Beet Necrotic Yellow Vein Virus Research Articles

Breeding for resistance to rhizomania in sugar beet: a review.

Currently rhizomania is the most important disease insugar beet worldwide, and attack can lead to seriousyield losses. The disease is caused by beet necroticyellow vein virus (BNYVV) that is transmitted by thesoil-borne fungus Polymyxa betae. Breeding sugarbeet cultivars with resistance to rhizomania isregarded as the most appropriate way to enablecontinued production of this crop in BNYVV-infestedfields and also to slow the spread of the disease.Breeding for resistance started with selection byscoring disease symptoms in field experiments. Thedevelopment of non-destructive greenhouse tests, withdetermination of the virus concentration in rootletsusing ELISA, has greatly improved the efficiency ofselection. In this paper the impact of scientificresearch on the progress in breeding cultivars withresistance to rhizomania is reviewed. This includesthe distribution, composition, and pathogenicity ofthe virus, the sources of resistance to virus andvector, the genetics of virus resistance, progresswith breeding methods, and the use of molecularmarkers and pathogen-derived resistance. The yieldsand quality characteristics of recently introducedresistant cultivars now equal those of the commercialsusceptible cultivars.

Changes in Ribonuclease and Glucose-6-Phosphate Dehydrogenase Activities Induced by Beet Necrotic Yellow Vein Virus in Sugar Beet

Activities of host ribonucleases and glucose-6-phosphate dehydrogenase were studied in three cultivars (Monosvalof, Steffi and Rimini) of sugar beet differing in their resistance to beet necrotic yellow vein virus (BNYVV). No differences were found in the susceptibility of cultivars to BNYVV between mechanically inoculated and Polymyxa betae (a natural fungal vector of the virus) infected plants, but the culmination of reproduction curves of BNYVV in mechanically inoculated plants was observed one week earlier than in plants inoculated by means of P. betae. The activities of ribonucleases corresponded with virus multiplication. In roots, activities of ribonucleases reached a maximum at day 7; in leaves, maximum activity was found at day 21 in cv. Monosvalof, and at day 14 in cv. Steffi. The relatively resistant cultivar Rimini showed much lower activities. The activity of glucose-6-phosphate dehydrogenase was only slightly increased at the time of culmination of the BNYVV reproduction curve in cvs. Monosvalof and Steffi.

RNA 3 Deletion Mutants of Beet Necrotic Yellow Vein Virus Do Not Cause Rhizomania Disease in Sugar Beets

ABSTRACT Two mutant strains of beet necrotic yellow vein virus (BNYVV) containing deletions in RNA 3 were obtained by single lesion transfers in Tetragonia expansa. The deleted regions encode either 94 or 121 amino acids toward the C-terminal part of the 25-kDa protein (P25). Wild-type and mutant virus strains were inoculated by Polymyxa betae to sugar beet seedlings of susceptible and partially resistant cultivars. No differences were found in virus content in rootlets between mutant and wild-type viruses or between susceptible and resistant cultivars after culture for 4 weeks in a growth cabinet. However, when virus-inoculated seedlings were grown in the field for 5 months, the wild-type virus caused typical rhizomania root symptoms (69 to 96% yield loss) in susceptible cultivars, but no symptoms (23% loss) developed in most plants of the resistant cultivar, and BNYVV concentrations in the roots were 10 to 20x lower in these plants than in susceptible plants. In contrast, the mutant strains caused no symptoms in susceptible or resistant cultivars, and the virus content of roots was much lower in both cultivars than in wild-type virus infections. Wild-type RNA 3 was not detectable in most of the taproots of a resistant cultivar without any symptoms, suggesting that replication of undeleted RNA 3 was inhibited. These results indicate that the P25 of BNYVV RNA 3 is essential for the development of rhizomania symptoms in susceptible cultivars and suggest that it may fail to facilitate virus translocation from rootlets to taproots in the partially resistant cultivar.

Specificity of TAS-ELISA for Beet Necrotic Yellow Vein Virus and Its Application for Determining Rhizomania Resistance in Field-Grown Sugar Beets.

Levels of beet necrotic yellow vein virus (BNYVV), as measured by triple-antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), were compared with biological evaluations in representative commercial and experimental sugar beet cultivars developed for production in the United States and ranging in their reactions to rhizomania from uniformly susceptible to highly resistant. TAS-ELISA was specific for BNYVV and did not react with related soilborne sugar beet viruses. Differences in absorbance (A405nm) values measured in eight cultivars closely correlated with the dosage and frequency of the Rz allele, which conditions resistance to BNYVV. A diploid (Rzrz) hybrid had a significantly lower absorbance value (less virus) than a similar triploid (Rzrzrz) hybrid. Cultivars that segregated (Rzrz:rzrz) had higher absorbance values than uniformly resistant (Rzrz) hybrids, as was expected. For all cultivars, absorbance values decreased as the season progressed. Absorbance value was significantly positively correlated with rhizomania disease index score and negatively correlated with individual root weight, plot root weight, and sugar yield. This information should be useful in resistance-breeding and -evaluation programs and in the sugar industry when considering cultivar choice, inoculum production, and future crop rotations.

