Strawberry Latent Ringspot Virus Research Articles

First Report of Tomato ringspot virus in Butterfly Bush (Buddleia davidii).

Leaves displaying bright yellow or light green line pattern symptoms were collected from individual, large, mature buddleias in a home garden in Clemson, SC, a botanical garden in Knoxville, TN, and a container-grown plant on sale in a retail home and garden store in Seneca, SC. Buddleias grown in the southeastern United States frequently display virus-like symptoms, but the line pattern symptom displayed by these plants was atypical of the mosaic, mottling, and leaf deformation seen when buddleias are infected with Alfalfa mosaic virus (AMV) or Cucumber mosaic virus (CMV) (2,4). Line pattern symptoms are frequently seen in woody species infected by ilarviruses or nepoviruses (2). No ilarviruses are reported to infect buddleia and only the nepovirus, Strawberry latent ringspot virus, which is restricted mainly to Europe, is reported to infect this species (1,2). The nepoviruses Tomato ringspot virus (ToRSV) and Tobacco ringspot virus (TRSV) are frequently found infecting plants of many species in the southeastern United States (3). Total RNA was extracted from the three symptomatic plants and used in reverse transcription-polymerase chain reactions (RT-PCR) to detect ToRSV and TRSV using primer pairs developed in this laboratory, which amplify regions around the amino terminus of the coat protein of the respective viruses. The expected amplification product for ToRSV of 327 base pairs was obtained from samples tested from each plant, and the nucleotide sequence of the product showed 96% identity with the corresponding fragment of GenBank Accession No. NC_003839 that the primers were designed to amplify. Repeated attempts to isolate a virus from symptomatic leaves using sap inoculation to Chenopodium amaranticolor Coste & Reyne, C. quinoa Willd, Nicotiana clevelandii Gray, and N. tabacum L. have failed. Repeated testing by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of leaves from the plant growing in Clemson consistently produced absorbance values at 405 nm in the range of 0.47 to 0.55 (mean of 8 separate samples per test) for symptomatic and asymptomatic leaves. The range of values for the positive control (ToRSV-G growing in N. clevelandii) was 1.3 to 1.5. The ranges of values for the noninfected controls (noninfected N. clevelandii and leaf tissue from a buddleia known to be infected with AMV and CMV but in which ToRSV or TRSV had never been detected by RT-PCR) were 0.102 to 0.104 and 0.102 to 0.106, respectively. The extraction buffer produced absorbance readings in the range of 0.098 to 0.102. RT-PCR of RNA extracted from other portions of the leaves used in the ELISA consistently amplified the 327-bp product from symptomatic leaves and from the positive control but not from noninfected control tissues. RNA from asymptomatic leaves on the infected plant also produced the 327-bp product in RT-PCR. Isolation of viruses from woody hosts is frequently difficult, and although, we have yet to succeed to confirm the association between the observed symptom and ToRSV, the evidence from PCR and ELISA would indicate ToRSV is present in these plants. To our knowledge, this is the first report of ToRSV, a member of the genus Nepovirus, in buddleia.

Detection of Strawberry latent ring spot virus in Leaves of Olive Trees in Italy using a One‐Step RT–PCR

AbstractA one‐step reverse transcription–polymerase chain reaction (RT–PCR) protocol was developed and used for the detection of Strawberry latent ring spot virus (SLRSV) in leaves of olive trees in Italy. The protocol was used to test for infection in samples from 87 trees and to correlate infection with leaf symptoms. SLRSV was found only in olives grown in Central Italy (the Marche and Umbria regions) and was detected in c. 15% of leaf samples. Most infected plants had narrow and twisted leaves, symptoms which seemed to occur in specific cultivars. The one‐step RT–PCR protocol is very rapid and sensitive and has the potential to be used on a mass scale for the diagnosis of SLRSV in olive. This virus should be considered an important pathogen in olive trees, even if its distribution in the field seems to be limited to well defined areas.

Simultaneous and co-operational amplification (Co-PCR): a new concept for detection of plant viruses

A new and highly sensitive method for the amplification of viral RNA targets from plant material has been developed and patented. This technique called Co-operational amplification (Co-PCR) can be carried out easily in a simple tetraprimer reaction based on the simultaneous action of four primers. The reaction process consists of the simultaneous reverse transcription of two different fragments from the same target, one containing the other; the production of four amplicons by the combination of the two pair of primers, one pair external to other; and the co-operational action of amplicons for the production of the largest fragment. The technique was used successfully, both in metal block and capillary air thermal cyclers for the detection of plant RNA viruses ( Cherry leaf roll virus, Strawberry latent ringspot virus, Cucumber mosaic virus, Plum pox virus and Citrus tristeza virus). The sensitivity observed is at least 100 times higher than that achieved with RT-PCR and similar to nested RT-PCR. Colorimetric detection was coupled with this methodology facilitating its introduction for routine indexing programs and for phytosanitary selection of virus-free plant material.

