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Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non‐dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self‐interaction and DNA binding to the promoters of unfolded protein response (UPR)‐related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants. [ABSTRACT FROM AUTHOR]

This document provides the conclusions of the pest survey card that was prepared in the context of the EFSA mandate on plant pest surveillance (M‐2020‐0114) at the request of the European Commission. The full pest survey card for beet necrotic yellow vein virus is published and available online in the EFSA Pest Survey Card gallery at the following link and will be updated whenever new information becomes available: https://efsa.europa.eu/plants/planthealth/monitoring/surveillance/beet-necrotic-yellow-vein-virus [ABSTRACT FROM AUTHOR]

\textbf{Background:}Rhizomania counts as the most important disease in sugar beet (\textit{Beta vulgaris} L.) for which no plant protection is available, leaving plant breeding as the only defence strategy at the moment. Five resistance genes have been detected on the same chromosome and further studies suggested that these might be different alleles at two resistance clusters. Nevertheless, it was postulated that rhizomania resistance might be a quantitative trait with multiple unknown minor resistance genes. Here, we present a first attempt at genomic prediction of rhizomania resistance in a population that was genotyped using single nucleotide polymorphism (SNP) markers. \newline \textbf{Methods:} First, genomic prediction was performed using all SNPs. Next, we calculated the variable importance for each SNP using machine learning and performed genomic prediction by including the SNPs incrementally in the prediction model based on their variable importance. Using this method, we selected the optimal number of SNPs that maximised the prediction accuracy. Furthermore, we performed genomic prediction with SNP pairs. We also performed feature selection with SNP pairs using the information about the variable importance of the single SNPs. \newline \textbf{Results:} From the four methods under investigation, the latter led to the highest prediction accuracy. These results lead to the following conclusions: (I) The genotypes that were resistant at all known resistance genes, provided the highest possible variation of virus concentrations that the machine can measure. Thus, it can be assumed that more genes must be involved in the resistance towards rhizomania. (II) We show that prediction models that include SNP interactions increased the prediction accuracy. \newline \textbf{Conclusions:} Altogether, our findings suggest that rhizomania resistance is a complex quantitative trait that is affected by multiple genes as well as their interaction.

The molecular interactions between Polymyxa betae, the protist vector of sugar beet viruses, beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania, and Beta vulgaris have not been extensively studied. Here, the transmission of BNYVV to sugar beet by P. betae zoospores was optimized using genetically characterized organisms. Molecular interactions of aviruliferous and viruliferous protist infection on sugar beet were highlighted by transcriptomic analysis. P. betae alone induced limited gene expression changes in sugar beet, as a biotrophic asymptomatic parasite. Most differentially expressed plant genes were down-regulated and included resistance gene analogs and cell wall peroxidases. Several enzymes involved in stress regulation, such as the glutathione-S-transferases, were significantly induced. With BNYVV, the first stages of the P. betae life cycle on sugar beet were accelerated with a faster increase of relative protist DNA level and an earlier appearance of sporangia and sporosori in plants roots. A clear activation of plant defenses and the modulation of genes involved in plant cell wall metabolism were observed. The P. betae transcriptome in the presence of BNYVV revealed induction of genes possibly involved in the switch to the survival stage. The interactions were different depending on the presence or absence of the virus. P. betae alone alleviates plant defense response, playing hide-and-seek with sugar beet and allowing for their mutual development. Conversely, BNYVV manipulates plant defense and promotes the rapid invasion of plant roots by P. betae. This accelerated colonization is accompanied by the development of thick-walled resting spores, supporting the virus survival. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Background: Rhizomania counts as the most important disease in sugar beet Beta vulgaris L. for which no plant protection is available, leaving plant breeding as the only defence strategy at the moment. Five resistance genes have been detected on the same chromosome and further studies suggested that these might be different alleles at two resistance clusters. Nevertheless, it was postulated that rhizomania resistance might be a quantitative trait with multiple unknown minor resistance genes. Here, we present a first attempt at genomic prediction of rhizomania resistance in a population that was genotyped using single nucleotide polymorphism (SNP) markers. Methods: First, genomic prediction was performed using all SNPs. Next, we calculated the variable importance for each SNP using machine learning and performed genomic prediction by including the SNPs incrementally in the prediction model based on their variable importance. Using this method, we selected the optimal number of SNPs that maximised the prediction accuracy. Furthermore, we performed genomic prediction with SNP pairs. We also performed feature selection with SNP pairs using the information about the variable importance of the single SNPs. Results: From the four methods under investigation, the latter led to the highest prediction accuracy. These results lead to the following conclusions: (I) The genotypes that were resistant at all known resistance genes, provided the highest possible variation of virus concentrations that the machine can measure. Thus, it can be assumed that more genes must be involved in the resistance towards rhizomania. (II) We show that prediction models that include SNP interactions increased the prediction accuracy. Conclusions: Altogether, our findings suggest that rhizomania resistance is a complex quantitative trait that is affected by multiple genes as well as their interaction.

