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The economic success of sugar beet production depends largely on the control of various pathogens and pests. Beet necrotic yellow vein virus (BNYVV) has historically been a major threat to sugar beet growers because of the high yield losses and the fact that the virus is transmitted by a soilborne protist vector that cannot be controlled. Resistance breeding began after the disease was first described in 1959 and led to the identification of two monogenic resistance genes. Rz1 was the first resistance gene to be introduced into all varieties, enabling sugar beet production under high disease pressure. However, the widespread use of a single resistance gene resulted in high selection pressure on the virus population. As a result, resistance‐breaking of Rz1 was first reported in the early 2000s in the United States and later in many other countries. The mechanism of resistance‐breaking is based on mutations in the viral pathogenicity factor P25 and the presence of an additional RNA5 species. The second resistance gene being used in breeding is Rz2, which is based on a different resistance mechanism and is highly effective at sites where Rz1 has been overcome by the virus. In addition, Rz2 was the first rhizomania resistance gene to be cloned, and the triple gene block I of BNYVV was identified as the corresponding avirulence gene. This article highlights that resistance breeding has been key to the control of rhizomania, but history also shows that re‐emergence of the disease due to resistance‐breaking remains a permanent threat. [ABSTRACT FROM AUTHOR]

Introduction: Beet necrotic yellow vein virus (BNYVV) is a common viral pathogen that causes considerable economic loss globally. In the present study, a commercial realtime PCR test system and custom loop mediated amplification primers were used to detect the virus in asymptomatic sugar beet samples. Methods: A total of 107 of 124 samples tested positive for the presence of the A type BNYVV coat protein gene. Near complete sequences of RNA-3 and RNA-4 were obtained using reverse transcription, followed by nanopore sequencing of 14 samples. Results and discussion: A comparison with available sequences, including previously published isolates Kas2 and Kas3 from Kazakhstan, identified RNA-3 as similar to such of the P-type isolates Puthiviers and Kas3. RNA-5 was not detected using real-time PCR or cDNA amplification. Unique variable sites were identified in the p25 protein sequence translated from RNA-3. Another virus, beet cryptic virus 2 (BCV2), was identified and sequenced in samples infected with BNYVV. With 85.28% genome coverage, the identified BCV2 samples were very similar to the previously reported isolates from Hungary and Germany [ABSTRACT FROM AUTHOR]

Sugar beet hybrids are essential in modern agriculture due to their superior yields, disease resistance, and adaptability. This study investigates the role of the Rz2 gene in conferring resistance to beet necrotic yellow vein virus (BNYVV) in 14 sugar beet hybrids cultivated in Kazakhstan, including local and European varieties. The Rz2 gene, encoding a CC-NB-LRR protein, is a known resistance factor against BNYVV. Using RT-qPCR, we assessed Rz2 expression and detected BNYVV in bait plants inoculated with virus-infested soil. Our findings identified two highly resistant varieties: the Kazakh cultivar ‘Abulhair’ and the French line 22b5006. Additionally, the Kazakh cultivar ‘Pamyati Abugalieva’ and the French hybrid ‘Bunker’ exhibited increased resistance, suggesting involvement of other resistance loci. Notably, the Danish hybrid ‘Alando’, despite resistance to rhizomania, did not effectively resist BNYVV, highlighting possible evasion of its genetic factors by local virus strains. Our results emphasize the importance of Rz2 in resistance breeding programs and advocate for further research on additional resistance genes and the genetic variability of BNYVV in Kazakhstan. This work pioneers the molecular evaluation of BNYVV resistance in sugar beet in Kazakhstan, contributing to sustainable disease management and improved sugar beet production.

IntroductionBeet necrotic yellow vein virus (BNYVV) is a common viral pathogen that causes considerable economic loss globally. In the present study, a commercial realtime PCR test system and custom loop mediated amplification primers were used to detect the virus in asymptomatic sugar beet samples.MethodsA total of 107 of 124 samples tested positive for the presence of the A type BNYVV coat protein gene. Near complete sequences of RNA-3 and RNA-4 were obtained using reverse transcription, followed by nanopore sequencing of 14 samples.Results and discussionA comparison with available sequences, including previously published isolates Kas2 and Kas3 from Kazakhstan, identified RNA-3 as similar to such of the P-type isolates Puthiviers and Kas3. RNA-5 was not detected using real-time PCR or cDNA amplification. Unique variable sites were identified in the p25 protein sequence translated from RNA-3. Another virus, beet cryptic virus 2 (BCV2), was identified and sequenced in samples infected with BNYVV. With 85.28% genome coverage, the identified BCV2 samples were very similar to the previously reported isolates from Hungary and Germany

