The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS–LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.
The Potyviridae family relies on a cap-independent translation mechanism to facilitate protein expression. The genomic architecture of the viral RNAs of the Potyviridae family resembles those of the animal picornaviruses. The viral genomes lack a 5′ cap structure. Instead, they have the viral protein VPg covalently linked to the 5′ end of the RNA. The viral RNAs code for a single large polyprotein, which is then cleaved into several functional subunits. With their common genome organization with the Picornaviridae, it has been largely assumed that the members of the plant Potyviridae family share similar translation mechanism. We will describe the remarkably diverse translational enhancers identified within the family and their unique mechanisms of translation, from internal recruitment of the ribosomes to ribosomal scanning from the 5′ end and the recruitment of the VPg in translation, which are unseen in their animal virus counterparts. The divergence among the potyviral translation enhancers is heightened with the recent discovery of Triticum mosaic virus, an atypical member of the Potyviridae family, for which its 5′ leader by far exceeds the typical length of plant viral leaders and contains features typically found in animal viruses. Much remains to be learned on how these highly divergent elements enable potyviruses, which include some of the most damaging plant viruses, to take over the host translation apparatus. While no clear consensus sequence, structure or mechanism has been reported yet among the potyviral elements, more thorough studies are need to fill in the gap of knowledge.
KeywordsPotyviridae;Cap-independent translation;5′ UTR;IRES;Ribosomal scanning;Viral genome-linked protein
Genome sequences of three episomal Banana streak MY virus (BSMYV) isolates sampled from triploid banana hybrids (Chini Champa: AAB; Malbhog: AAB and Monthan: ABB), grown in North-East and South India are reported in this study by sequence-independent improved rolling circle amplification (RCA). RCA coupled with restriction fragment length polymorphism revealed diverse restriction profiles of five BSMYV isolates. Nucleotide substitution rates of BSMYV subpopulation and Banana streak OL virus subpopulation was 7.13 × 10−3 to 1.59 × 10−2 and 2.65 × 10−3 to 5.49 × 10−3, respectively, for the different coding regions. Analysis of the genetic diversity of banana and sugarcane badnaviruses revealed a total of 32 unique recombination events among banana and sugarcane badnaviruses (inter BSV–SCBV), in addition to the extensive recombination with in banana streak viruses and sugarcane bacilliform viruses (intra-BSV and intra-SCBV). Many unique fragments were shown to contain similar ruminant sequence fragments which indicated the possibility that the two groups of badnaviruses or their ancestors to colonise same host before making the host shift. The distribution of recombination events, hot-spots (intergenic region and C-terminal of ORF3) as well as cold-spots (distributed in ORF3) displayed the mirroring of recombination traces in both group of badnaviruses. These results support the hypothesis of relatedness of banana and sugarcane badnaviruses and the host and geographical shifts that followed the fixation of the species complex appear to be a recent event.
Viral pathogens are a major threat to rice production worldwide. Although RNA interference (RNAi) is known to mediate antiviral immunity in plant and animal models, the mechanism of antiviral RNAi in rice and other economically important crops is poorly understood. Here, we report that rice resistance to evolutionarily diverse viruses requires Argonaute18 (AGO18). Genetic studies reveal that the antiviral function of AGO18 depends on its activity to sequester microRNA168 (miR168) to alleviate repression of rice AGO1 essential for antiviral RNAi. Expression of miR168-resistant AGO1a in ago18 background rescues or increases rice antiviral activity. Notably, stable transgenic expression of AGO18 confers broad-spectrum virus resistance in rice. Our findings uncover a novel cooperative antiviral activity of two distinct AGO proteins and suggest a new strategy for the control of viral diseases in rice.
Sites of virus attachment and entry in the vector offer key sites for disruption of transmission.
Viral capsid protein fused to a toxin is a promising vector control strategy.
Transgenic expression of viral proteins in plants can block virus acquisition and transmission.
RNA interference has potential for disruption of virus transmission and elimination of the vector.
Proteomic tools can be used to identify candidate vector proteins that function in virus transmission.