Abstract Background In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. Methods To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. Results We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. Conclusions ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial.

Background: In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. Methods: To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. Results: We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. Conclusions: ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial. [ABSTRACT FROM AUTHOR]

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is characterized by excessive lateral root (LR) formation. Auxin-mediated degradation of Aux/IAA transcriptional repressors stimulates gene regulatory networks leading to LR organogenesis and involves several Aux/IAA proteins acting at distinctive stages of LR development. Previously, we showed that BNYVV p25 virulence factor interacts with BvIAA28, a transcriptional repressor acting at early stages of LR initiation. The evidence suggested that p25 inhibits BvIAA28 nuclear localization, thus, de-repressing transcriptional network leading to LR initiation. However, it was not clear whether p25 interacts with other Aux/IAA proteins. Here, by adopting bioinformatics, in vitro and in vivo protein interaction approaches we show that p25 interacts also with BvIAA2 and BvIAA6. Moreover, we confirmed that the BNYVV infection is, indeed, accompanied by an elevated auxin level in the infected LRs. Nevertheless, expression levels of BvIAA2 and BvIAA6 remained unchanged upon BNYVV infection. Mutational analysis indicated that interaction of p25 with either BvIAA2 or BvIAA6 requires full-length proteins as even single amino acid residue substitutions abolished the interactions. Compared to p25-BvIAA28 interaction that leads to redistribution of BvIAA28 into cytoplasm, both BvIAA2 and BvIAA6 remained confined into the nucleus regardless of the presence of p25 suggesting their stabilization though p25 interaction. Overexpression of p25-interacting partners (BvIAA2, BvIAA6 and BvIAA28) in Nicotiana benthamiana induced an auxin-insensitive phenotype characterized by plant dwarfism and dramatically reduced LR development. Thus, our work reveals a distinct class of transcriptional repressors targeted by p25. [ABSTRACT FROM AUTHOR]

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is characterized by excessive lateral root (LR) formation. Auxin-mediated degradation of Aux/IAA transcriptional repressors stimulates gene regulatory networks leading to LR organogenesis and involves several Aux/IAA proteins acting at distinctive stages of LR development. Previously, we showed that BNYVV p25 virulence factor interacts with BvIAA28, a transcriptional repressor acting at early stages of LR initiation. The evidence suggested that p25 inhibits BvIAA28 nuclear localization, thus, de-repressing transcriptional network leading to LR initiation. However, it was not clear whether p25 interacts with other Aux/IAA proteins. Here, by adopting bioinformatics, in vitro and in vivo protein interaction approaches we show that p25 interacts also with BvIAA2 and BvIAA6. Moreover, we confirmed that the BNYVV infection is, indeed, accompanied by an elevated auxin level in the infected LRs. Nevertheless, expression levels of BvIAA2 and BvIAA6 remained unchanged upon BNYVV infection. Mutational analysis indicated that interaction of p25 with either BvIAA2 or BvIAA6 requires full-length proteins as even single amino acid residue substitutions abolished the interactions. Compared to p25-BvIAA28 interaction that leads to redistribution of BvIAA28 into cytoplasm, both BvIAA2 and BvIAA6 remained confined into the nucleus regardless of the presence of p25 suggesting their stabilization though p25 interaction. Overexpression of p25-interacting partners (BvIAA2, BvIAA6 and BvIAA28) in Nicotiana benthamiana induced an auxin-insensitive phenotype characterized by plant dwarfism and dramatically reduced LR development. Thus, our work reveals a distinct class of transcriptional repressors targeted by p25.

