvae and adults are able to transmit but the virus is not retained after moulting, nor is it passed to progeny through the egg. Nematodes acquire virus from infected plants after feeding for about 1 hour and retain the ability to transmit for many weeks in soil without host plants. Different TBRV isolates often have different vector species. Thus, in the UK, isolates of TBRV from Scotland are transmitted most efficiently by L. elongatus, whilst those in England (and also on the mainland of Europe) are transmitted largely by L. attenuatus.2

The virus is also transmitted through seeds of infected plants, often with a high frequency, especially in some crop species and weeds. This enables the virus to be dispersed over a wide area.6 Additionally, the virus can be dispersed by transport of soil containing TBRV-infected nematodes and/or TBRV-infected seed. In perennial plants, virus may be distributed in material vegetatively propagated from infected plants.2

TBRV can cause severe disease in some raspberry, strawberry and peach cultivars in some localities but the incidence of such infections is often small. Yield loss in crops is difficult to quantify but, although significant in some cultivars of some crops, it is probably of only local importance.2

Pathology

Infected weed and crop plants may show few or no symptoms especially in the year of infection or when infection occurs through the seed. Nevertheless, plant growth in such plants may be impaired. Where infection occurs through nematode transmission, this often appears as patches of poor growth which slowly extend in size each year. Depending on the cultivar, natural infection in Rubus and Fragaria may induce chlorotic mottling and/or ringspots in leaves. In potato, leaves may develop black necrotic spots.9 In celery, Sambucus nigra and some other shrubs, leaves may show bright-yellow vein-clearing. Symptoms are generally most obvious in plants in early spring growth and are less noticeable during more rapid growth in summer.10 TBRV is readily transmitted by inoculation of sap to many herbaceous test plants but mechanical inoculation of virus from woody plants should be made in 2% (v/v) nicotine sulfate (pH 9.3). In test species such as Chenopodium quinoa, C. amaranticolor and Nicotiana clevelandii, TBRV induces chlorotic or necrotic local lesions and systemic necrosis, depending on the virus isolate. Such symptoms, although indicative of virus infection, are not diagnostic for TBRV, and other tests, such as serology or nucleic acid hybridization, are necessary to establish unequivocally the presence of TBRV. Serological tests are generally the most convenient and ELISA is probably the most sensitive. However, because of the serological variability of TBRV isolates, antisera to each of the two main serotype groups should be used. ELISA has been successfully used to detect TBRV directly in plants such as grapevine, raspberry and strawberry.11,2

Current Research

New research has been conducted where two generic PCR protocols were developed to detect nepoviruses in subgroups A and B using degenerate primers designed to amplify part of the RNA-dependent RNA polymerase (RdRp) gene. It was observed that detection sensitivity and specificity could be improved by adding a 12-bp non-complementary sequence to the 5' termini of the forward, but not the reverse, primers. The optimized PCR protocols amplified a specific product (~340bp and ~250bp with subgroups A and B, respectively) from all 17 isolates of the 5 virus species in subgroup A and 3 species in subgroup B tested. The primers detect conserved protein motifs in the RdRp gene and it is anticipated that they have the potential to detect unreported or uncharacterized nepoviruses in subgroups A and B.12


Another current research was conducted where several different isolates of TBRV were collected in Poland from cucumber, tomato, potato and black locust plants. Biological tests showed some differences in the range of infected plants and the type of symptoms,which was the basis for selection of seven of the most biologically different TBRV isolates. According to the sequence of TBRV-MJ, several primer pairs were designed and almost the entire sequence of both genomic RNAs was amplified. The RT-PCR products derived from all tested TBRV isolates were digested by restriction enzymes. On the basis of the restriction patterns, the variable and the conserved regions of the TBRV genome were defined and the relationship between the Polish TBRV isolates established.13