Plant-infecting viruses are transmitted by a diverse array of organisms including insects, mites, nematodes, fungi, and plasmodiophorids. Virus interactions with these vectors are diverse, but there are some commonalities. Generally the infection cycle begins with the vector encountering the virus in the plant and the virus is acquired by the vector. The virus must then persist in or on the vector long enough for the virus to be transported to a new host and delivered into the plant cell. Plant viruses rely on their vectors for breaching the plant cell wall to be delivered directly into the cytosol. In most cases, viral capsid or membrane glycoproteins are the specific viral proteins that are required for transmission and determinants of vector specificity. Specific molecules in vectors also interact with the virus and while there are few-identified to no-identified receptors, candidate recognition molecules are being further explored in these systems. Due to the specificity of virus transmission by vectors, there are defined steps that represent good targets for interdiction strategies to disrupt the disease cycle. This review focuses on new technologies that aim to disrupt the virus–vector interaction and focuses on a few of the well-characterized virus–vector interactions in the field. In closing, we discuss the importance of integration of these technologies with current methods for plant virus disease control.
Abstract: Chromosomal translocations in wheat derived from alien species are a valuable source of genetic diversity that have provided increases in resistance to various diseases and improved tolerance to abiotic stresses in wheat. These alien genomic segments can also affect multiple traits, with a concomitant ability to alter yield potential in either a positive or negative fashion. The aim of this work was to characterize the effects on yield of two types of translocations, namely T4-derived translocations from Thinopyrum ponticum, carrying the leaf rust resistance gene Lr19, and the TC14 translocation from Th. intermedium, carrying the barley yellow dwarf virus resistance gene Bdv2, in Australian adapted genetic backgrounds and under Australian conditions. A large range of germplasm was developed by crossing donor sources of the translocations into 24 Australian adapted varieties producing 340 genotypes. Yield trials were conducted in 14 environments to identify effects on yield and yield components. The T4 translocations had a positive effect on yield in one high yielding environment, but negatively affected yield in low-yielding environments. The TC14 translocation was generally benign, however, it was associated with a negative impact on yield and reduced height in two genetic backgrounds. The translocation was also associated with a delayed maturity in several backgrounds. The T4 translocations results were consistent with previously published data, whilst this is the first time that such an investigation has been undertaken on the TC14 translocation. Our data suggests a limited role for each of these translocations in Australia. The T4 translocations may be useful in high yielding environments, such as under irrigation in NSW and in the more productive high rainfall regions of south-eastern Australia. Traits associated with the TC14 translocation, such as BYDV resistance and delayed maturity, would make this translocation useful in BYDV-prone areas that experience a less pronounced terminal drought (e.g., south-eastern Australia).
ABSTRACT Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach.
In plants, symptom recovery is usually accompanied by antiviral RNA silencing.
RNA silencing mechanisms include slicing and/or translation repression of viral RNAs.
DNA viruses are targeted by post-transcriptional and transcriptional gene silencing.
RNA silencing may contribute to symptom remission by regulating plant gene expression.
The natural outcome of some plant–virus interactions is symptom recovery, which is characterized by the emergence of asymptomatic leaves following a systemic symptomatic infection. Symptom recovery is generally accompanied with reduced virus titers and sequence-specific resistance to secondary infection and has been linked with the induction of antiviral RNA silencing. Recent studies have revealed an unsuspected diversity of silencing mechanisms associated with symptom recovery in various host-virus interactions, including degradation or translation repression of viral RNAs and in the case of DNA viruses, transcriptional arrest of viral minichromosomes. RNA silencing may also contribute to symptom alleviation by regulating plant gene expression. In this review, we discuss the evidence supporting the role of various RNA silencing mechanisms in symptom recovery. We also discuss how a delicate equilibrium between RNA silencing and virus counter-defense responses in recovered leaves may help maintain virus titers at levels below the threshold required for symptom induction.
With increasing winter temperatures, Barley yellow dwarf virus (BYDV) is expected to become an increasing problem in maize cultivation in Germany. Earlier studies revealed that BYDV has a negative impact on maize performance. Molecular markers would accelerate the development of BYDV resistant maize. Therefore, the objectives of this study were (i) the identification of quantitative trait loci (QTL) for BYDV resistance in five connected segregating maize populations in a field experiment and (ii) their comparison with the QTL detected under greenhouse conditions.