DEVELOPMENT OF A NESTED PCR PROTOCOL FOR DETECTION OF OLIVE-INFECTING VIRUSES IN CRUDE EXTRACTS

A reverse transcription nested polymerase chain reaction (RT-n-PCR) was developed and applied to the detection of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Olive latent virus-1 (OLV-1), Olive latent virus-2 (OLV-2), Olive latent ringspot virus (OLRSV) and Strawberry latent ringspot virus (SLRV) in naturally infected olive cuttings. The method was more sensitive and reliable than conventional RT-PCR. This increased sensitivity allowed virus detection in samples that were negative to the first round of RT-PCR amplification. Coupled with simple sample preparation from cortical scrapings of olive cuttings, this protocol should be useful in routine testing for olive viruses.

Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees

A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses ( Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3′ digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that ∼1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.

DETECTION OF NEMATODE–TRANSMITTED NEPOVIRUSES BY THE NOVEL, ONE-TUBE AMPLIDET RNA ASSAY

ISHS XVIII International Symposium on Virus and Virus-like Diseases of Temperate Fruit Crops - Top Fruit Diseases DETECTION OF NEMATODE–TRANSMITTED NEPOVIRUSES BY THE NOVEL, ONE-TUBE AMPLIDET RNA ASSAY

CERTIFICATION SCHEMES

CERTIFICATION SCHEMES

Nucleotide sequence of the coat protein genes of citrus mosaic virus

The sequence of the 3′-terminal 2201 nucleotides of RNA2 of citrus mosaic virus (CiMV) was determined. The sequence contains a long open reading frame (ORF) of 1989 nucleotides in the virus-sense strand and a 3′ untranslated region of 212 nucleotides upstream of a poly(A) tail. The N-terminal amino acid sequence of the two coat proteins was determined by Edman degradation and the corresponding coding regions were identified in the polyprotein. The larger coat protein with M r 48 122 is encoded upstream of the smaller one with M r 24 172. The coat proteins are apparently cleaved from the polyprotein at an Arg-Gly and a Thr-Asn bond. Although CiMV has properties in common with the comoviruses and nepoviruses, there is no significant sequence homology between the coat proteins of CiMV and those of either group. Furthermore, the coat proteins of CiMV lack homology with those of strawberry latent ringspot virus (SLRSV), which has been reported to be more distantly related to the como- and nepoviruses. This lack of homology distinguishes CiMV from the como- and nepoviruses, SLRSV and other viruses.

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG ‘initiation’ codon sequences added at the 5′-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5′ S/G site and a TAG ‘stop’ codon sequence added at the 3′-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.

Strawberry latent ringspot virus in lilies

Strawberry latent ringspot virus was detected in the oriental lily hybrid ‘Stargazer’ grown in Israel from bulbs imported from Europe. Virus infection was associated with asymmetrical opening of flowers. Virus identification was based on electron microscopy, host range and serology.

Sequence analysis and location of capsid proteins within RNA 2 of strawberry latent ringspot virus

The nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98.8K. The N-terminal amino acid sequences of virion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97% identical to that of SLRSV-H. Except in the 3' and 5' terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.

Nucleotide sequence of the coat protein genes of strawberry latent ringspot virus: lack of homology to the nepoviruses and comoviruses.

The sequence of the 3'-terminal 2424 nucleotides of RNA-2 of the flowering cherry strain of strawberry latent ringspot virus (SLRV) was determined from cDNA clones. The sequence contains a reading frame in the virus-sense strand of 2070 nucleotides, a 3' untranslated region of 552 nucleotides and a 3'-terminal poly(A) tract. The positions of the two coat proteins of SLRV within the reading frame were determined from sequence data obtained by N-terminal sequencing using Edman degradation. The larger coat protein with an M(r) of 43K is located 5' of the smaller coat protein of 27K, and the two proteins are apparently cleaved at a Ser-Gly bond. Although there are numerous similarities between SLRV and the nepoviruses and comoviruses, there is no significant homology between the SLRV coat proteins and the coat proteins of either group. Furthermore, the hydropathy profiles of the SLRV coat proteins are unlike those of either group. No comparisons could be made with the fabaviruses owing to lack of sequencing information. This lack of homology suggests that SLRV is more distantly related to the nepoviruses and comoviruses than has been considered previously.

