Barley Yellow Dwarf virus-PAV Research Articles

First Record of Barley yellow striate mosaic virus Affecting Wheat Summer-Nurseries in Syria.

A limited survey to identify virus diseases affecting wheat in summer nurseries in agricultural stations in southern Syria was conducted during October 2002. A total of 94 bread and durum wheat samples with symptoms suggestive of virus infection (stripping, stunting, and yellowing) were collected. All samples were tested for the presence of four viruses by tissue-blot immunoassay (2) at the Virology Laboratory of ICARDA, Aleppo, Syria using the following polyclonal antibodies: Barley stripe mosaic virus (BSMV); Barley yellow dwarf virus-PAV (BYDV-PAV) and Wheat streak mosaic virus (WSMV) from the Virology Laboratory at ICARDA; and Barley yellow striate mosaic virus (BYSMV) isolated from Italy (BYSMV-Italy) and provided by M. Conti, Instituto di Fitovirologia applicata, Turino, Italy. Serological results obtained indicated that BYSMV was the most commonly encountered virus (78.7%) followed by BYDV-PAV (22.3%), whereas, BSMV and WSMV were not detected in any of the samples tested. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, purified BYSMV preparations were observed to contain a 47-kDa structural protein typical of the N protein of Rhabdoviruses that reacted strongly with three BYSMV antisera (BYSMV-Italy, BYSMV-Lebanon [4], and BYSMV-Morocco [1]). Samples that reacted with BYSMV antisera were transmitted from wheat to wheat, barley, and oat plants by the planthopper Laodelphax striatella (Fallen) (Hemiptera: family Delphacidae) in a persistent manner, and the major symptoms of BYSMV on cereal crops were stripping and stunting. BYDV-PAV has been reported from Syria earlier (3) but to our knowledge, this is the first report of BYSMV affecting wheat in Syria.

Molecular diversity of the coat protein-encoding region of Barley yellow dwarf virus-PAV and Barley yellow dwarf virus-MAV from Latvia and Sweden.

The sequence variability of Barley yellow dwarf virus-PAV (PAV) and Barley yellow dwarf virus-MAV (MAV) was studied by comparing 502 nucleotides from the coat protein-encoding region of six isolates from Latvia and four from Sweden. The diversity within MAV was low (>97% sequence identity), also when compared to isolates from USA and China. In contrast, the variability among PAV isolates was greater and phylogenetic analysis including isolates of a wide geographic origin detected two major clusters, of which both contained isolates from Latvia and Sweden. A new distinct variant of BYDV-PAV was discovered in Latvia, and because of the sequence difference it is proposed to belong to a new species (BYDV-OYV).

Barley yellow dwarf virus and Cereal yellow dwarf virus Quantification by Real-Time Polymerase Chain Reaction in Resistant and Susceptible Plants

ABSTRACT Reliable detection and quantification of barley and cereal yellow dwarf viruses (YDVs) is a critical component in managing yellow dwarf diseases in small grain cereal crops. The method currently used is enzyme-linked immunosorbent assay (ELISA), using antisera against the coat proteins that are specific for each of the various YDVs. Recently, quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR) has been used to detect bacterial and viral pathogens and to study gene expression. We applied this technique to detect and quantify YDVs using primers specific for Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) coat protein genes because of the higher sensitivity of RT-PCR and the advantage of using a real-time PCR instrument. This Q-RT-PCR was used to detect BYDV and CYDV, and to examine disease development in a resistant wheatgrass, a resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV were detected as early as 2 and 6 h, respectively, in susceptible oat compared with detection by ELISA at 4 and 10 days postinoculation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV RNA in both oat and wheat, which may account for PAV being more prevalent and causing more severe viral disease than CYDV. Q-RT-PCR is reproducible, sensitive, and has the potential to be used for examining yellow dwarf disease and as a rapid diagnostic tool for YDVs.

Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real time RT-PCR

One of the major factors determining the incidence of Barley yellow dwarf virus (BYDV) on autumn-sown cereals is the viruliferous state of immigrant winged aphids. This variable is assessed routinely using the enzyme-linked immunosorbant assay (ELISA). However, the threshold for virus detection by ELISA can lead to false negative results for aphids carrying less than 10 6 particles. Although molecular detection techniques enabling the detection of lower virus quantities in samples are available, the relatively laborious sample preparation and data analysis have restricted their use in routine applications. A gel-free real-time one-step reverse transcription polymerase chain reaction (RT-PCR) protocol is described for specific detection and quantitation of BYDV-PAV, the most widespread BYDV species in Western Europe. This new assay, based on TaqMan® technology, detects and quantifies from 10 2 to 10 8 BYDV-PAV RNA copies. This test is 10 and 10 3 times more sensitive than the standard RT-PCR and ELISA assays published previously for BYDV-PAV detection and significantly improves virus detection in single aphids. Extraction of nucleic acids from aphids using either phenol/chloroform or chelatin resin-based protocols allow the use of pooled samples or of a small part (up to 1/1600th) of a single aphid extract for efficient BYDV-PAV detection.

Biolistic-mediated inoculation of immature wheat embryos with Barley yellow dwarf virus-PAV

Successful mechanical inoculation of plant with viruses requires an efficient method to introduce the viral pathogen into the appropriate cells of the plant. Barley yellow dwarf virus-PAV (BYDV-PAV, Luteovirus), transmitted naturally by aphids, must be inoculated into the phloem tissue to infect systemically inoculated hosts. The particle bombardment method used widely for nucleic acid transfer into plant tissues was adapted to inoculate immature embryos of winter and spring wheat cultivars with either BYDV-PAV particles or viral full-length RNAs. DAS-ELISA and RT-PCR were carried out on extracts of developed leaves at 7 weeks post-bombardment and revealed that up to 14% of bombarded embryos produced BYDV-infected wheat plants. This is the first report of an aphid-free inoculation method for BYDV.

Effect of mutations in Barley yellow dwarf virus genomic RNA on the 5' termini of subgenomic RNAs.

To investigate the role in transcription initiation of sequences upstream of the promoter for subgenomic RNA2 (sgRNA2) of Barley yellow dwarf virus PAV, deletion and insertion clones were constructed and the 5' termini of all three sgRNAs were determined. Deletion and insertion of sequences upstream of the transcription start site of sgRNA2 altered the position and nucleotide used to initiate transcription of sgRNA2, but not of sgRNAs 1 or 3. Since the promoter for sgRNA2 is downstream of the mutations, these results show that sequences outside of the core promoter can influence transcription start site selection from the sgRNA2 promoter.

A single copy of a virus‐derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus

Summary Barley yellow dwarf virus-PAV (BYDV-PAV) is the most serious and widespread virus of cereals worldwide. Natural resistance genes against this luteovirus give inadequate control, and previous attempts to introduce synthetic resistance into cereals have produced variable results. In an attempt to generate barley with protection against BYDV-PAV, plants were transformed with a transgene designed to produce hairpin (hp)RNA containing BYDV-PAV sequences. From 25 independent barley lines transformed with the BYDV-PAV hpRNA construct, nine lines showed extreme resistance to the virus and the majority of these contained a single transgene. In the progeny of two independent transgenic lines, inheritance of a single transgene consistently correlated with protection against BYDV-PAV. This protection was rated as immunity because the virus could not be detected in the challenged plants by ELISA nor recovered by aphid feeding experiments. In the field, BYDV-PAV is sometimes associated with the related luteovirus Cereal yellow dwarf virus-RPV (CYDV-RPV). When the transgenic plants were challenged with BYDV-PAV and CYDV-RPV together, the plants were susceptible to CYDV-RPV but immune to BYDV-PAV. This shows that the immunity is virus-specific and not broken down by the presence of CYDV. It suggests that CYDV-RPV does not encode a silencing-suppressor gene or that its product does not protect BYDV-PAV against the plant's RNAi-like defence mechanism. Either way, our results indicate that the BYDV-PAV immunity will be robust in the field and is potentially useful in minimizing losses in cereal production worldwide.

Molecular and Biological Characterization of a Trackable Illinois Isolate of Barley yellow dwarf virus-PAV.