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) causes rhizomania disease in sugar beet (Beta vulgaris), which is controlled since more than two decades by cultivars harboring the Rz1 resistance gene. The development of resistance-breaking strains has been favored by a high selection pressure on the soil-borne virus population. Resistance-breaking is associated with mutations at amino acid positions 67-70 (tetrad) in the RNA3 encoded pathogenicity factor P25 and the presence of an additional RNA component (RNA5). However, natural BNYVV populations are highly diverse making investigations on the resistance-breaking mechanism rather difficult. Therefore, we applied a reverse genetic system for BNYVV (A type) to study Rz1 resistance-breaking by direct agroinoculation of sugar beet seedlings. The bioassay allowed a clear discrimination between susceptible and Rz1 resistant plants already four weeks after infection, and resistance-breaking was independent of the sugar beet Rz1 genotype. A comprehensive screen of natural tetrads for resistance-breaking revealed several new mutations allowing BNYVV to overcome Rz1. The supplementation of an additional RNA5 encoding the pathogenicity factor P26 allowed virus accumulation in the Rz1 genotype independent of the P25 tetrad. This suggests the presence of two distinct resistance-breaking mechanisms allowing BNYVV to overcome Rz1. Finally, we showed that the resistance-breaking effect of the tetrad and the RNA5 is specific to Rz1 and has no effect on the stability of the second resistance gene Rz2. Consequently, double resistant cultivars (Rz1+Rz2) should provide effective control of Rz1 resistance-breaking strains. Our study highlights the flexibility of the viral genome allowing BNYVV to overcome host resistance, which underlines the need for a continuous search for alternative resistance genes. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) causes rhizomania disease in sugar beet (Beta vulgaris), which is controlled since more than two decades by cultivars harboring the Rz1 resistance gene. The development of resistance-breaking strains has been favored by a high selection pressure on the soil-borne virus population. Resistance-breaking is associated with mutations at amino acid positions 67-70 (tetrad) in the RNA3 encoded pathogenicity factor P25 and the presence of an additional RNA component (RNA5). However, natural BNYVV populations are highly diverse making investigations on the resistance-breaking mechanism rather difficult. Therefore, we applied a reverse genetic system for BNYVV (A type) to study Rz1 resistance-breaking by direct agroinoculation of sugar beet seedlings. The bioassay allowed a clear discrimination between susceptible and Rz1 resistant plants already four weeks after infection, and resistance-breaking was independent of the sugar beet Rz1 genotype. A comprehensive screen of natural tetrads for resistance-breaking revealed several new mutations allowing BNYVV to overcome Rz1. The supplementation of an additional RNA5 encoding the pathogenicity factor P26 allowed virus accumulation in the Rz1 genotype independent of the P25 tetrad. This suggests the presence of two distinct resistance-breaking mechanisms allowing BNYVV to overcome Rz1. Finally, we showed that the resistance-breaking effect of the tetrad and the RNA5 is specific to Rz1 and has no effect on the stability of the second resistance gene Rz2. Consequently, double resistant cultivars (Rz1+Rz2) should provide effective control of Rz1 resistance-breaking strains. Our study highlights the flexibility of the viral genome allowing BNYVV to overcome host resistance, which underlines the need for a continuous search for alternative resistance genes.

Rhizomania caused by the Beet necrotic yellow vain virus (BNYVV) is the most damaging disease of sugar beet worldwide. Antibody-based resistance was used as an efficient strategy to generate transgenic sugar beet plants resistant to the virus through the expression of single-chain variable fragments (scFv) directed against a major coat protein of the virus, P21. Four anti-P21 scFv fusion proteins were targeted towards different subcellular compartments including the cytosol, apoplast, and mitochondrial outer membrane where P21 is probably located. Transgenic sugar beet plants showed levels of resistance correlated with the expression levels of scFv antibody when they were inoculated by rhizomania-infested soil. Although transgenic plants expressing scFv in various locations showed comparable titers of the virus, those expressing scFv targeted towards the apoplast or cytosol carrying the stabilizing KDEL tetrapeptide showed the highest rates of resistance against the virus. Further work may lead to rhizomania resistance. [ABSTRACT FROM AUTHOR]