A New Host Record: Strawberry Latent Ringspot Virus Isolated From Flowering Cherry.

Strawberry latent ringspot virus (SLRV) was isolated from flowering cherry (Prunus serrulata sensu lato) trees in close proximity to each other in Auckland, New Zealand. The virus was not isolated from any of 390 flowering cherry trees tested from four other regions in the North Island. The virus was identified by host range, particle morphology, RNA and protein content and by serology. This is the first record of SLRV in flowering cherry.

Sap‐transmissible viruses in flowering cherry in New Zealand

Abstract Six sap‐transmitted viruses were identified during a study of 434 flowering cherries in the North Island of New Zealand. These included Prunus necrotic ringspot ilarvirus (PNRSV), apple mosaic ilarvirus (ApMV), flowering cherry virus B (FCVB), strawberry latent ringspot virus (SLRV), prune dwarf ilarvirus (PDV), and flowering cherry virus I (FCVI). Of these, ApMV, FCVB, SLRV, and FCVI were new records for this host. FCVB and FCVI appear to be newly described viruses with characteristics of members of the bromovirus and ilarvirus groups respectively. The most common virus was PNRSV (30.6%); the other viruses ranged in incidence from 10.2% (FCVB) to 0.5% (PDV). A further 43 virus isolates were categorised into nine classes based on symptoms in Chenopodium quinoa and cucumber, and were not further characterised in this study. Repeated sampling of 30 flowering cherry trees during late winter and early spring showed that the enzyme‐linked immunosorbent assay (ELISA) was more sensitive for detecting PNRSV infection of flowering cherries than sap transmission, but that neither method was absolutely reliable.

The nucleotide sequence of a satellite RNA associated with strawberry latent ringspot virus

The nucleotide sequence of a satellite RNA associated with a strawberry isolate (H) of strawberry latent ringspot nepovirus (SLRSV) was determined from cDNA copies and the 5' end sequence was deduced from directly sequenced virion RNA. At the 3' end a poly(A) sequence was identified. A long open reading frame encoding a polypeptide of 331 amino acids (M(r) 36488) was determined. Sequence comparisons showed that SLRSV satellite RNA has no extensive homology with other sequences in the GenEmbl and Swiss-Prot databases.

Virusbefall in wildbeständen des Schwarzen Holunders (Sambucus nigra L.)

Summary During a period of five years, numerous elder ( Sambucus nigra L.) shrubs wild-growing in different regions of Brandenburg, Sachsen-Anhalt and Saxony countries were surveyed for the presence of sap-transmissible viruses in order to evaluate their importance as natural virus sources. Out of altogether 1.049 shrubs tested, 192 were found to be virus-infested. Among these, 80 shrubs were infected by cherry leaf roll virus, 67 shrubs by a virus resembling elderberry carlavirus, 28 shrubs by cherry leaf roll virus in mixture with tomato black ring virus, 12 shrubs by tomato black ring virus solely, 2 shrubs by arabis mosaic virus, 2 shrubs by cucumber mosaic virus, and one shrub by tomato bushy stunt virus. Strawberry latent ringspot virus was not detected in elder shrubs tested. It could be clearly demonstrated that in regions with light soils nepoviruses were dominating, whereas in regions with loess soil and high aphid populations aphid-transmissible viruses such as the virus resembling elderberry carlavirus or cucumber mosaic virus were prevailing.

Wildgehölze aus der familie Rosaceae als natürliche reservoire von viren des kern- und steinobstes