In consecutive annual statewide surveys of the incidence of Barley yellow dwarf viruses (BYDVs) in Illinois wheat and oat fields, 27 BYDV-PAV-like isolates were identified. Using polymerase chain reaction (PCR), the coat protein regions of all 27 isolates were analyzed for restriction fragment length polymorphisms. The PCR products of two isolates, one from each year, had restriction fragment profiles after digestion with HaeIII that differed from the other isolates. The nucleotide sequences of the coat protein regions of a laboratory isolate, BYDV-PAV-IL (PAV-IL), two of the isolates with the common restriction profile, and the two isolates with polymorphic profiles were more than 98% identical. The relatively rare isolate identified during the first year was designated BYDV-PAV-DK1 (PAV-DK1) and further characterized biologically. PAV-DK1 and PAV-IL did not differ significantly in symptom expression, but did differ significantly in rates of transmission by two of the three biotypes of Rhopalosiphum padiexamined. Since PAV-DK1 does not occur in high levels in the state of Illinois, and its PCR products have a unique restriction enzyme profile, it has the potential to be used as a traceable isolate in field epidemiological experiments.

Assessment of Transmission Ability of Barley Yellow Dwarf Virus-PAV Isolates by Different Populations of Rhopalosiphum padi and Sitobion avenae

Populations of two aphid species from different geographic regions of Morocco were tested for their ability to transmit five barley yellow dwarf virus-PAV (BYDV-PAV) type isolates using Clintland 64 oat as the test plant. Transmission efficiencies were determined for 10 sub-populations of Rhopalosiphum padi and 12 sub-populations of Sitobion avenae. After a short acquisition access period (AAP) of 4 h, all populations transmitted the virus but with different efficiencies. R. padi (Rp-S) and S. avenae (Sa-S) collected in the Settat region were the most efficient vectors, with transmission rates of 38% and 27%, respectively. R. padi (Rp-C) collected at Chaouen and S. avenae (Sa-B) at Berkane, were poor transmitters with respective vectoring abilities of 20% and 16%. These four sub-populations were chosen to study the acquisition of BYDV-PAV and the retention of virus within aphids in more detail. The transmission after two AAPs of 4 h and 48 h were compared. Starved aphids given a 4 h AAP had significantly higher transmission efficiencies than non-starved aphids. However, after a 48 h AAP, no difference was observed in the transmission between starved and non-starved aphids. Intraspecific variability was also detected by means of serial transfers of individual aphids after the given AAP. Following the first day of serial transfers, no differences were observed in transmission efficiency and virus titers for sub-populations within each species acquiring the virus during 48 h, but there was significant variation when the virus was acquired in 4 h. The levels of PAV antigen retained by aphids fed on healthy plants declined rapidly during the first day after acquisition, but remained fairly constant during the next 5–7 days depending on the length of the AAP. Virus antigen could be detected by ELISA in Rp-S and Sa-S for up to 11 days of serial transfer, but it was shown that aphids could retain and transmit BYDV-PAV for at least 3 weeks.

Severity of infection of Moroccan barley yellow dwarf virus PAV isolates correlates with variability in their coat protein sequences

SummaryBiological properties and deduced amino acid sequences of the overlapping coat and 17kDa proteins were compared for 12 barley yellow dwarf virus PAV‐type isolates collected at different locations in Morocco. The biological variability of the 12 isolates was investigated by inoculation to a set of as many as eight barley cultivars and an oat cultivar. Significant biological variability was observed among the Moroccan PAV–type isolates, with the isolates falling into two clusters. Cluster 1 contained isolates MA9501, MA9502, MA9504, MA9512 and MA9513, and cluster 2 the MA9415, MA9505, MA9508, MA9511, MA9514, MA9516 and MA9517. Isolates of cluster 2 produced more severe symptoms and stunting than did those of cluster 1 when inoculated to appropriate index plants. In parallel, the nucleic acid sequences were obtained for the region containing the cistron coding for the coat protein (CP) and 17 kDa protein for all 12 isolates by reverse transcription followed by the polymerase chain reaction. Sequence homology grouping yielded the same two clusters of the isolates as found by measuring biological variability. Comparisons of the CP sequences between the two clusters revealed 87.4% to 90.5% nucleotide identity and 82.6% to 87.6% amino acid identity. Comparison with other sequenced isolates demonstrated that the CP's of cluster 1 were closely related to the Australian (Vic‐PAV), the North American (NY–PAV, P–PAV), and Japanese (JPN–PAV) PAV–type isolates. The CP's of cluster 2, on the other hand, were closely related to the North American PAV–129 isolate. Finally, an RFLP test on CP gene–derived PCR products has been developed which distinguishes between the two clusters of the PAV–type isolates.