Over the last three decades, the control of Beet necrotic yellow vein virus (BNYVV) in sugar beet has relied on the cultivation of varieties harbouring the Rz1 resistance gene. The strong selection pressure on BNYVV has favoured the development of resistance‐breaking strains that display mutations at amino acid positions 67–70 (tetrad) in the pathogenicity factor P25 and strains possessing an additional RNA component (RNA5). To evaluate the durability of Rz1 resistance, it is essential to monitor the ongoing virulence development in BNYVV populations. Therefore, we characterized BNYVV populations from different fields in Europe suspected to possess resistance‐breaking properties. The ability to overcome Rz1 resistance could be confirmed for several populations in greenhouse bait plant tests using susceptible and resistant genotypes. These populations were able to accumulate similar virus concentrations in both genotypes. Most of the populations harboured multiple P25 tetrad variants, but there was no obvious effect of the sugar beet genotype on their composition. The strong selection pressure on P25 was reflected by the overall high number of P25 haplotypes with several amino acids under positive selection. Two different RNA5 species closely related to either the French or the Asian RNA5 type were additionally identified in some populations. Finally, high‐throughput sequencing revealed the formation of genetic reassortments between different virus types, but the overall sequence diversity within BNYVV populations was relatively low and not affected by the genotype. Our results highlight the ongoing adaption of BNYVV towards Rz1 resistance, stressing the importance of alternative resistance sources. [ABSTRACT FROM AUTHOR]

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Sugar beet hybrids are essential in modern agriculture due to their superior yields, disease resistance, and adaptability. This study investigates the role of the Rz2 gene in conferring resistance to beet necrotic yellow vein virus (BNYVV) in 14 sugar beet hybrids cultivated in Kazakhstan, including local and European varieties. The Rz2 gene, encoding a CC-NB-LRR protein, is a known resistance factor against BNYVV. Using RT-qPCR, we assessed Rz2 expression and detected BNYVV in bait plants inoculated with virus-infested soil. Our findings identified two highly resistant varieties: the Kazakh cultivar 'Abulhair' and the French line 22b5006. Additionally, the Kazakh cultivar 'Pamyati Abugalieva' and the French hybrid 'Bunker' exhibited increased resistance, suggesting involvement of other resistance loci. Notably, the Danish hybrid 'Alando', despite resistance to rhizomania, did not effectively resist BNYVV, highlighting possible evasion of its genetic factors by local virus strains. Our results emphasize the importance of Rz2 in resistance breeding programs and advocate for further research on additional resistance genes and the genetic variability of BNYVV in Kazakhstan. This work pioneers the molecular evaluation of BNYVV resistance in sugar beet in Kazakhstan, contributing to sustainable disease management and improved sugar beet production.

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]

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, the most serious sugar beet disease worldwide. Since the first finding in Japan in 1969, BNYVV became widespread throughout Hokkaido in a few decades and led to the introduction of Rz1‐resistant sugar beet cultivars in the 1990s. Here, we report the historical progress of the BNYVV epidemic in Hokkaido from 1969 to 2019. Previous analysis on samples from 1991 showed that BNYVV isolates were classified into three strains (named O, D, and T) based on the RNA3‐encoded p25 gene. The O‐type viruses were widely detected in Hokkaido, while the D‐ and T‐type viruses were detected in limited areas. The RNA5, encoding the p26 gene, was initially contained in some D‐ and O‐type isolates but not in any T‐type isolates. Interestingly, recent sample analysis revealed that RNA5‐containing T‐type viruses, seemingly more virulent than the other two strains, were widely detected in Hokkaido. Additionally, a small group of virus isolates harbouring a new p25 gene (named C) was found in limited areas. These results suggest that the T‐type viruses, which accompanied RNA5, have been preferentially spread from a limited area to other districts over the last few decades and that this spread might be strongly associated with the recent introduction of Rz1‐resistant sugar beet cultivars. BNYVV‐positive samples also contained mainly beet soil‐borne virus and traces of beet virus Q, both of which are the first to be recorded in Japan. [ABSTRACT FROM AUTHOR]

Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots. [ABSTRACT FROM AUTHOR]

Agrobacterium rhizogenes–mediated transformation of sugar beet hairy roots expressing single-chain variable fragment (scFv) was exploited to evaluate the efficacy of four antibody-based constructs for interfering with the Beet necrotic yellow vein virus infection. The scFv specific to a major coat protein of virus, p21, was targeted to various cellular compartments including the cytosol (pIC and pICC constructs), apoplast (pIA), and mitochondrion (pIM). After mechanical virus inoculation, most of the hairy root clones expressing scFv in the cytosol displayed low virus titers while the majority of transgenic hairy root clones accumulated antibody in outer membrane of mitochondria or apoplast were infected. This hairy root system provided an efficient and rapid approach to initially investigating root disease resistance like rhizomania prior to transform whole recalcitrant plants such as sugar beet.