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled‐coil and leucine‐rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV‐encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)‐like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) is causal agent of rhizomania disease, which is the most devastating viral disease in sugar beet production leading to a dramatic reduction in beet yield and sugar content. The virus is transmitted by the ubiquitous distributed soil-borne plasmodiophoromycete Polymyxa betae that infects the root tissue of young sugar beet plants. Rz1 is the major resistance gene widely used in most sugar beet varieties to control BNYVV. The strong selection pressure on the virus population promoted the development of strains that can overcome Rz1 resistance. Resistance-breaking has been associated with mutations in the RNA3-encoded pathogenicity factor P25 at amino acid positions 67–70 (tetrad) as well as with the presence of an additional RNA component (RNA5). However, respective studies investigating the resistance-breaking mechanism by a reverse genetic system are rather scarce. Therefore, we studied Rz1 resistance-breaking in sugar beet using a recently developed infectious clone of BNYVV A-type. A vector free infection system for the inoculation of young sugar beet seedlings was established. This assay allowed a clear separation between a susceptible and a Rz1 resistant genotype by measuring the virus content in lateral roots at 52 dpi. However, mechanical inoculation of sugar beet leaves led to the occurrence of genotype independent local lesions, suggesting that Rz1 mediates a root specific resistance toward BNYVV that is not active in leaves. Mutation analysis demonstrated that different motifs within the P25 tetrad enable increased virus replication in roots of the resistant genotype. The resistance-breaking ability was further confirmed by the visualization of BNYVV in lateral roots and leaves using a fluorescent-labeled complementary DNA clone of RNA2. Apart from that, reassortment experiments evidenced that RNA5 enables Rz1 resistance-breaking independent of the P25 tetrad motif. Finally, we could identify a new resistance-breaking mutation, which was selected by high-throughput sequencing of a clonal virus population after one host passage in a resistant genotype. Our results demonstrate the feasibility of the reverse genetic system for resistance-breaking analysis and illustrates the genome plasticity of BNYVV allowing the virus to adapt rapidly to sugar beet resistance traits.

Rhizomania is one of the most devastating diseases of sugar beet worldwide. The disease poses a serious threat to Moroccan production and it is capable of significantly decreasing quality and yield of sugar beet plantations. The long-term survival of its fungal vector (Polymyxa betae) in soil makes it a very difficult disease to manage. Therefore, this study investigated the potential of a non-pathogenic fungal Fusarium oxysporum strain Fo47 to control Polymyxa betae. This biocontrol agent was applied as soil treatment, seed treatment, or a combination of the both treatments. A bio-test was performed on treated soil. After four weeks of culture, the roots of sugar beet seedlings were retrieved and analyzed by the DAS-ELISA test. Results indicated that F. oxysporium Fo47 reduced the activity and survival of P. betae when compared to a reference biocontrol agent Trichoderma harzianum, which only revealed significant in reducing the viral load of Beet Necrotic Yellow Vein Virus (BNYVV) as seed treatment. The non-pathogenic Fusarium oxysporum Fo47 was more effective as soil treatment and allowed almost the same reduction of BNYVV virus concentration as T. harzianum 908. Therefore, our findings emphasizes that the performance of the biocontrol agent depends on the method of application.