Plant diseases are responsible for major economic losses in the agricultural industry worldwide. Monitoring plant health and detecting pathogen early are essential to reduce disease spread and facilitate effective management practices. DNA-based and serological methods now provide essential tools for accurate plant disease diagnosis, in addition to the traditional visual scouting for symptoms. Although DNA-based and serological methods have revolutionized plant disease detection, they are not very reliable at asymptomatic stage, especially in case of pathogen with systemic diffusion. They need at least 1–2 days for sample harvest, processing, and analysis. Here, we describe modern methods based on nucleic acid and protein analysis. Then, we review innovative approaches currently under development. Our main findings are the following: (1) novel sensors based on the analysis of host responses, e.g., differential mobility spectrometer and lateral flow devices, deliver instantaneous results and can effectively detect early infections directly in the field; (2) biosensors based on phage display and biophotonics can also detect instantaneously infections although they can be integrated with other systems; and (3) remote sensing techniques coupled with spectroscopy-based methods allow high spatialization of results, these techniques may be very useful as a rapid preliminary identification of primary infections. We explain how these tools will help plant disease management and complement serological and DNA-based methods. While serological and PCR-based methods are the most available and effective to confirm disease diagnosis, volatile and biophotonic sensors provide instantaneous results and may be used to identify infections at asymptomatic stages. Remote sensing technologies will be extremely helpful to greatly spatialize diagnostic results. These innovative techniques represent unprecedented tools to render agriculture more sustainable and safe, avoiding expensive use of pesticides in crop protection.
Virus infections have the potential to reduce biomass yields in energy crops, including Panicum virgatum (switchgrass). As a first step towards managing virus-induced biomass reduction, deep sequencing was used to identify viruses associated with mosaic symptoms in switchgrass. Two sequences with homology to mastreviruses were identified. Total DNA extracted from switchgrass varieties ‘Dewey Blue’ and ‘Cloud Nine’ was used as template to amplify mastrevirus DNA by the rolling-circle method. Complete mastrevirus genome sequences were obtained from cloned amplicons. The two nucleotide sequences were 88 % identical to each other but only 56–57 % identical to the closest relatives in the genus Mastrevirus. Predicted amino acid sequences of the coat protein, replication-associated protein A, replication-associated protein, and putative movement protein encoded by the two mastrevirus-like sequences were 95 %, 79 %, 79 %, and 87 % identical to each other, respectively, and 46–48 %, 31 %, 31 %, and 42–48 % identical to those of the closest mastrevirus relatives. Based on a genome-wide identity threshold of 75 % set by the International Committee on Taxonomy of Viruses and phylogenetic analyses, the two virus sequences appear to represent a new mastrevirus species. The mastrevirus is tentatively named switchgrass mosaic-associated virus 1 (SgMaV-1) and is the first mastrevirus reported from North America.
Sorghum mosaic virus (SrMV), a causal agent of the destructive sugarcane mosaic disease, has a global presence. An isolate of SrMV infecting a commercially-grown sugarcane plant was recovered from the Hainan province of China. The virions were visualized by an electron microscope, and the coat proteins (CP) were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tandem mass spectrometry. Discrepancies between the CP predicted and actual amino acid sequences were noted, which confounded the phylogenetic assignment of the isolate. The apparent variations may have physiological effects on the pathogenicity and virulence of SrMV.
Keywords SrMV; Sugarcane mosaic disease; Coat protein
‘YZ05-51’ (Reg. No. CV-161, PI 673444) sugarcane (a complex hybrid of Saccharum spp.) was developed through cooperative research conducted by the Yunnan Sugarcane Research Institute (YSRI) of Yunnan Academy of Agricultural Sciences, and Yunnan Yunzhe Technology Development Co. Ltd. It was released to growers in mainland China on 1 Aug. 2013. YZ05-51 was selected from the cross Yacheng 90-56 × ‘ROC 23’ planned by YSRI and crossed at Yacheng Sugarcane Breeding Station (YSBS) of the Guangzhou Sugar Industry Research Institute, Yacheng, Hainan Province of mainland China in January 2005. The female parent (Yacheng 90-56) is an advanced clone developed by YSBS. The male parent (ROC23) is a cultivar developed by the Taiwan Sugar Research Institute. YZ05-51 was selected from a five-stage selection program in YSRI and tested in the eighth China Regional Trial for Sugarcane Varieties at 14 locations in mainland China for 2 yr. YZ05-51 was released by the China Committee of Sugarcane Variety Release because of its higher cane yield compared with ROC16 and ROC22 (two reference cultivars) (7.1 and 1.1%, respectively), its higher sugar yield compared with ROC16 and ROC22 (10.6 and 2.7%, respectively), and its resistance to drought, high resistance to smut (caused by Ustilago scitaminea H. & P. Sydow), and moderate resistance to mosaic (Sorghum mosaic virus and Sugarcane mosaic virus).