Rhizomania is one of the most damaging sugar beet diseases and is caused by Beet necrotic yellow vein virus (BNYVV). In order to interfere with viral propagation, the expression of recombinant antibody strategy was employed. The coding sequence of a single-chain variable fragment (scFv) specific to the main BNYVV coat protein, P21, was targeted to the cytosol, apoplast or outer membrane of mitochondrion of Nicotiana benthamiana plants. Also, an endoplasmic reticulum retention signal peptide, KDEL, was added at the C-terminal of the scFv protein which presumably stabilizes it in cytoplasm. Although the accumulation levels of scFv could not be correlated to the levels of the inhibition of viral accumulation, the titer of the virus in all groups of transgenic plants was significantly lower than the wild type ones when grown in BNYVV-infested soil. No significant differences were found in the number of resistant plants targeting P21-scFv in either putative subcellular locations.

Rhizomania is one of the most damaging and widespread diseases in the major sugar beet (Beta vulgaris L. subsp. vulgaris) growing regions worldwide. The causal agent, Beet necrotic yellow vein virus, is transmitted by a plasmodiophorid vector, Polymyxa betae Keskin. In this study, the inhibition of BNYVV multiplication was evaluated, under field conditions, in six sugar beet transgenic events generated based on gene silencing of the major coat protein gene (CP21). Phenotypic criteria, including scoring for disease symptoms and an immunoassay for viral accumulation, indicated that the transgenic events were less susceptible to rhizomania and had lower virus titre rates than non-transgenic plants of the susceptible cultivar '9597'. Disease severity and virus titres were comparable to the resistant cultivar 'Dorothea', which carries the Rz1-resistance gene and was bred through traditional means. This study provides a proof of concept for the effectiveness of the gene-silencing mechanism for introducing rhizomania resistance in sugar beet at the field level. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) generally has a four‐segmented positive‐sense RNA genome (RNAs 1–4), but some European and most Asian strains have an additional segment, RNA5. This study examined the effect of RNA5 and RNA3 on different sugar beet cultivars using a Polymyxa‐mediated inoculation system under field and laboratory conditions. In field tests, the degree of sugar yield served as an index for assessing the virulence of BNYVV strains. Japanese A‐II type isolates without RNA5 caused mostly 15%–90% sugar yield reductions, depending on the susceptibility of sugar beet cultivars, whereas the isolates with RNA5 induced more than 90% yield losses in the seven susceptible cultivars, but small yield losses in one Rz1‐resistant and Rizor cultivars. However, a laboratory‐produced isolate containing RNA5 but lacking RNA3 caused higher yield losses in Rizor than in susceptible plants, and induced scab‐like symptoms on the root surface of both susceptible and resistant plants. In laboratory tests, A‐II type isolates without RNA5 had low viral RNA accumulation levels in roots of Rizor and Rz1‐resistant plants at early stages of infection, but in the presence of RNA5, viral RNA3 accumulation levels increased remarkably. This increased RNA3 accumulation was not observed in roots of the WB42 accession with the Rz2 gene. In contrast, the presence of RNA3 did not affect RNA5 accumulation levels. Collectively, this study demonstrated that RNA5 is involved in the development of scab‐like symptoms and the enhancement of RNA3 accumulation, and suggests these characteristics of RNA5 are associated with Rz1‐resistance breaking. [ABSTRACT FROM AUTHOR]

The Beet necrotic yellow vein virus (BNYVV) causes rhizomania in sugar beet (Beta vulgaris L.), which is one of the most destructive diseases in sugar beet worldwide. In breeding projects towards resistance against BNYVV, the enzyme-linked immunosorbent assay (ELISA) is used to determine the virus concentration in plant roots and, thus, the resistance levels of genotypes. Here, we present a simulation study to generate 10,000 small samples from the estimated density functions of ELISA values from susceptible and resistant sugar beet genotypes. We apply receiver operating characteristic (ROC) analysis to these samples to optimise the cutoff values for sample sizes from two to eight and determine the false positive rates (FPR), true positive rates (TPR), and area under the curve (AUC). We present, furthermore, an alternative approach based upon Bayes factors to improve the decision procedure. The Bayesian approach has proven to be superior to the simple cutoff approach. The presented results could help evaluate or improve existing breeding programs and help design future selection procedures based upon ELISA. An R-script for the classification of sample data based upon Bayes factors is provided.

