Beet Chlorosis Virus Research Articles

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.

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.

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.

First Report on the Natural Occurrence of Beet chlorosis virus in Poland.

Yellowing symptoms on sugar beet (Beta vulgaris L.) are caused by several viruses, especially those belonging to the genus Polerovirus of the family Luteoviridae, including Beet mild yellowing virus (BMYV) and Beet western yellows virus (BWYV), and recently, a new species, Beet chlorosis virus (BChV), was reported (2). To identify Polerovirus species occurring in beet crops in Poland and determine their molecular variability, field surveys were performed in the summer and autumn of 2005. Leaves from symptomatic beet plants were collected at 26 localities in the main commercial sugar-beet-growing areas in Poland that included the Bydgoszcz, Kutno, Lublin, Poznań, Olsztyn, and Warszawa regions. Enzyme-linked immunosorbent assay (ELISA) tests (Loewe Biochemica GmbH, Sauerlach, Germany) detected poleroviruses in 23 of 160 samples (approximately 20 samples from each field). Multiplex reverse-transcription polymerase chain reaction (RT-PCR) (1) (GE Healthcare S.A.-Amersham Velizy, France) confirmed the presence of poleroviruses in 13 of 23 samples. Nine of twenty sugar beet plants gave positive reactions with BChV-specific primers and three with primers specific to the BMYV P0 protein. Two isolates reacted only with primer sets CP+/CP, sequences that are highly conserved for all beet poleroviruses. Leaf samples collected from three plants infected with BChV were used as inoculum sources for Myzus persicae in transmission tests to suitable indicator plants including sugar beet, red beet (Beta vulgaris L. var. conditiva Alef.), and Chenopodium capitatum. All C. capitatum and beet plants were successfully infected with BChV after a 48-h acquisition access period and an inoculation access period of 3 days. Transmission was confirmed by the presence of characteristic symptoms and by ELISA. Amino acid sequences obtained from each of four purified (QIAquick PCR Purification kit, Qiagen S.A., Courtaboeuf, France) RT-PCR products (550 and 750 bp for CP and P0, respectively) were 100% identical with the CP region (GenBank Accession No. AAF89621) and 98% identical with the P0 region (GenBank Accession No. NP114360) of the French isolate of BChV. To our knowledge, this is the first report of BChV in Poland.

Distribution and properties of geographically distinct isolates of sugar beet yellowing viruses

From a total of 261 yellow sugarbeet leaves collected from 10 countries representing three continents, the incidence and distribution of strains of Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) and Beet yellows virus (BYV) were analysed using serological and molecular methods. BMYV was found in all countries except Greece, and more frequently in the northern and western areas of Europe, whereas BYV predominated in Turkey, Spain, Greece, the USA and Chile. BChV, originally found in the USA and the UK in 1989, was identified in France, Spain, the Netherlands and Chile. Nine sugar beet poleroviruses, plus a reference isolate of Turnip yellows virus (TuYV, syn. Beet western yellows virus), were further characterized and compared. Isolates obtained from sugar beet infected this species, but not oilseed rape or lettuce; all isolates except one infected Capsella bursa‐pastoris. The coat‐protein sequences of these isolates were highly similar, with the consensus sequence representing 89% of nucleotide residues. Within the coat‐protein gene, two regions were identified that could represent specific epitopes to which monoclonal antibody BYDV‐PAV‐IL‐1 could bind; this antibody is used to distinguish beet poleroviruses in ELISA. Comparison of the sequences at the 5′ end showed that sequence homology existed only between isolates with the same host range. The first sequence data of polerovirus isolates from Chile are presented, showing that the coat protein and the 5′ end of their genomes are highly similar to those of BMYV isolates found in Europe. Chilean polerovirus isolates may have been imported from the northern hemisphere in sugar beet breeding material.

Beet poleroviruses: close friends or distant relatives?

SUMMARY Taxonomy: There are three members of the genus Polerovirus (family Luteoviridae) that induce yellowing of sugar beet: Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) and Beet western yellows virus-USA (BWYV-USA, Fig. 1). Non-beet-infecting isolates of BWYV found particularly within Europe have now been re-named Turnip yellows virus (TuYV). Species-specific antibodies are unavailable, but the viruses can be distinguished by RT-PCR using primers specifically designed to the 5' end of their respective genomes. Physical properties: The isometric virus particles are approximately 26 nm in diameter and the genome consists of a single strand of positive sense RNA that utilizes almost all known plant virus gene expression strategies (initiation bypass, translational frameshifting and readthrough, synthesis of subgenomic RNA and proteolytic processing). Many members of the Chenopodiaceae are susceptible, including commercial crops of sugar beet (Beta vulgaris), red beet and spinach. Experimental hosts include Montia perfoliata, Nicotiana benthamiana and Arabidopsis thaliana. Sugar beet infected with beet poleroviruses show patches of chlorosis on the older leaves 4-6 weeks post-infection; these areas expand until the whole leaf becomes yellow and older leaves then tend to thicken and become brittle. Beet poleroviruses are transmitted in a persistent (circulative, non-propagative) manner by several different aphid species, Myzus persicae being the most important vector.