Rice stripe disease is caused by the rice stripe virus (RSV) which is transmitted by the small brown plant hopper (SBPH). This disease significantly restricts rice productivity in East Asia. Major RSV resistance quantitative trait loci (QTLs) have been shown to be located on chromosome 11 in several resistant cultivars. However, the molecular identities of these QTLs are largely unknown. In this study, we identified the serine/threonine protein kinase OsPBL1 ( O RYZA S ATIVA ARABIDOPSIS PB S1-L IKE 1), a potential resistance gene to rice stripe disease, by reverse genetic screening with T-DNA insertional mutant lines. The OsPBL1 protein is highly conserved among various monocots and dicots, including Arabidopsis. Specifically, OsPBL1 exhibits 67 % amino acid sequence identity to AtPBS1 (AVRPPHB SUSCEPTIBLE1), a positive regulator of effector-triggered immunity (ETI) in Arabidopsis. Moreover, we show here that OsPBL1 transcripts are abundantly expressed in the leaves of Dongjin seedlings—an RSV-resistant—under normal growth conditions. Exogenous treatment with defense-related phytohormones such as cytokinin and salicylic acid increased the expression of OsPBL1. This gene is also modulated by the circadian rhythm. Furthermore, OsPBL1, OsPR1b, and OsPR2 transcripts were up-regulated to higher levels by SBPH treatment, which was specifically observed in RSV-resistant varieties. The OsPBL1 protein was found to localize to the nucleus and to be cleaved upon attack with either healthy or infected SBPHs. Taken together, our data indicate that OsPBL1 undergoes various transcriptional and post-translational modifications upon SBPH and/or RSV attack, similar to Arabidopsis PBS1. We propose that OsPBL1 is involved in antiviral defense signaling pathways in rice.
Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in G. nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G. nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G. nigrifrons transcripts will facilitate the study of gene function and pathogen transmission, and may provide approaches for developing novel targets of RNAi-based pest control.
Maize (Zea mays L.) is a promising new world cereal crop throughout the world including Bangladesh. The crop is seriously affected by different viral diseases. Maize dwarf mosaic disease is one of them caused by a number of viruses. Most of the viruses attacking maize are member of the genus Potyvirus under the family Potyviridae such as Maize Dwarf Mosaic Virus (MDMV), Sugarcane Mosaic Virus (SCMV), Johnsongrass Mosaic Virus (JGMV), Sorghum Mosaic Virus (SrMV) etc. Maize Chlorotic Dwarf Virus (MCVD) is another devastating virus belongs to the genus Waikavirus of the family Sequiviridae. Resistance to another member of the family Potyviridae, Wheat Streak Mosaic Virus (WSMV), is conferred by Wsm gene in maize inbred lines Pa405. The resistance was governed by three alleles (Wsm1, Wsm2 and Wsm3) in a dose dependent manner up to a certain degree. These alleles or closely linked genes were previously shown to confer resistance to the potyviruses infecting maize. In Bangladesh, MDMV and MCDV are severely reducing maize production but no genotype has been identified yet as a source of resistant gene. The study was started with the objective to screen nine locally available maize lines carrying Wsm gene using Single Sequence Repeat (SSR) marker. Morphological data was recorded at field maturity to study genetic diversity. Maize plants were inoculated with virus and scoring on symptom development was performed at 7, 10 and 14 dpi (days post inoculation) to calculate infection percentage and Area Under Disease Progress Curve (AUDPC). Finally molecular detection for desired gene was performed using three sets of SSR markers. The result indicated that five genotypes among nine carried Wsm gene, but functional/durable resistance was observed for only three genotypes (BHM-7, V-92 and Uttaran) at a certain level. BHM-5 and Duranta showed non-functional resistance despite the presence of Wsm gene. BHM-7 (BARI Hybrid Maize 7) was noticed as the best one for good yielding performance, carrying Wsm gene and showing resistance against MDMV. No genotype was found to govern resistance against MCDV. Intensive study is needed to find out the fact responsible for such behavior of maze genotypes.
The 3′ tRNA-like structure (3′TLS) conserved among three genomic RNAs of brome mosaic virus is multifunctional.
BMV TLS harbors recognition signals for minus-strand initiation by viral RdRp.
BMV TLS functions in RNA recombination and as a nucleation motif for virion assembly.
The 3′ untranslated region in each of the three genomic RNAs of Brome mosaic virus (BMV) is highly homologous and contains a sequence that folds into a tRNA-like structure (TLS). Experiments performed over the past four decades revealed that the BMV 3′ TLS regulates many important steps in BMV infection. This review summarizes in vitro and in vivo studies of the roles of the BMV 3′ TLS functioning as a minus-strand promoter, in RNA recombination, and to nucleate virion assembly.