Rhizomania is one of the most damaging sugar beet diseases and is caused by Beet necrotic yellow vein virus (BNYVV). In order to interfere with viral propagation, the expression of recombinant antibody strategy was employed. The coding sequence of a single-chain variable fragment (scFv) specific to the main BNYVV coat protein, P21, was targeted to the cytosol, apoplast or outer membrane of mitochondrion of Nicotiana benthamiana plants. Also, an endoplasmic reticulum retention signal peptide, KDEL, was added at the C-terminal of the scFv protein which presumably stabilizes it in cytoplasm. Although the accumulation levels of scFv could not be correlated to the levels of the inhibition of viral accumulation, the titer of the virus in all groups of transgenic plants was significantly lower than the wild type ones when grown in BNYVV-infested soil. No significant differences were found in the number of resistant plants targeting P21-scFv in either putative subcellular locations. [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.

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]

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]

The plasmodiophorid Polymyxa betae is considered as the only natural transmitting agent of Beet necrotic yellow vein virus (BNYVV), the most devastating agent of sugar beet fields throughout the world. To evaluate for the first time the genetic diversity of P. betae isolated from different autumn and spring sugar beet fields, and also to detect the presence of virus in these isolates, susceptible sugar beet plants (cv. Regina) were grown in soil samples collected from 10 different regions of Iran. P. betae detection was carried out using root microscopic observations, DAS-ELISA, and PCR-based methods. Results showed the presence of plasmodiophorids in all soil samples. Complementary assays also revealed the presence of viruses in soil samples collected from Khorasan Razavi, Fars, Hamadan and Kermanshah regions. The genetic diversity was evaluated through comparing glutathione-S-transferase nucleotide sequences amplified by PCR with the Internal Transcribed Spacers region. Results showed no significant differences in nucleotide sequences between virus-bearing and virus-free isolates of P. betae.

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.

Rhizomania, şeker pancarının en zararlı hastalıklarından biridir. Hastalık, enfeksiyon şiddetine bağlı olarak, şeker pancarında % 90’a varan kök verimi ve % 70’e varan şeker verimi kayıplarına yol açmaktadır. Türkiye’de Marmara, Orta Karadeniz ve İç Anadolu’nun bazı bölgelerinde hastalığın varlığı rapor edilmiş ve hastalık, dünyada olduğu gibi diğer bölgelere giderek yayılmaktadır. Bu hastalığa pancar nekrotik sarı damar virüsü BNYVV yol açmakta ve Polymyxa betae fungusu vektörlük etmektedir. Yayılma esas olarak, bulaşık toprak veya şeker pancarının taşınmasıyla olmaktadır. Hastalığın mücadelesi, bulaşık ekim alanlarında yalnızca kültürel tedbirler alınarak yapılmaktadır. Günümüzde, en etkili kontrol tedbiri, kısmen dayanıklı şeker pancarı çeşitlerini kullanmaktır. Soğuk topraklara erken ekim, aşırı sulamanın azaltılması, toprak sıkışmasından kaçınılması ve münavebe uygulaması gibi yardımcı tedbirler topraktaki virus seviyesini düşürebilmektedir. Bu nedenle, bulaşık alanların büyük ekonomik kayıplara yol açmadan tespit edilmesi ve kültürel tedbirlerin alınması büyük önem taşımaktadır. Üç yıllık münavebe uygulamasına göre, 1996-1998, 1999-2001 ve 2002-2004 yıllarında olmak üzere, üçer yıllık üç dönem halinde 17 şeker fabrikasının 102 bölgesinin şeker pancarı ekim alanlarında sürveyler yapılarak, şeker pancarı kök örnekleri toplanmıştır. Bu örnekler DAS-ELISA ile test edilmiş ve BNYVV’nin yaygınlığı belirlenmiştir. Buna gore, 1996-1998 döneminde 4 fabrikada toplam % 19.30 46200 ha , 1999-2001 döneminde 9 fabrikada % 31.42 94140 ha ve 2002-2004 döneminde 15 fabrikada % 48.66 203640 ha Rhizomania yaygınlığı tespit edilmiştir