Beet necrotic yellow vein virus (BNYVV) is the cause of rhizomania, an important disease of sugar beet around the world. The multipartite genome of the BNYVV contains four or five single-stranded RNA that has been used to characterize the virus. Understanding genome composition of the virus not only determines the degree of pathogenicity but also is required to development of resistant varieties of sugar beet. Resistance to rhizomania has been conferred to sugar beet varieties by conventional breeding methods or modern genome engineering tools. However, over time, viruses undergo genetic alterations and develop new variants to break crop resistance. Here, we report the occurrence of genetic reassortment and emergence of new variants of BNYVV among the isolates of Thrace and Asia Minor (modern-day Turkey). Our findings indicate that the isolates harbor European A-type RNA-2 and RNA-3, nevertheless, RNA-5 is closely related to East Asian J-type. Furthermore, RNA-1 and RNA-4 are either derived from A, B, and P-types or a mixture of them. The RNA-5 factor which enhance the pathogenicity, is rarely found in the isolates studied (20%). The creation of new variants of the virus emphasizes the necessity to develop new generation of resistant crops. We anticipate that these findings will be useful for future genetic characterization and evolutionary studies of BNYVV, as well as for developing sustainable strategies for the control of this destructive disease. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) is causal agent of rhizomania disease, which is the most devastating viral disease in sugar beet production leading to a dramatic reduction in beet yield and sugar content. The virus is transmitted by the ubiquitous distributed soil-borne plasmodiophoromycete Polymyxa betae that infects the root tissue of young sugar beet plants. Rz1 is the major resistance gene widely used in most sugar beet varieties to control BNYVV. The strong selection pressure on the virus population promoted the development of strains that can overcome Rz1 resistance. Resistance-breaking has been associated with mutations in the RNA3-encoded pathogenicity factor P25 at amino acid positions 67–70 (tetrad) as well as with the presence of an additional RNA component (RNA5). However, respective studies investigating the resistance-breaking mechanism by a reverse genetic system are rather scarce. Therefore, we studied Rz1 resistance-breaking in sugar beet using a recently developed infectious clone of BNYVV A-type. A vector free infection system for the inoculation of young sugar beet seedlings was established. This assay allowed a clear separation between a susceptible and a Rz1 resistant genotype by measuring the virus content in lateral roots at 52 dpi. However, mechanical inoculation of sugar beet leaves led to the occurrence of genotype independent local lesions, suggesting that Rz1 mediates a root specific resistance toward BNYVV that is not active in leaves. Mutation analysis demonstrated that different motifs within the P25 tetrad enable increased virus replication in roots of the resistant genotype. The resistance-breaking ability was further confirmed by the visualization of BNYVV in lateral roots and leaves using a fluorescent-labeled complementary DNA clone of RNA2. Apart from that, reassortment experiments evidenced that RNA5 enables Rz1 resistance-breaking independent of the P25 tetrad motif. Finally, we could identify a new resistance-breaking mutation, which was selected by high-throughput sequencing of a clonal virus population after one host passage in a resistant genotype. Our results demonstrate the feasibility of the reverse genetic system for resistance-breaking analysis and illustrates the genome plasticity of BNYVV allowing the virus to adapt rapidly to sugar beet resistance traits. [ABSTRACT FROM AUTHOR]

Summary: Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana. A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22, ARF17/18, and TIR, were down-regulated. GRF1, which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus-induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin, and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana. These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.

Summary: Rhizomania of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is characterized by excessive lateral root (LR) formation leading to dramatic reduction of taproot weight and massive yield losses. LR formation represents a developmental process tightly controlled by auxin signaling through AUX/IAA‐ARF responsive module and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcriptional network. Several LBD transcription factors play central roles in auxin‐regulated LR development and act upstream of EXPANSINS (EXPs), cell wall (CW)‐loosening proteins involved in plant development via disruption of the extracellular matrix for CW relaxation and expansion. Here, we present evidence that BNYVV hijacks these auxin‐regulated pathways resulting in formation LR and root hairs (RH). We identified an AUX/IAA protein (BvAUX28) as interacting with P25, a viral virulence factor. Mutational analysis indicated that P25 interacts with domains I and II of BvAUX28. Subcellular localization of co‐expressed P25 and BvAUX28 showed that P25 inhibits BvAUX28 nuclear localization. Moreover, root‐specific LBDs and EXPs were greatly upregulated during rhizomania development. Based on these data, we present a model in which BNYVV P25 protein mimics action of auxin by removing BvAUX28 transcriptional repressor, leading to activation of LBDs and EXPs. Thus, the evidence highlights two pathways operating in parallel and leading to uncontrolled formation of LRs and RHs, the main manifestation of the rhizomania syndrome. [ABSTRACT FROM AUTHOR]

