Mild Yellowing Virus Research Articles

Closely Related Poleroviruses Depend on Distinct Translation Initiation Factors to Infect Arabidopsis thaliana

In addition to being essential for translation of eukaryotic mRNA, translation initiation factors are also key components of plant-virus interactions. In order to address the involvement of these factors in the infectious cycle of poleroviruses (aphid-transmitted, phloem-limited viruses), the accumulation of three poleroviruses was followed in Arabidopsis thaliana mutant lines impaired in the synthesis of translation initiation factors in the eIF4E and eIF4G families. We found that efficient accumulation of Turnip yellows virus (TuYV) in A. thaliana relies on the presence of eIF (iso)4G1, whereas Beet mild yellowing virus (BMYV) and Beet western yellows virus-USA (BWYV-USA) rely, instead, on eIF4E1. A role for these factors in the infectious processes of TuYV and BMYV was confirmed by direct interaction in yeast between these specific factors and the 5' viral genome-linked protein of the related virus. Although the underlying molecular mechanism is still unknown, this study reveals a totally unforeseen situation in which closely related viruses belonging to the same genus use different translation initiation factors for efficient infection of A. thaliana.

Genetic Structure of the Aphid,Chaetosiphon fragaefolii, and Its Role as a Vector of theStrawberry yellow edge virusto a Native Strawberry,Fragaria chiloensisin Chile

The monoecious anholocyclical aphid, Chaetosiphon fragaefolii (Cockerell) (Homoptera: Aphididae), was collected on a native strawberry, Fragaria chiloensis (L.) Duchesne (Rosales: Rosaceae) from different sites in Chile. The presence of this aphid was recorded during two consecutive years. F. chiloensis plants were collected from seven natural and cultivated growing areas in central and southern Chile. Aphids were genotyped by cross-species amplification of four microsatellite loci from other aphid species. In addition, the aphid borne virus Strawberry mild yellow edge virus was confirmed in F. chiloensis plants by double-antibody sandwich ELISA and RT-PCR. Genetic variability and structure of the aphid populations was assessed from the geo-referenced individuals through AMOVA and a Bayesian assignment test. The presence of C. fragaefolii, during the two-year study was detected in only four of the seven sites (Curepto, Contulmo, Chilián and Cucao). Genetic variation among these populations reached 19% of the total variance. When assigning the individuals to groups, these were separated in three genetic clusters geographically disjunct. Of the seven sampled sites, six were positive for the virus by RT-PCR, and five by double-antibody sandwich ELISA . The incidence of the virus ranged from 0–100%. Presence of the virus corresponded with the presence of the aphid in all but two sites (Chilian and Vilches). The greatest incidence of Strawberry mild yellow edge virus was related to the abundance of aphids. On the other hand, sequences of the coat protein gene of the different virus samples did not show correspondence with either the genetic groups of the aphids or the sampling sites. The genetic structure of aphids could suggest that dispersal is mainly through human activities, and the spread to natural areas has not yet occurred on a great scale.

Occurrence of aphidborne viruses in southernmost South American populations of Fragaria chiloensis ssp. chiloensis

Wild and cultivated Fragaria chiloensis ssp. chiloensis (Fcc) plants were collected at different locations in southern Chile in order to determine the current viral status of this native strawberry. The following aphidborne viruses (ABVs): Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV), Strawberry crinkle virus (SCV) and Strawberry vein banding virus (SVBV), were found in wild and cultivated Fcc plants, but severe symptoms were not associated with viral infection. Furthermore, partial gene sequences of these ABV isolates were determined and displayed a high degree of conservation with virus isolates reported previously. In addition, partial gene sequences of SCV and SVBV from southernmost South American populations of Fcc are described for the first time. High‐throughput parallel sequencing (Illumina) of double‐stranded RNA was used to provide viral profiles of Fcc from different locations. Although strong evidence of novel viruses affecting Fcc was not found, it was confirmed that ABVs are the most frequent viruses affecting this subspecies. The information provided will help in the development of high‐quality molecular tools for virus detection and control in Fcc.