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]

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Field experiments were conducted at the medium heavy luvisol under the unirrigated conditions at the locality with continental climate in the years 2002 and 2003. Foliar preparations ´Avit 35´ and ´Humix univerzál plus´ were sprayed at the sugar beet leaves during the vegetation. These liquid foliar preparations contain growth stimulators (humates, ethanolamine, ureasalicylate) enriched by macroelements and microelements. Two sugar beet varieties (STRUBEDIECKMANN) were involved in the experiment: a rhizomania sensitive one (´Swing´) and a rhizomania tolerant one (´Takt´). The experimental locality was without BNYVV infection. Infl uence of foliar preparations in interaction with other factors (weather conditions, variety) on sugar beet yield and quality parameters (root yield, digestion, molasses forming substances, refi ned sugar yield) was evaluated in the experiment. Foliar preparations high signifi cantly increased the root yield and digestion in the year with suffi cient rainfalls and decreased molasses substances content in roots in both experimental years. There were high signifi cant differences in digestion between the varieties. Rhizomania tolerant variety (´Takt´) reached better yield and quality parameters than the rhizomania sensitive variety (´Swing´) in conditions without rhizomania infection.

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]

Large changes introduced in the sugar beet production technology in the Vojvodina Province over last 40 years resulted in changes in the etiology and harmfulness of different agents of sugar beet root diseases. Improvements in cultivation practices reduced the harmfulness of some diseases while increased the harmfulness of others. Some disease agents became obsolete, but others gained importance. New agents of root diseases were found. The most frequent damages, persisting over long periods of time were caused by seedling damping-off, Fusarium root rot, charcoal root rot, parasitic (Rhizomania) and non-parasitic root bearding. The parasitic damping-off caused by several fungal species but most frequently by Phoma betae occurred at the time when multigerm seeds were used in combination with extensive cultural practices. The agents of seedling diseases completely lost their significance as the consequence of switching to fungicide - treated monogerm seeds, earlier planting and improved soil tillage. In the period of intensive use of agricultural chemicals, seedling damping-off occurred frequently due to the phytotoxic action of chemicals (insecticides, herbicides and mineral fertilizers). In some years, frosts caused damping- off of sugar beet seedlings on a large scale in the Vojvodina Province. Poor sugar beet germination and emergence were frequently due to spring droughts. Sometimes they were due to strong winds. The occurrence of Fusarium root rot and charcoal root rot intensified on poor soils. Fusariosis symptoms were exhibited as plant wilting and different forms of root rot. In recent years root tip rot has occurred frequently in the first part of the growing season causing necrosis and dying of plants. Lateral roots tended to proliferate from the healthy tissue, giving the root a bearded appearance similar to Rhizomania. Fusarium oxysporum was the most frequent agent of this fusariosis. F. graminearum, F. equiseti, F. solani have also been identified in recent years as the agent of root rot, but its importance was much lower. Charcoal root rot and plant wilting (Macrophomina phaseolina) have caused extensive damages in sugar beets, especially under the conditions of severe drought and high temperatures in summer. In some years, it was the dominant agent of root rot. Mixed infections caused by fungi from the genera Fusarium and M. phaseolina were encountered frequently. The extent of damage caused by these diseases was reduced by improved pro- duction technology. Rhizomania of sugar beet (caused by beet necrotic yellow vein virus) was identified in Serbia in the 1970s. Results of recent investigations have shown that BNYVV is widespread in Vojvodina, since the virus was found on 36,7% (24,674 ha) of acreages from 67,213 ha of total sugar beet acreages inspected on incidence of BNYVV in the period from 1997 to 2004 year. In the last few years, the occurrence of Rhizoctonia root rot (Rhizoctonia solani) was registered in some localities in Vojvodina.

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]

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.

Beet necrotic yellow vein virus (BNYVV) was shown to occur in sugar beets in three of six regions surveyed in Lithuania. Characterizing the coat protein (CP) and p25 genes by RT-PCR and sequencing showed the presence of A and B types of BNYVV isolates. The p25 protein of Lithuanian BNYVV isolates contained a distinct amino acid tetrad (residues 67-70) with AYHR associated with a B type isolate and AHHG with two A type isolates. These sequence features were consistent with phylogenetic grouping of isolates based on CP and p25 gene sequences. This is the first characterization of BNYVV isolates in Lithuania. [ABSTRACT FROM AUTHOR]