Rhizomania is one of serious threat to sugar beet production in Morocco and in several parts of the world. This disease led to a statistically significant decrease in the quality and yield of sugar beet plantations. Therefore, this study aimed at comparing the efficacy of six commonly used RNA extraction methods for the detection, recovery of RNA of beet necrotic yellow vein virus (BNYVV) and removal of amplification inhibitors by reverse transcription-polymerase chain reaction (RT-PCR). The efficiency of these extraction methods was then compared to that of a commercial isolation kit with high content of phenolic compounds. The results showed that the extraction with the lithium chloride technique, the commercial kit, and direct and membrane spotting crude extract methods were found effective in yielding a higher purity and a higher concentration of RNA when compared to the other tested methods. Extraction with the lithium chloride technique and the Qiagen kit (RNeasy Plant Mini Kit) allowed the most intense band, whereas the CTAB method has generated the least intense band. Furthermore, the silica capture extraction method did not yield any RNA after extraction and electrophoresis. Consequently, it was concluded that, of these six methods, the lithium chloride technique and the Qiagen kit are the most appropriate for the extraction of viral RNA from sugar beet samples prior to RT-PCR for detecting BNYVV. [ABSTRACT FROM AUTHOR]

Rhizomania disease, caused by the Beet necrotic yellow vein virus (BNYVV), is considered as one of the major constraints for sugar beet production, worldwide. As a result of the introgression of major resistance genes (Holly, Rz2) in commercially available sugar beet varieties, the virus has endured strong selection pressure since the 90s'. Understanding the virus response and diversity to sugar beet resistance is a key factor for a sustainable management of only few resistance genes. Here we report rhizomania surveys conducted in a rhizomania hot spot, the Pithiviers area (France) during a 4-year period and complementary to the study of Schirmer et al. ( 2005 ). The study aimed at evaluating the intra- and inter-field BNYVV diversity in response to different sources of resistance and over the growing season. To follow rhizomania development over the sugar beet growing season, extensive field samplings combined with field assays were performed in this study. The evolution of the BNYVV diversity was assessed at intra- and inter-field levels, with sugar beet cultivars containing different resistance genes (Rz1, Rz1 + Heterodera schachtii resistance and Rz1Rz2). Intra-field diversity was analyzed at the beginning and the end of the growing season of each field. From more than one thousand field samples, the simultaneous presence of the different A, B and P types of BNYVV was confirmed, with 21 variants identified at positions 67–70 of the p25 tetrad. The first variant, AYHR, was found most commonly followed by SYHG. Numerous mixed infections (9.93% of the samples), mostly of B-type with P-type, have also been evidenced. Different tetrads associated with the A- or B-type were also found with a fifth RNA-genome component known to allow more aggressiveness to BNYVV on sugar beet roots. Cultivars with Rz1+Rz2 resistant genes showed few root symptoms even if the BNYVV titre was quite high according to the BNYVV type present. The virus infectious potential in the soil at the end of the growing season with such cultivars was also lower despite a wider diversity at the BNYVV RNA3 sequence level. Rz1+Rz2 cultivars also exhibited a lower presence of Beet soil-borne virus (BSBV), a P. betae-transmitted Pomovirus. Cultivars with Rz1 and nematode (N) resistance genes cultivated in field infected with nematodes showed lower BNYVV titre than those with Rz1 or Rz1+Rz2 cultivars. Overall, the population structure of BNYVV in France is shown to be different from that previously evidenced in different world areas. Implications for long-term management of the resistance to rhizomania is discussed. [ABSTRACT FROM AUTHOR]

Two members of the Benyviridae family and genus Benyvirus , Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa . These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants. [ABSTRACT FROM AUTHOR]