First Report of Strawberry necrotic shock virus in China.

Strawberry necrotic shock virus (SNSV) is an economically important viral pathogen that infects Fragaria and Rubus spp. SNSV was first identified in the 1950s and early studies indicated that SNSV was a strain of Tobacco streak virus (TSV). Recently, it was shown that SNSV was a distinct virus based on molecular characterization (2). Currently, SNSV is a tentative member of the Ilarvirus genus in the Bromoviridae family. In 2008, a small sampling survey for SNSV was done in Heilongjiang Province of China, and 15 strawberry samples were collected from symptomless strawberries in a home garden that had more than 5 years of strawberry cultivation history. Total nucleic acid was extracted from strawberry leaflets by modified cetyltrimethylammoniumbromide methods (3). Reverse transcription (RT)-PCR was operated with the published primer pair CPbeg F/CPend R (2). Amplified DNA fragments with the predicted size were obtained only in one strawberry sample, which was further cloned and sequenced. The sequence (GenBank Accession No. HQ830017) was closely related and highly homologous (89.7 to 98.5% identity) to that of viral isolates (GenBank Accession Nos. AY363228-AY363242) from Fragaria and Rubus spp. Phylogenetic analysis based on nucleotide sequence of the coat protein gene was done with the neighbor-joining method of MEGA 4.0 software. The result showed that all the isolates of SNSV fell into two distinct clades. The Chinese isolate formed one small clade with Japanese isolate 1291. The isolate was also transmitted to Chenopodium quinoa by mechanical inoculation in the greenhouse, and the symptom of chlorotic mottling could be found in C. quinoa and detected by RT-PCR. To determine whether the sample was infected by other strawberry viruses, RT-PCR assays with the published primer pairs SVBVdeta/SVBVdetb, SMoVdeta/SMoVdetb, and SMYEVdeta/SMYEVdetb were also performed for detection of Strawberry vein banding virus, Strawberry mottle virus, and Strawberry mild yellow edge virus using total nucleic acid extracted from the SNSV-positive sample as a template (1). The result indicated that it had been also infected by Strawberry mild yellow edge virus, although no visible symptoms were observed. To our knowledge, this is the first report of SNSV in strawberry in China. Additional work is needed to elucidate the biological characterization and significance of the finding.

Strawberry mild yellow edge potexvirus from Strawberry in Korea

Symptoms induced in the leaves of strawberry (Fragaria x ananassa Duch.), 'Seolhyang' and 'Eyeberry', were mosaic, distortion and black colored edge on leaves at Nonsan area, one of the important production areas in Korea. Electron microscopy by quick-dip revealed the flexuous rod-shape particles having about 550-600 nm length. Cytoplasmic inclusion bodies composed of aggregated virus particles were observed frequently in mesophyll parenchyma and epidermal cells for the leaves of strawberry. The specific primers amplifying products of 635 bp and 729 bp were developed for RT-PCR detection of Strawberry mild yellow edge virus (SMYEV). Nucleotide identity of the CP gene of SMYEV was 92.8-99.2% with those of other SMYEV isolates from Gen-Bank database.

Detection and discrimination of members of the family Luteoviridae by real-time PCR and SYBR ® GreenER™ melting curve analysis