Sequences of the 3′ Halves of the Genomes of Barley Yellow Dwarf Virus-PAV cpA Isolates that Vary in Symptom Severity

Barley yellow dwarf virus (BYDV)-PAV isolates from USA have been separated into two distinct clusters (Chay et al. (1996) Virology 219: 57–65; Chay et al. (1996) Phytopathology 86: 370–377). Following this finding we have shown that BYDV-PAV is divided into two groups cpA and cpB based on their coat protein gene sequence, and distinct host preferences (Mastari et al. (1998) Phytopathology 88: 818–821). We have sequenced the complete 3′ half of the genomes of two lethal and two mild cpA isolates and compared them with those of several known PAV cpA isolates to assess variability and locate potential determinants of severity. Open reading frames (ORFs) 3, 4, 5, 6 and the 3′ untranslated regions had different percent homologies between isolates: ORF5 (92–97%), ORF3 (88–98%) 3′-translational enhancer (87–100%) ORF4 (85–99%), 3′ untranslated region (72–97%) and ORF6 (61–99%). In contrast to the mild isolates, the field-lethal isolates (FHv1 and FHv2) fell into the same cluster, regardless of the genomic region analysed. The isolates FHv1 and FHv2 differed from mild isolates by eight amino acid substitutions in ORFs 3 and 4, and insertions in ORF5. Four amino acid substitutions in the 17-kDa protein encoded by ORF4 caused a change in local net charge in the field-lethal isolates. Two insertions of four amino acids were identified in the C-terminal half of ORF5 of the field-lethal isolates, but were not present systematically in all lethal isolates analysed. The potential relationships of these differences in predicted amino acid sequences to disease severity are discussed.

Extreme Reduction of Disease in Oats Transformed with the 5′ Half of the Barley Yellow Dwarf Virus-PAV Genome

ABSTRACT Barley yellow dwarf viruses (BYDVs) are the most serious and widespread viruses of oats, barley, and wheat worldwide. Natural resistance is inadequate. Toward overcoming this limitation, we engineered virus-derived transgenic resistance in oat. Oat plants were transformed with the 5' half of the BYDV strain PAV genome, which includes the RNA-dependent RNA polymerase gene. In experiments on T2- and T3-generation plants descended from the same transformation event, all BYDV-inoculated plants containing the transgene showed disease symptoms initially, but recovered, flowered, and produced seed. In contrast, all but one of the BYDV-PAV-inoculated nontransgenic segregants died before reaching 25 cm in height. Although all of the recovered transgenic plants looked similar, the amount of virus and viral RNA ranged from substantial to undetectable levels. Thus, the transgene may act either by restricting virus accumulation or by a novel transgenic tolerance phenomenon. This work demonstrates a strategy for genetically stable transgenic resistance to BYDVs that should apply to all hosts of the virus.

Asymmetrical Distribution of Barley Yellow Dwarf Virus PAV Variants Between Host Plant Species

ABSTRACT A large epidemiological study of the genetic variation of barley yellow dwarf virus (BYDV) serotype PAV involving different host plant species was conducted. French BYDV PAV isolates were collected from barley and ryegrass, and their capsid protein gene sequences characterized using restriction fragment length polymorphism, single-strand conformation polymorphism, and sequence analyses. The data show that BYDV PAV isolates from five different continents are separated into two distinct groups named cpA and cpB, which are distributed irrespective of geographical location. Amino acid identity of the capsid proteins ranged from 93 to 99.5% in group cpA and from 95 to 99.5% in group cpB, while this value was only from 82 to 88% between the groups. Moreover, isolates from each group were found preferentially (up to 98%) in one of the two plant species examined. These results show that host plant species play a role in isolate selection and maintenance and that they contribute to the genetic diversity of BYDV PAV.