It was also determined that efficient replication of the in vitro transcripts from cloned cDNA of tomato black ring virus satellite RNA-encoded requires the 48K satellite RNA-encoded protein.Tomato black ring virus isolate L supports the multiplication of a large satellite RNA of 1376 nt which has no common features with the 2 genomic RNAs except for the terminal motif 5’ VPg UUFAAAA and a 3’ poly(A) tail. The TBRV sat-RNA contains an ORF for a protein of 48K which is translated both in vitro and in vivo. To determine the function of the 48K protein the effect of different mutations introduced in the ORF of the cDNA clone on the capacity of transcripts to multiply Chenopodium quinoa plants or protoplasts were inoculated along with the genomic RNAs were studied. Transcripts in which nucleotides were substituted within the 5’ proximal region of the ORF multiplied poorly even when the modification conserved the 48K protein sequence. This suggested that this portion of the ORF contained cis-acting RNA sequences. Transcripts with alterations in the internal region of the ORF retained their multiplication capacity provided the mutation didn’t destroy the ORF or modify the length of the protein expressed. The absence of multiplication in plants of transcripts unable to express the 48K protein and their inability to replicate in protoplasts suggested strongly that the sat-RNA translation product itself was implicated in the replication of sat-RNA.14

References

1Schmelzer, K. 1963. Phytopathologische Zeitschrift. v. 46, p. 235

2Murant, A.F. 1970. CMI/AAB Description of Plant Viruses No.38, p.4

3Greif, C., Hemmer, O., and Fritsch, C. 1988. Nucleotide sequence of tomato black ring virus RNA-1. Journal of General Virology, v. 69, p.1517-1529.

4Randles, J.W., Harrison, B.D., and Murant, A.F., 1977. Packaging and biological activity of the two essential RNA species of tomato black ring virus. Journal of General Virology, v. 36, p.187-193.

5Harrison, B.D., Mowat, W.P., Taylor, C.E. 1961. Transmission of a strain of tomato black ring virus by Longidorus elongates (Nematoda). Virology, v. 14, p. 480-485

6Lister, R.M.; Murant, A.F. 1967. Seed-transmission of nematode-borne viruses. Annals of Applied Biology v. 59, p. 49-62.

7Fritsch, C., Koenig, I., Murant, A.F., Raschke, J.H., and Mayo, M.A. 1984. Comparisons among satellite RNA species from five isolates of tomato black ring virus and one isolate of myrobalan latent ringspot virus. Journal of General Virology, v. 65, p. 289-294.

8Gall, O.L., Lanneau, M., Candresse,T., and Dunez, J. 1995. The nucleotide sequence of the RNA-2 of an isolate of the English serotype of tomato black ring virus: RNA recombination in the history of nepoviruses. Journal of General Virology, v. 76, p. 1279-1283.

9Murant, A.F. 1987. Raspberry ringspot and associated diseases of Rubus caused by raspberry ringspot and tomato black ring viruses. In: Virus diseases of small fruits (Ed. By Converse, R.H.), p. 211-220.

10Schmelzer, K. 1966 Studies on viruses of ornamental and wild woody plants. 5. Virus disease of Populus and Sambucus. Phytopathologische Zeitschrift v. 55, p. 317-351.

11Bercks, R. 1963 Serological cross-reactions between isolates of the tomato black ring spot virus. Phytopathologische Zeitschrift v. 46, p.97-100.

12Wei, T., Clover, G. 2008. Use of primers with 5’ non-complementary sequences in RT-PCR for the detection of nepovirus subgroups A and B. Journal of Virological Methods, v. 153. p. 16-21.

13Jonczyk, M., Borodynko, N., Pospieszny, H. 2004. Restriction analysis of genetic variability of Polish isolates of Tomato black ring virus. Acta Biochimica Polonica, v. 51, p. 673-681

14Hemmer, O., Oncino, C., and Fritsch, C. 1993. Efficient replication of the in vitro transcripts from cloned cDNA of tomato black ring virus satellite requires the 48K satellite RNA-encoded protein. Virology, v. 194, p. 800-806