This study investigated the suitability of a two step real-time RT-PCR melting curve analysis as a tool for the detection and discrimination of nine species in the plant virus family Luteoviridae, being Soybean dwarf virus [SbDV], Bean leafroll virus [BLRV], Beet chlorosis virus [BChV], Beet mild yellowing virus [BMYV], Beet western yellows virus [BWYV], Cereal yellow dwarf virus-RPV [CYDV-RPV], Cucurbit aphid-borne yellows virus [CABYV], Potato leafroll virus [PLRV] and Turnip yellows virus [TuYV]. Melting temperature and shape of the melting peak were analysed for 68 bp and 148 bp coat protein gene amplicons using SYBR ® GreenER™ fluorescent dye. Specific melting peaks with unique melting temperature were observed for the various species of the family Luteoviridae using the 68 bp amplicon, but not with the 148 bp amplicon. Due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of the species CYDV-RPV and TuYV. Nevertheless, discrimination between species was achieved for SbDV, BLRV, BChV, BMYV, CABYV and either PLRV or BWYV. Melting curve analysis, in this study, is a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection.

A generic RT‐PCR assay for the detection of Luteoviridae

This study, using RT‐PCR, is the first comprehensive assessment since 1991 of a generic detection method for the Luteoviridae. Thirteen Luteoviridae species were detected using three separate sets of low‐degeneracy generic primers with RT‐PCR to amplify 68‐, 75‐ and 129/156‐bp regions of the Luteoviridae coat‐protein gene. Species detected include all members of the genus Luteovirus [Barley yellow dwarf virus (BYDV)‐PAV, BYDV‐PAS, BYDV‐MAV (129 and/or 156 bp amplicons), Soybean dwarf virus, Bean leafroll virus (68 bp amplicon)] and eight of nine species from the genus Polerovirus [Beet western yellows virus, Beet chlorosis virus, Beet mild yellowing virus, Turnip yellows virus, Potato leafroll virus, Cucurbit aphid‐borne yellows virus, Cereal yellow dwarf virus‐RPV (68‐bp amplicon) and Sugarcane yellow leaf virus (75‐bp amplicon)]. These primers were not able to detect Carrot red leaf virus, Sweet potato leaf speckling virus (both belong to unassigned Luteoviridae) and Pea enation mosaic virus‐1 (genus Enamovirus). A synthetic positive control containing all primer sequence priming sites was designed to facilitate this method as a generic tool for use with a variety of host plants. The Luteoviridae primers described in this study present a simple infection‐detection tool of benefit to biosecurity authorities in nursery‐stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as‐yet undiscovered species within the Luteovirus and Polerovirus genera.

P0 proteins of European beet-infecting poleroviruses display variable RNA silencing suppression activity

Post-transcriptional gene silencing (PTGS), or RNA silencing, is one of the key mechanisms of antiviral defence used by plants. To counter this defence response, viruses produce suppressor proteins that are able to inhibit the PTGS pathway or to interfere with some of its function. The aim of this study was to evaluate the RNA silencing suppressor (RSS) activity of P0 proteins from selected European isolates of the beet-infecting poleroviruses beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) using two different experimental systems: (i) agro-infiltration of Nicotiana benthamiana green fluorescent protein-positive plants and (ii) mechanical inoculation of Chenopodium quinoa using a beet necrotic yellow vein virus (BNYVV, genus Benyvirus) RNA3-based replicon. The results demonstrated that P0 of most BMYV isolates exhibited RSS activity, although at various efficiencies among isolates. Conversely, P0 of BChV isolates displayed no RSS activity in either of the two systems under the experimental conditions used. These results are the first reported evidence that P0 proteins of two closely related beet poleroviruses show strain-specific differences in their effects on RNA silencing.