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is a yield-limiting sugar beet disease that was observed to influence root resistance to freezing in storage. Thus, studies were conducted to gain a better understanding of the influence of BNYVV and freezing on sugar beet roots to improve pile management decisions. Roots from five commercial sugar beet cultivars (one susceptible and four resistant to BNYVV) were produced in fields under high and trace levels of rhizomania pressure and subjected to storage using five temperature regimes ranging from 0 to -4.4°C for 24 h. After cold treatment, eight-root samples were stored in a commercial indoor storage building (set point 1.1°C) for 50 days in 2014 and 57 days in 2015. Internal root temperature, frozen and discolored tissue, and moisture and sucrose loss were evaluated. The air temperature at 0, --1.1, and -2.2°C matched internal root temperature but internal root remained near -2.2°C when air temperature was dropped to -3.3 and -4.4°C. In a susceptible cultivar produced under high rhizomania pressure, the percentage of frozen tissue increased (P < 0.0001) from an average of 0 to 7% at 0, -1.1, and -2.2°C up to 16 to 63% at -3.3°C and 63 to 90% at -4.4°C, depending on year. Roots from the susceptible cultivar produced under low rhizomania pressure and those from the resistant cultivars from both fields only had elevated (P ≤0.05) frozen tissue at -4.4°C in 15 of 18 cultivar-year combinations. Frozen tissue was related to discolored tissue ( r² = 0.91), weight loss (r² = 0.12 to 0.28), and sucrose reduction (r² = 0.69 to 0.74). Consequently, BNYVV will not only lead to yield and sucrose loss in susceptible sugar beet cultivars but also to more frozen root tissue as temperatures drop below -2.2°C. Based on these observations, the air used to cool roots in nonfrozen sugar beet piles throughout the winter should not drop below -2.2°C to maximize sucrose retention. [ABSTRACT FROM AUTHOR]

The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the 'core' nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this 'core' sequence resides a conserved "coremin" motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing "coremin" at its 5' end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S. cerevisiae ribonuclease mutants identified the 5'-to-3' exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 'core' sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle. [ABSTRACT FROM AUTHOR]

Rhizomania is one of the most devastating diseases of sugar beet worldwide. The disease poses a serious threat to Moroccan production and it is capable of significantly decreasing quality and yield of sugar beet plantations. The long-term survival of its fungal vector (Polymyxa betae) in soil makes it a very difficult disease to manage. Therefore, this study investigated the potential of a non-pathogenic fungal Fusarium oxysporum strain Fo47 to control Polymyxa betae. This biocontrol agent was applied as soil treatment, seed treatment, or a combination of the both treatments. A bio-test was performed on treated soil. After four weeks of culture, the roots of sugar beet seedlings were retrieved and analyzed by the DAS-ELISA test. Results indicated that F. oxysporium Fo47 reduced the activity and survival of P. betae when compared to a reference biocontrol agent Trichoderma harzianum, which only revealed significant in reducing the viral load of Beet Necrotic Yellow Vein Virus (BNYVV) as seed treatment. The non-pathogenic Fusarium oxysporum Fo47 was more effective as soil treatment and allowed almost the same reduction of BNYVV virus concentration as T. harzianum 908. Therefore, our findings emphasizes that the performance of the biocontrol agent depends on the method of application. [ABSTRACT FROM AUTHOR]