Barley yellow dwarf virus-PAV RNA does not have a VPg.

The genus Luteovirus is divided into two subgroups. It has already been shown that a genome-linked protein (VPg) is covalently attached to the 5'-end of the genomic RNA of subgroup II luteoviruses, but whether a VPg is present or absent on the genomic RNA of subgroup I luteoviruses is unknown. In the present work, a simple and sensitive method was developed for the detection of the VPg on virion genomic RNA. This method was used to show that a VPg is not attached to the 5'-end of the RNA genome of barley yellow dwarf virus-PAV which is a member of subgroup I luteoviruses.

Diversity Among Isolates Within the PAV Serotype of Barley Yellow Dwarf Virus

Several barley yellow dwarf virus (BYDV) isolates collected from viruliferous aphids in upstate New York were identified as PAV serotypes, based on their reaction with a polyclonal antiserum to NY-PAV. Four of six isolates examined were distinguished from the NY-PAV type isolate of BYDV by their failure to react with a PAV-specific monoclonal antibody in enzyme-linked immunosorbent assay (ELISA) and by restriction fragment length polymorphisms of polymerase chain reaction-amplified viral sequences. The capsid protein amino acid sequence of one of these four isolates, designated PAV-129, was less similar to that of NY-PAV (86.5% similar) than NY-PAV is to two other isolates, serotyped as PAV, from Indiana (98% similar) or Australia (97% similar). In biological comparisons of PAV-129 and NY-PAV, the efficiency of transmission by two aphid species, Rhopalosiphum padi and Sitobion avenae, was not significantly different; however, PAV-129 caused more severe symptoms when inoculated to a variety of oat genotypes. In addition, the growth and grain yield of 'Ogle', a spring oat considered resistant to the BYDV PAV serotype was significantly reduced when infected with PAV-129.

Complete Nucleotide Sequence of the Japanese Isolate of Barley Yellow Dwarf Virus-PAV Serotype.

The complete nucleotide sequence of the genomic RNA of the Japanese isolate of barley yellow dwarf virus-PAV serotype (BYDV Jpn-PAV) was determined. The genome of BYDV Jpn-PAV was comprised of 5667 nucleotides and had six open reading frames (ORF 1 to ORF 6) on the virus sense strand. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of the Australian (Vic-PAV) and the North American (P-PAV) PAV isolates and the PS1 isolate of MAV serotype (MAV-PS1). Comparisons of the corresponding ORFs showed that the proposed RNA replicase domains (ORF 1 and ORF 2) of all the isolates shared more than 97% amino acid homology. Sequence comparison between Jpn-PAV and MAV-PS1 showed considerable dissimilarity in the ORF 3 to 6. Among the overall genomes of Jpn-PAV and two other PAV isolates, an apparent sequence diversity was found in the ORF 6 domain. The ORF 1 to 5 of Jpn-PAV shared a high degree of amino acid homology with those of Vic- and P-PAV isolates, whereas ORF 6 had only 61% and 78% identity with the respective ORF 6 of Vic-PAV and P-PAV. From sequence comparisons, Jpn-PAV appears to be more closely related to the North American and European PAV isolates than to the Australian isolates.

Genes and cis-Acting Sequences Involved in Replication of Barley Yellow Dwarf Virus-PAV RNA

Deletion and point mutation analyses were employed to determine gene products and cis-acting signals involved in translation, replication, and encapsidation of barley yellow dwarf virus (PAV serotype) RNA in oat protoplasts. Of the six open reading frames (ORFs), only ORFs 1 and 2, which include the putative RNA-dependent RNA polymerase gene, were required for replication. In vitro translation of these mutants revealed that sequence upstream of the shifty heptanucleotide was required for ribosomal frameshifting, and that a 3′-translational enhancer stimulated translation more efficiently when located in closer proximity to the translated ORFs. Deletion of the coat protein gene reduced the accumulation of genomic but not subgenomic RNA. The carboxy-terminally extended form of the coat protein, produced by readthrough of its stop codon, was not required for encapsidation. Although the ORF6 product was not necessary, cis-acting RNA signals in and around ORF6 were required for RNA replication. Defective RNAs harboring various deletions were not replicated in trans by the co-inoculated wild-type helper genome, suggesting that replication of PAV RNA may be coupled to translation.