FIRST REPORT OF STRAWBERRY MILD YELLOW EDGE VIRUS IN ARGENTINA

Numerous viruses have been mentioned infecting strawberry in a single or mixed infection. Many of them do not induce distinct symptoms in commercial cultivars, and often the only signs of infection are loss of vigor and reduced yield. Strawberry mild yellow edge virus (SMYEV) is one of the most widespread virus diseases of cultivated strawberry. Most of the grown cultivars are tolerant to infection with this virus and they do not show symptoms. However, infections by severe strains and mixed infection with other viruses lead to decline and to the development of virus symptoms. In strawberry production areas in Tucuman province, Argentina, strawberry plants were detected with dwarfism, stocky plants, mosaic, mottle, and leaf distortion. Plants were serologically tested for SMYEV by ELISA (Agdia kit). Of 21 symptomatic plants analyzed, 8 were positive (38%). Simultaneously, 454 symptomless plants were tested and 12 of them were found positive (2.6%). ELISA positive plants were further analyzed by immunocapture reverse transcription seminested polymerase chain reaction (IC-RT-seminested PCR) with specific primers for SMYEV. IC was performed with inmunoglobuline from Agdia kits. RT-seminested PCR reaction was done using three specific primers A, B, and C. Using this technique a fragment of the expected size was amplified. After graft inoculation of Fragaria vesca and F. virginiana indicator clones, necrosis, mosaic, epinasty, chlorosis, and distorted leaves were observed, along with other symptoms. Further research is being conducted to identify other possible viruses infecting this crop.

DETECTION OF STRAWBERRY VIRUSES IN EGYPT

As part of a USAID-MERC funded project, 'Disease-indexing and mass propagation of superior strawberry cultivars', an effort was made to evaluate the virus status of strawberries in Egypt. Diagnostic reverse transcription-polymerase chain reaction (RT-PCR) tests for Strawberry mottle virus, Strawberry crinkle virus, Strawberry vein banding virus (SVBV), Strawberry mild yellow edge virus (SMYEV), Strawberry chlorotic fleck virus, Strawberry necrotic shock virus, Strawberry latent ringspot virus, Apple mosaic virus, Fragaria chiloensis latent virus, Strawberry pallidosis associated virus (SPaV) and Beet pseudo yellows virus (BPYV) were developed and/or evaluated at the USDA-ARS laboratory in Corvallis, Oregon. Positive controls for the testing consisted of dried leaf samples of strawberries infected with each of the above viruses, shipped to Egypt and Israel under import permits. Collection of strawberry test samples was done in production fields (cultivars unknown) and from nuclear stock plants grown in Egypt ('Tamar' and 'Yael' originating from Israel). RNA extraction was carried out using Qiagen kits or a previously described protocol. Reverse transcription and polymerase chain reactions were carried as per manufacturer's recommendations. Amplicons were visualized after separation on an agarose gel and staining with ethidium bromide. The RT-PCR detection of viruses from RNA extracted from positive controls that were vacuum dried was successful for several but not all of the viruses used in this study. Thus, extracted RNA that can be shipped under ethanol may be a better positive control that dried tissue samples. Material carried back to the USA from Egypt as extracted RNA under ethanol was not impacted by storage at room temperature during transport. Initial testing of strawberry material from fields in Egypt showed that SMYEV, SVBV, BPYV and SPaV were detected in field samples. The plants tested from the nuclear stocks tested negative for each of the viruses tested.

DIAGNOSIS AND MOLECULAR ANALYSIS OF STRAWBERRY VIRUSES IN CHINA

Losses of fruit yield and runner production in commercial strawberries due to infection by viruses are high. In this paper, the system of detection of strawberry viruses by PCR was studied, sequences of Chinese isolates were analyzed, and the virus elimination methods were compared and evaluated with detection of viruses by PCR in strawberry plants. Total nucleic acid extracted from strawberry leaves by a modified CTAB method is suitable as a template for virus detection by RT-PCR. The method of detection of strawberry RNA and DNA viruses by multiplex RT-PCR using total nucleic acid as a template was established. Both Strawberry mottle virus (SMoV) and Strawberry mild yellow edge virus (SMYEV) were eliminated effectively from strawberry plants by culture of 0.2 mm or 0.5 mm length of shoot tip, while the application of thermotherapy to in vitro strawberry plants only eliminated SMoV. The 930 bp segment in 3' terminal region of SMYEV genome and the 574 bp segment located in the region of coat protein gene of SVBV were amplified respectively. The results of sequence analysis indicate that there are the special isolates of SMYEV and Strawberry vein banding virus (SVBV) in China.