Aim. This study was conducted to determine the prevalence of Beet necrotic yellow vein virus (BNYVV) circulating in agrocenoses of Ukraine and to develop some methods for detecting and identifying thе virus. Methods. The classical virology and molecular biological methods were used for the diagnosis and identification of viral pathogens, in particular, electron microscopy, plant-indicator method, polymerase chain reaction (PCR). The BNYVV was purified and all Koch's postulates to improve infectious nature of the disease were fulfilled. To investigate the plant cell responses to the virus infections we performed histochemical analysis. Results. The study contains results on various methods used for the diagnosis of BNYVV. Polyclonal rabbit sera, obtained as a result of virus immunization contained high-titre antibodies. Specific antiviral antibodies can be used for a serological diagnostic system development. The presence of BNYVV has also been confirmed by electron microscopy and mechanical inoculation of the indicator plant. It was established significant decreasing of the cell-wall-bound peroxidase concentration in phloem cells of conducting beams of sugar beet infected with BNYVV. At the same time, the accumulation of free isoenzymes in protoplasts was observed. Such a redistribution of peroxidase molecules in cell compartments is accompanied by a significant increase in their activity. Conclusions. The survey of agrocenoses in Cherkassy region showed sugar beets are infected with both Beet necrotic yellow vein virus and Beet mosaic virus. The presence of a mixed infection in plants was confirmed by PCR. Indicator plants using in bio-test detection of the virus produced typical BNYVV symptoms. It was shown the possibility of rapid BNYVV diagnosis by the histochemical method. It is recommended to examine the virus presence both in leaves and in root crops of sugar beet plants. [ABSTRACT FROM AUTHOR]

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV. [ABSTRACT FROM AUTHOR]

Sugar beet can be infected by many different viruses that can reduce yield; beet necrotic yellow vein virus (BNYVV) is one of the most economically important viruses of this crop plant. This report describes a new reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for identification of BNYVV. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection (without RNA extraction) of BNYVV was developed here and compared with DAS-ELISA and RT-LAMP assays. Our results show that the IC-RT-LAMP assay is a highly reliable alternative assay for identification of BNYVV. [ABSTRACT FROM AUTHOR]

Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV), the agent of rhizomania disease, causes severe economic losses in sugar beet fields in all over the world. The virus is transmitted by a plasmodiophorid vector, Polymyxa betae Keskin. Twenty soil samples, collected from sugar beet fields in northern and central parts of Turkey during surveys in 2004 and 2005 and known to be infested with viruliferous cultures of P. betae carrying BNYVV, were selected and used in this study. Sample selection was made according to symptom expression of beet seedlings in preliminary bait plant tests and locations of the soil samples that accurately represent the region from which they were taken. Total RNAs were extracted from sugar beet plants grown in these soils and used to amplify RNA-2 (nt. 19-1088) and RNA-3 (nt. 50-1268) of BNYVV by reverse transcription polymerase chain reaction (RT-PCR) method. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified products showed that most of BNYVV isolates studied were A-type strain, however, two isolates did not exactly match the band profile of A-type strain. Additionally, the presence of BNYVV RNA-5 component was investigated by RTPCR using the primers specific for P26 coding region. Four samples belonging to three provinces were found to be involving RNA-5 segment (20%). [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus(BNYVV) is the most devastating pathogen of sugar beet worldwide. This virus has been reported in the majority of sugar beet growing regions of Iran as well. For the present study, we collected samples from different sugar beet varieties with suspected symptoms of BNYVV from the main important sugar beet growing regions in eight provinces of Iran. Infection of collected samples to BNYVV was tested by ELISA and RT-PCR. Upon testing of 167 collected samples of BNYVV suspected through ELISA and RT-PCR, 115 (68.9%) were infected. Different incidences of BNYVV through surveyed provinces may represent the presence of diverse infective viral sources or resistance genes in tested sugar beet varieties which need further attempts to develop control strategies. Results also showed that BNYVV has been recently distributed throughout some surveyed regions. Otherwise, trace infection or resistance to BNYVV infection in some varieties of distinct regions may represent proper sources of resistance to BNYVV. [ABSTRACT FROM PUBLISHER]

Rhizomania is the most damaging and widespread disease of the sugar beet crop and is caused by Beet necrotic yellow vein virus vectored by the fungus Polymyxa betae. The only disease management tool used is resistant varieties. In the last 30 years, 'Rizor' and 'Holly' (Rz1) resistances have been the most widely used. Despite the lack of information, a common origin for both types of resistance was hypothesized by some breeders. The aim of this study was to assess the phylogenetic relationship between Rizor and Rz1 by means of SNP analysis. Fifty leaf samples of each were genotyped with a fingerprinting panel of 192 SNPs, using the QuantStudio 12K Flex system coupled with Taqman OpenArray technology. Analysis of molecular variance and principal coordinate analysis confirmed that Rizor and Rz1 cannot be distinguished as separate sources of resistance. [ABSTRACT FROM AUTHOR]