Summary Wild-growing woody plants of species taxonomically related to cultivated pome and stone fruits were surveyed for the presence of fruit tree viruses in order to determine their importance as natural virus sources. Among wild-growing roses ( Rosa canina L., R. corymbifera Borckh. , R. elliptica Tausch , R. rubiginosa L.) no plants investigated were infected by ilarviruses (Prunus necrotic ringspot virus, prune dwarf virus) or nepoviruses (especially Arabis mosaic virus). Wild-growing apple trees ( Malus sylvestris Mill .) proved to be not infected by closteroviruses (apple chlorotic leaf spot virus) or by the apple Spy decline agent. In wild-growing mountain ash ( Sorbus aucuparia L. em. Hedl .) neither closteroviruses (apple chlorotic leaf spot virus, apple stem grooving virus) nor ilarviruses (apple mosaic virus) were detectable, although 7 % of the trees investigated were infested by the mountain ash ringspot disease. Mazzard cherries ( Prunus avium (L.) L. var. avium ) were free from nepoviruses (Arabis mosaic virus, cherry leaf roll virus, raspberry ringspot virus, strawberry latent ringspot virus, tomato black ring virus), but tomato bushy stunt virus was present in this species with 1.2%. Wild-growing European dwarf cherry ( Prunus fruticosa Pall .) proved to be free from ilarviruses (Prunus necrotic ringspot virus, prune dwarf virus), while escaped sour cherry shrubs ( Prunus cerasus L.) were shown to contain Prunus necrotic ringspot virus at a rate of 40%. Wild-growing or escaped rose, apple, mountain ash, mazzard cherry or European dwarf cherry plants therefore seem not to be dangerous virus sources for cultivated apples and cherries, because of lack of virus infestation. The main danger of virus spread seems to originate from cultivated pome and stone fruit trees itself.

Purification and properties of lucerne Australian symptomless virus, a new virus infecting lucerne in Australia

SUMMARYA virus with isometric particles c. 26–28 nm in diameter isolated from naturally infected lucerne (Medicago sativa) in Australia and reported there to be a strain of lucerne Australian latent virus (LALV), is shown to be a distinct virus. The virus, called lucerne Australian symptomless (LASV), was mechanically transmitted to 10 of 22 plant species inoculated, but only induced symptoms in three Chenopodium species and Gomphrena globosa. Virus particles occurred in relatively low concentrations in plant sap, and the virus could not be reliably maintained in culture by serial transmission to plants during winter (October‐April). During the summer, sap of infected C. quinoa remained infective after diluting 10‐2 but not 10‐3, after heating for 10 min at 50 but not 55 oC and after storage for 24 days (the longest period tested) at 20, 4 and ‐15 oC. LASV was seed‐borne to 6% of C. quinoa seedlings. Partially purified preparations of virus particles contained one nucleoprotein component with a sedimentation coefficient of c. BOS. Particles contained two polypeptide species of estimated mol. wts 26 000 and 40 000, and two ssRNA species which, when denatured in glyoxal, had apparent mol. wts of 2–5 times 106 and 1–4 times 106. The infectivity of virus RNA was abolished by incubation with proteinase K. Purified particles of LASV reacted with homologous antiserum (gel diffusion titre 1:256) but not with antiserum to LALV or to 13 other plant viruses with isometric particles including arracacha B (AVB), broad bean wilt, rubus Chinese seed‐borne (RCSV) and strawberry latent ringspot (SLRV) viruses, and five comoviruses.These properties distinguish LASV from LALV and from all recognised nepoviruses and comoviruses. Its closest affinities are with SLRV, RCSV and possibly AVB; these viruses may comprise a distinct virus group or nepovirus subgroup.

Strawberry Latent Ringspot Virus Associated with a New Disease of Olive in Central Italy

Strawberry Latent Ringspot Virus Associated with a New Disease of Olive in Central Italy

Host range, purification and some properties of rubus Chinese seed‐borne virus

SUMMARYA previously undescribed virus, for which the name rubus Chinese seed‐borne virus (RCSV) is proposed, was isolated from a single, symptomless plant of an unidentified Rubus species grown from seed collected in the wild in the People's Republic of China, Experimentally RCSV infected 23 out of 39 spp. in six out of eight families. The virus was seed‐transmitted in Chenopodium quinoa (100%) and Nicotiana bigelowii (27%). RCSV was not transmitted by the nematodes Xiphinema diversicaudatum or X. index.The particles of RCSV were isometric, c. 30 nm in diameter with some penetrated by negative stains. In thin sections particles were found in double walled tubular structures with an outer membrane enclosing one or more tubules. In crude extracts some particles were found within single‐walled tubules. Two virus‐associated bands were seen in sucrose density gradients of purified preparations. The upper band was not infective and consisted of penetrated particles apparently devoid of nucleic acid. The lower, infective band was resolved into two components, of density 1.452 and 1.461 g/ml, in caesium chloride isopycnic gradients. There were two polypeptides (mol. wts c. 47 000 and 25 200 daltons) and two nucleic acid species (one of mol. wt c. 1.4 × 106 daltons; the second was poorly defined by the methods used but was of higher molecular weight).RCSV was distantly related serologically (6–7 SDI) to the type isolate of strawberry latent ringspot virus (SLRV) and also reacted with antisera to serologicaly distinct grape and olive isolates of SLRV. It did not react with antisera to 10 other isometric viruses.