Decision making in controlling virus yellows of sugar beet in the UK

Virus yellows is an important disease affecting yield in sugar beet in the UK. Myzus persicae (Sulzer) is the most effective and efficient aphid vector of the three viruses causing the disease: beet yellows virus, beet mild yellowing virus and beet chlorosis virus. Control of virus yellows disease is thus focused on the study and control of this aphid species. UK national surveys of virus yellows began in 1946 and these data helped to formulate disease forecasting schemes to optimise control. Over the years, in addition to improvements in farm hygiene, periodic changes and developments in control of the disease have occurred. To accommodate these important developments, virus yellows forecasting schemes have evolved accordingly. The most recent version has been adapted to take account of the current widespread use of imidacloprid seed treatment. Its application offers potential to optimise the rational use of aphicides such as imidacloprid so as to benefit beet growers and the environment by reducing prophylactic use of seed treatment.

The effects of Beet mild yellowing virus and Beet chlorosis virus on the yield of UK field‐grown sugar beet in 1997,1999 and 2000

SummaryThe separate effects of the aphid‐transmitted poleroviruses; Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV), on the yield of field‐grown sugar beet were studied following different inoculation dates from May to July in 1997,1999 and 2000. Each sugar beet plant within the appropriate plots was infected with virus using at least 10 wingless viruliferous Myzus persicae per plant. In all 3 years, overall yield losses caused by BMYV were negatively correlated with time of infection with early season (May) inoculations causing 18–27% losses in sugar yield but late season losses only 4–15%. BChV decreased the sugar yield and sugar content of beet following early season inoculations, although the effects on sugar yield were more variable (range 8–24%) and the virus appeared to be less damaging compared to BMYV. However, inoculations with BChV in July of each year caused greater root and sugar losses than inoculations with BMYV at that time. Both poleroviruses increased the sodium content of the roots early in the season, although neither virus had an effect on potassium levels at any stage.

Improved detection and differentiation of poleroviruses infecting beet or rape by multiplex RT-PCR

Three distinct species of virus inducing yellowing of beet, Beet mild yellowing virus (BMYV), Brassica yellows virus (BrYV, synonym BWYV) and Beet chlorosis virus (BChV) have been characterised from the genus Polerovirus. Until recently, no available tools were available to allow accurate and reliable distinction of the three species. Based on previous nucleotide sequence alignments and phylogenetic studies, we show that the use of molecular methods enabled the discrimination of these three beet Polerovirus species, but with differences in efficiency and specificity. Primers CP+ and CP− encompassing ORF-3, which encodes the coat protein, allowed the amplification by RT-PCR of a fragment of 563 bp for all isolates. Molecular methods such as SSCP or RFLP were able to discriminate these fragments by utilizing the differences in sequence. However, SSCP is a highly sensitive technique and was not suitable for the distinction of the Polerovirus species, because all isolates tested displayed a unique pattern. Analysis of the ORF3 RT-PCR products, digested with SmaI, RsaI and AccI restriction enzymes revealed four distinct patterns specific for the three species. However, point mutations can alter the RFLP patterns, making the interpretation of the results difficult. Primers were designed to amplify specifically sequences corresponding to ORF-0 of the three viral species. By using the three new sets of ORF-0 specific primers and CP+/CP− primers in a single multiplex RT-PCR, the detection and discrimination of the three beet Polerovirus species was possible in infected plants. The multiplex RT-PCR method provides a reliable and highly sensitive method to detect and identify viral species and will be of great interest for epidemiological studies of beet poleroviruses.

Biological, Serological, and Molecular Variability Suggest Three Distinct Polerovirus Species Infecting Beet or Rape

Yellowing diseases of sugar beet can be caused by a range of strains classified as Beet mild yellowing virus (BMYV) or Beet western yellows virus (BWYV), both belonging to the genus Polerovirus of the family Luteoviridae. Host range, genomic, and serological studies have shown that isolates of these viruses can be grouped into three distinct species. Within these species, the coat protein amino acid sequences are highly conserved (more than 90% homology), whereas the P0 sequences (open reading frame, ORF 0) are variable (about 30% homology). Based on these results, we propose a new classification of BMYV and BWYV into three distinct species. Two of these species are presented for the first time and are not yet recognized by the International Committee on Taxonomy of Viruses. The first species, BMYV, infects sugar beet and Capsella bursa-pastoris. The second species, Brassica yellowing virus, does not infect beet, but infects a large number of plants belonging to the genus Brassica within the family Brassicaceae. The third species, Beet chlorosis virus, infects beet and Chenopodium capitatum, but not Capsella bursa-pastoris.