Control of start codon choice on a plant viral RNA encoding overlapping genes.

The signals that control initiation of translation in plants are not well understood. To dissect some of these signals, we used a plant viral mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype, the coat protein (CP) and overlapping 17K open reading frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relative to the AUG codons in efficiency of translation initiation were investigated by translation of sgRNA1 mutants in a cell-free extract and by expression of a reporter gene from mutant sgRNA1 leaders in protoplasts. The effects of mutations that disrupted and restored secondary structure encompassing the CP AUG independently of, and in combination with, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary structure. Secondary structure had an overall inhibitory effect on translation of both ORFs. In general, the "Kozak rules" of start codon preference predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To explain this observation, we propose a new model in which pausing of the ribosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structure in controlling translation initiation should be of value for understanding expression of any plant gene and in the design of artificial constructs.

Occurrence of barley yellow dwarf virus serotypes MAV and RMV in over-summering grasses

Over-summering grasses were collected in the south-west of Western Australia in 1991 and 1992 and tested by ELISA using serotype specific polyclonal antibodies for presence of barley yellow dwarf virus (BYDV) serotypes MAV, PAV and RPV (1991) or MAV, PAV, RPV and RMV (1992). In 1991, 33 samples from 33 sites were tested and MAV was detected in four of 12 samples of Pennisetum clandestinum found infected with BYDV. Presence of MAV was confirmed by retesting these samples using MAV specific monoclonal antibodies. In 1992, 802 samples from 16 grass species were collected from 579 sites in six regions. BYDV was detected in 214 (27%) samples at 178 (31%) sites. MAV was found in 50% and RMV in 38% of infected samples. Both were found either alone or in mixed infections with each other or with PAV and/or RPV; all four serotypes were found in 18 (8%) infected samples. The most important hosts were four perennials: Cynodon dactylon, Eragrostis curvula, Paspalum dilatatum and P. clandestinum. Eight other perennial and five annual grass species were also infected. MAV was most commonly found in C. dactylon and P. clandestinum and RMV in P. dilatatum. All four serotypes were present in the six regions sampled, but the relative proportions of the serotypes found varied from region to region. This paper represents the first extensive survey of MAV and RMV serotypes of BYDV in over-summering grasses in Australia. Aphid species found during this survey infesting over-summering grasses were Rhopalosiphum padi,R. maidis and Hysteroneura setariae. In the Mediterranean type climate of the south-west of Western Australia, BYDV and aphids on over-summering perennial grasses constitute the main reservoir of infection and means of spread to cereal crops. Factors favouring grass survival over summer result in increased cereal crop infection.

Use of group-specific primers and the polymerase chain reaction for the detection and identification of luteoviruses

A general diagnostic assay for a number of distinct luteoviruses was developed using the polymerase chain reaction (PCR) and restriction enzyme analysis. Two minimally degenerate, group-specific primers were derived from previously published RNA sequences of three luteoviruses. This primer pair generated specific PCR fragments of about 530 bp from extracts of plants infected with potato leafroll virus, beet western yellows virus, or New York barley yellow dwarf virus (BYDV) serotypes MAV, PAV, RMV, RPV and SGV, which span much of the respective viral coat protein gene. Each virus was easily distinguished from the others by restriction enzyme analysis of the amplified DNA products. Samples from BYDV-infected oat and wheat collected in Nebraska were identified as containing PAV-like serotypes; micro-heterogeneity was detected in several samples. This method provides a rapid, sensitive and relatively inexpensive means of luteovirus detection and identification. It is the first test capable of simultaneously detecting all five BYDV serotypes.