Aphid transmissibility of different European beet polerovirus isolates

Different field isolates of the ‘beet poleroviruses’ Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV) (genus Polerovirus, family Luteoviridae) collected in France and Poland were evaluated for transmissibility from and to sugar beet plants by different aphid species. In general, both BMYV and BChV were efficiently transmitted by Myzus persicae and by a French clone of Macrosiphum euphorbiae. In contrast, transmissibility of the two poleroviruses by an English clone of M. euphorbiae was evidently weaker, although the aphid samples contained the virus as demonstrated by RT-PCR. None of the BMYV or BChV isolates was transmitted by Aphis fabae or Myzus ascalonicus. In attempting to correlate biological properties with molecular variations, the RT proteins were sequenced. Some amino acid point variations, presumably affecting aphid transmissibility, were identified.

Potentilla reptansis an Alternative Host for Two Strawberry Viruses

Strawberry [Fragaria x ananassa Duchesne = Potentilla x ananassa (Rozier) Mabb] has accumulated a variety of viruses in its relatively short history. Strawberry crinkle virus and Strawberry mild yellow edge virus are transmitted obligately by the aphid Chaetosiphon fragaefolii (Cockerell), which also occasionally infests other species of Potentilla. Several wild strawberry species have been shown previously to be hosts of the viruses. Leaves infected with these strawberry viruses were grafted into plants of UC-5, an indicator strawberry cultivar, and then UC-5 leaves were grafted into Potentilla reptans. Leaves from P. reptans were subsequently grafted back to uninfected UC-5 plants to demonstrate their potential as reservoirs for the virus. UC-5, Fragaria vesca and P. reptans plants were indexed for strawberry viruses by end-point reverse transcriptase PCR subsequent to back grafting.

우리나라에서 발생하는 딸기 바이러스병(2007-2008)

Virus disease surveys of strawberries cultivated and preserved as germplasm resources in Korea was conducted during 2007-2008. Virus detection was conducted by RT-PCR using total RNAs extracted from strawberry samples. We detected the infection with Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV), Strawberry vein banding virus (SVBV) and Strawberry pallidosis associated virus (SPaV) while no infection with Strawberry crinkle virus (SCV), Strawberry necrotic shock virus (SNSV), Strawberry latent ring spot virus (SLRSV) and Arabis mosaic virus (ArMV) was observed. The infection rate of virus disease on 4 cultivars including Seolhyang, Maehyang, Gumhyang, and Dahong, bred in Korea, was 0.1, 1.9, 0, and 0%, respectively. Surprisingly, however, cultivar Red Peal introduced from Japan in 1997 revealed 48.3% virus infection rate. SMYEV, SMoV and SPaV were also identified in strawberries growing in the farm fields of Korea. In the field, however, SMYEV was the most predominant virus (97.4%) among those 3 identified viruses. SVBV was detected only in strawberry kept as a germplasm.

Certification scheme for strawberry

Certification scheme for strawberry

Beet mild yellowing virus resistance derived from wild and cultivated Beta germplasm

AbstractSugar beet (Beta vulgaris ssp. vulgaris L.) has a relatively narrow genetic base and several programmes have sought to evaluate the potential for introducing novel traits from wild and cultivated Beta germplasm into the crop. In particular, resistance to important sugar beet diseases has been identified within individual Beta accessions. We report here the successful transfer of resistance to Beet mild yellowing virus (BMYV) from garden beet, fodder beet and leaf beet accessions to progeny populations in initial crosses with sugar beet. Twelve plant populations derived from different Beta accessions were inoculated with viruliferous aphids carrying BMYV and the virus content of individual plants subsequently quantified by an ELISA test. Seven populations were significantly more resistant than a control sugar beet cultivar (P ≤ 0.05). BMYV resistance was successfully inherited in BC1 and BC2 generations, suggesting that resistance could potentially be introgressed from these sources into elite sugar beet lines.