Background: Rhizomania is one of the most devastating diseases of sugar beet. It is caused by Beet necrotic yellow vein virus (BNYVV) transmitted by the obligate root-infecting parasite Polymyxa betae. Beta macrocarpa, a wild beet species widely used as a systemic host in the laboratory, can be rub-inoculated with BNYVV to avoid variation associated with the presence of the vector P. betae. To better understand disease and resistance between beets and BNYVV, we characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using the Illumina sequencing platform. Results: The overall de novo assembly of cDNA sequence data generated 75,917 unigenes, with an average length of 1054 bp. Based on a BLASTX search (E-value ≤ 10−5) against the non-redundant (NR, NCBI) protein, Swiss-Prot, the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, there were 39,372 unigenes annotated. In addition, 4,834 simple sequence repeats (SSRs) were also predicted, which could serve as a foundation for various applications in beet breeding. Furthermore, comparative analysis of the two transcriptomes revealed that 261 genes were differentially expressed in infected compared to control plants, including 128 up- and 133 down-regulated genes. GO analysis showed that the changes in the differently expressed genes were mainly enrichment in response to biotic stimulus and primary metabolic process. Conclusion: Our results not only provide a rich genomic resource for beets, but also benefit research into the molecular mechanisms of beet- BNYV Vinteraction. [ABSTRACT FROM AUTHOR]

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is an important disease affecting sugar beet. Control is achieved through planting of resistant varieties; however, following the introduction of Rz1 , new pathotypes that overcome resistance have appeared. To understand how BNYVV overcomes resistance, we examined quantitative protein differences during compatible and incompatible interactions when sugar beet is infected with either a traditional A-type strain or with an Rz1 resistance breaking strain. Proteomic data suggest distinct biochemical pathways are induced during compatible and incompatible sugar beet interactions with BNYVV. Pathways including the detoxification of reactive oxygen species, UB/proteasome, and photosynthesis should be studied in more depth to characterize roles in symptom development. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) is a serious threat to the sugar beet industry worldwide. However, little information is available regarding the genetic diversity and population structure of BNYVV in China. Here, we analyzed multiple sequences from four genomic regions ( CP , RNA3, RNA4 and RNA5) of a set of Chinese isolates. Sequence analyses revealed that several isolates were mixed infections of variants with different genotypes and/or different p25 tetrad motifs. In total, 12 distinct p25 tetrads were found in the Chinese BNYVV population, of which four tetrads were newly identified. Phylogenetic analyses based on four genes ( CP , RNA3- p25 , RNA4- p31 and RNA5- p26 ) in isolates from around the world revealed the existence of two to four groups, which mostly corresponded to previously reported phylogenetic groups. Two new subgroups and a new group were identified from the Chinese isolates in p25 and p26 trees, respectively. Selection pressure analysis indicated that there was a positive selection pressure on the p25 from the Chinese isolates, but the other three proteins were under a negative selection pressure. There was frequent gene flow between geographically distant populations, which meant that BNYVV populations from different provinces were not geographically differentiated. [ABSTRACT FROM AUTHOR]

Rhizomania is one of the most damaging and widely spread diseases in major sugar beet growing regions of the world. The causal agent, beet necrotic yellow vein virus ( BNYVV), is transmitted via the fungus Polymyxa betae, which retains it in the field for years. In this study, an RNA silencing mechanism was employed to induce resistance against rhizomania using intron-hairpin RNA (ihp RNA) constructs. These constructs were based on sequences of the BNYVV 5′-untranslated region of RNA-2 or the flanking sequence encoding P21 coat protein, with different lengths and orientations. Both transient and stable transformation methods produced effective resistance against rhizomania correlated with the transgene presence. Among the constructs, those generating ihp RNA structures with small intronic loops produced the highest frequencies of resistant events. The inheritance of transgenes and resistance was confirmed over generations in stably transformed plants. [ABSTRACT FROM AUTHOR]