Abstract A database and website (http://www.ictvonline. org/taxonomyReleases.asp) have been established where the history of changes in virus taxonomy from 1971 to the present day can easily be traced. Each change is linked to a source document confirming the change or, for most changes since 2002, to the taxonomic proposal approved by the International Committee on Taxonomy of Viruses (ICTV).
The genomes of a large number of highly diverse novel circular DNA viruses from a wide range of sources have been characterised in recent years, including circular single-stranded DNA (ssDNA) viruses that share similarities with plant-infecting ssDNA viruses of the family Geminiviridae. Here, we describe six novel circular DNA viral genomes that encode replication-associated (Rep) proteins that are most closely related to those of either geminiviruses or gemycircularviruses (a new group of ssDNA viruses that are closely related to geminiviruses). Four possible viral genomes were recovered from Bromus hordeaceus sampled in New Zealand, and two were recovered from B. hordeaceus and Trifolium resupinatum sampled in France. Two of the viral genomes from New Zealand (one from the North Island and one from the South Island each) share >99 % sequence identity, and two genomes recovered from B. hordeaceus and T. resupinatum sampled in France share 74 % identity. All of the viral genomes that were recovered were found to have a major open reading frame on both their complementary and virion-sense strands, one of which likely encodes a Rep and the other a capsid protein. Although future infectivity studies are needed to identify the host range of these viruses, this is the first report of circular DNA viruses associated with grasses in New Zealand.
The complete genome of Barley yellow striate mosaic virus is obtained.
A novel nested gene is found in the genome of BYSMV.
The nested gene is translated from the mRNA via a leaky scanning mechanism.
The nested gene encodes a small hydrophobic protein.
The siRNAs derived from BYSMV is cloned and analyzed.
Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, causes serious crop losses in agriculture. Here, we have cloned the BYSMV-derived small interfering RNAs (siRNAs), assembled the siRNAs and used RT-PCR to reconstruct the BYSMV genome. The genome consists of 12,706 nucleotides and encodes ten predicted genes from the antigenomic strand. The major BYSMV structural proteins share identities ranging from 35% to 62% with northern cereal mosaic virus (NCMV) counterparts. A notable difference is that BYSMV contains three transcriptional units residing between the P and M genes compared with four units in the corresponding region of NCMV. Unexpectedly, the middle mRNA in this region encodes gene5 nested in an alternative frame within gene4 via a leaky scanning mechanism. The gene5 encodes a small hydrophobic protein targeting to the endoplasmic reticulum (ER). To our knowledge, this is the first report of nested gene in plant rhabdoviruses.
Soil-borne wheat mosaic virus (SBWMV) was detected in Poland for the first time in 2010 in the Southern Greater Poland and in the following years in the Lower Silesia and in the Pomerania regions. All found isolates induced mild symptoms of disease. Stunting without typical leaf mosaic of infected plants was observed. A low concentration of the virus in plant sap caused troubles in diagnostics. Difficulties in interpreting of obtained ambiguous results of ELISA test and RT-PCR technique were the main problem. The specific primers and real-time RT-PCR conditions were developed to improve sensitivity and of this assay for the detection of SBWMV. It can be used when the virus concentration in infected plants is very low and the standard ELISA test and RT-PCR technique failed.
Wirus odglebowej mozaiki pszenicy (Soil-borne wheat mosaic virus, SBWMV) wykryto w Polsce po raz pierwszy w 2010 roku, w południowej Wielkopolsce, a w kolejnych latach na Dolnym Śląsku i na Pomorzu. Wszystkie wykryte izolaty powodowały łagodny przebieg choroby. Obserwowano jedynie zahamowanie wzrostu chorych roślin bez typowych objawów mozaiki liści. Niska koncentracja cząstek wirusowych w soku porażonych roślin powodowała kłopoty w diagnostyce. Główny problem polegał na trudnościach w interpretacji otrzymywanych często niejednoznacznych wyników zastosowanego testu ELISA oraz techniki RT-PCR (reverse transcription – polymerase chain reaction). Dzięki opracowaniu specyficznych starterów oraz warunków przebiegu reakcji odwrotnej transkrypcji i łańcuchowej polimerazy w czasie rzeczywistym (real-time RT-PCR) wykazano przydatność tej techniki do wykrywania SBWMV i do weryfikacji wątpliwych wyników.
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