Biological and Molecular Characterization of an American Sugar Beet-Infecting Beet western yellows virus Isolate.

Three aphid-transmitted viruses belonging to the Polerovirus genus, Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV), and Beet western yellows virus (BWYV), have been described as pathogens of sugar beet. We present the complete biological, serological, and molecular characterization of an American isolate of Beet western yellows virus (BWYV-USA), collected from yellow beet leaves. The biological data suggested that BWYV-USA displayed a host range similar to that of BMYV, but distinct from those of BChV and the lettuce and rape isolates of Turnip yellows virus. The complete genomic RNA sequence of BWYV-USA showed a genetic organization and expression typical of other Polerovirus members. Comparisons of deduced amino acid sequences showed that P0 and the putative replicase complex (P1-P2) of BWYV-USA are more closely related to Cucurbit aphid-borne yellows virus (CABYV) than to BMYV, whereas alignments of P3, P4, and P5 showed the highest homology with BMYV. Intraspecific and interspecific phylogenetic analyses have suggested that the BWYV-USA genome may be the result of recombination events between a CABYV-like ancestor contributing open reading frame (ORF) 0, ORF 1, and ORF 2, and a beet Polerovirus progenitor providing the 3' ORFs, with a similar mechanism of speciation occurring for BMYV in Europe. Results demonstrate that BWYV-USA is a distinct species in the Polerovirus genus, clarifying the nomenclature of this important group of viruses.

Development of a multiplex immunocapture-RT-PCR for simultaneous detection of BMYV and BChV in plants and single aphids

A multiplex immunocapture-reverse transcription-polymerase chain reaction protocol (mIC-RT-PCR) was successfully developed to improve the detection of Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV) in plants and aphids in single and mixed infections. Viral particles from plant and aphid extracts were enriched by antibody-capture and lysed by heating to release the viral RNA. During the RT-PCR step, 5′ end sequences specific to each virus were amplified and the products analysed by gel electrophoresis; the PCR products corresponding to BMYV and BChV were 440 and 348 bp respectively. The test was evaluated on single aphids carrying BMYV, BChV or both viruses and the results demonstrated that the mIC-RT-PCR is specific and sensitive. Its sensitivity was found to be 10 5 times higher than the TAS-ELISA routinely used for detecting BMYV and BChV and 10 4 times better than RT-PCR when both viruses were present. Eliminating the antibody-capture step to simplify the technique did not affect the sensitivity of the test and a procedure using microtitre plates was developed to allow simultaneous processing of large numbers of samples.

Detection of Strawberry RNA and DNA Viruses by RT‐PCR Using Total Nucleic Acid as a Template

Abstract Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV), three of strawberry viruses can cause a severe loss in commercial strawberry worldwide. In this paper, total nucleic acid from strawberry leaves was compared with total RNA as the template for detection of the above viruses both by single and multiplex reverse transcription polymerase chain reaction (RT‐PCR). Total nucleic acid extract with the modified CTAB method and total RNA extracts with both the modified RNeasy method and the modified CTAB method examined by electrophoresis resulted in a high quality ribosomal 28S and 18S RNA when loaded on an agarose gel. When total RNA and total nucleic acid were used as templates, detection results of SMYEV and SMoV were identical and the amplified band intensity was nearly the same whether by single or duplex RT‐PCR. Using total nucleic acid as the template, the detection results of SMYEV and SMoV by multiplex RT‐PCR were in a good concord with that amplified by single and duplex RT‐PCR, and for SVBV this was similar to the result of detection that amplified by direct PCR using pure DNA as the template. This study demonstrates that total nucleic acid extracted from strawberry leaves by a modified CTAB method is suitable as a template for virus detection by RT‐PCR. This makes investigation of the epidemiology of strawberry viruses much easier.

Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.