Inheritance of resistance to beet necrotic yellow vein virus in Beta vulgaris conferred by a second gene for resistance

Rhizomania is a serious disease of sugar beet, caused by beet necrotic yellow vein virus (BNYVV). The disease can only be controlled by the use of resistant cultivars. The accession Holly contains a single dominant gene for resistance, called Rz. The identification of a locus for resistance that differs from Rz would provide possibilities to produce cultivars with multiple resistance to BNYVV. Inheritance of resistance to BNYVV was studied by screening progenies of crosses between resistant plants of the accessions Beta vulgaris subsp. maritima WB42 and B. vulgaris subsp. vulgaris Holly-1-4 or R104. Observed and expected segregation ratios were compared to elucidate whether the resistance genes in the three accessions are alleles or situated on different loci. STS markers, linked to the genes for resistance, were used to study the segregation in more detail. The results demonstrated that the genes for resistance to BNYVV inHolly-1-4 and WB42 are closely linked. The gene for resistance in R104 is at the same locus as in Holly-1-4, and also closely linked to the gene in WB42. As the Holly resistance gene has been named Rz, the name Rz2 is proposed to refer to the resistance gene in WB42. Consequently, the gene Rz should be referred to as Rz1.

Evidence of Cross-Protection Between Beet Soilborne Mosaic Virus and Beet Necrotic Yellow Vein Virus in Sugar Beet.

A high degree of reciprocal cross-protection occurred in sugar beets between beet soilborne mosaic virus (BSBMV) and beet necrotic yellow vein virus (BNYVV) in greenhouse experiments. The incidence of cross-protection depended on the interval between inoculations with protecting and challenging virus; longer inoculation intervals enhanced the incidence of cross-protection. Cross-protection was most effective when the inoculation interval was between 5 and 10 days, a period during which virus accumulated to a maximum level in plants singly infected with BSBMV or BNYVV. Results obtained by enzyme-linked immunosorbent assay (ELISA) and Western blotting were consistent and indicated that cross-protection affected viral capsid protein accumulation. RNA of both protecting and challenging viruses was detected in doubly infected plants by using reverse transcription-polymerase chain reaction (RT-PCR), indicating that RNA of the challenge virus was present in protected plants, but capsid protein was undetected by serological tests. BSBMV is a closely related but serologically distinct virus that provides an excellent system for studies of cross-protection with BNYVV.

Effects of structural modifications upon the accumulation in planta of replicons derived from beet necrotic yellow vein virus RNA 3.

Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3' and 5' 'core' replication origins (promoters) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting 'replicon' can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to approximately 240 nucleotides of sequence from the region immediately upstream of the core 3'-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral 'spacer' sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3'-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons.

Transgenic plants expressing the TGB1 protein of peanut clump virus complement movement of TGB1-defective peanut clump virus but not of TGB1-defective beet necrotic yellow vein virus

The triple gene block (TGB) of peanut clump virus (PCV) is indispensable for cell-to-cell movement. To determine if the TGB gene arrangement is necessary for viral multiplication, we introduced the p51 gene corresponding to the first PCV TGB protein into Nicotiana benthamiana plants; high-level expression of P51 was obtained in regenerated transformed plants. Two lines of the R5 generation of transgenic plants were used for complementation experiments. To this end deficient PCV RNA 2 transcripts (p51 deletion mutant, RNA 2Δ51) which could not multiply in the whole plant were co-inoculated with wild-type PCV RNA 1 transcripts into P51-transgenic plants. Viral multiplication was observed in 19%–67% of the transgenic plants, depending upon the line. However, no trans-complementation was observed when P51-transgenic plants were inoculated with beet necrotic yellow vein virus defective in the corresponding TGB1 protein, the P42 protein.

Selection of beet necrotic yellow vein virus specific single-chain Fv antibodies from a semi-synthetic combinatorial antibody library

Methods for the generation of monoclonal antibodies against plant viruses are limited because current hybridoma techniques do not allow efficient exploitation of the immune repertoire. Moreover, the immunization procedures often lead to a bias towards an immunodominant contaminant in the immunogen preparation and not to the plant virus itself. The selection of six different single-chain antibody variable fragments (scFv) against beet necrotic yellow vein virus from a semi-synthetic human combinatorial antibody library showed the feasibility of the phage display system. No bias towards minor contaminants in the purified virus preparation was observed in ELISA, as all the selected scFvs reacted only with beet necrotic yellow vein virus infected plant homogenates. In addition, two of the isolated beet necrotic yellow vein virus-specific scFvs could be produced in E. coli as a scFv fusion protein with alkaline phosphatase, and were applied in ELISA as specific ready to use antibody-enzyme conjugates. Because of their specificity, these antibodies have potential to be used as reagents in sensitive diagnostic assays for routine testing for beet necrotic yellow vein virus in sugar beets.

Variation in Pathogenicity and Multiplication of Beet Necrotic Yellow Vein Virus (BNYVV) in Relation to the Resistance of Sugar-beet Cultivars

Some partially resistant cultivars varied in their response to beet necrotic yellow vein virus (BNYW), which could be due to the occurrence of different pathotypes. In the past three different types of BNYVV could be identified. Since in the field no consistent cultivar×virus source interaction could be detected, greenhouse trials were carried out under more standardised conditions, starting with a similar initial density of BNYVV. Cultivars with different degrees of resistance varied in their response to various types of beet necrotic yellow vein virus (BNYVV). The B type appeared to be less damaging than the A and P types. The virus content in the tap roots and the ratio of the virus content in tap roots to that in lateral roots were both higher in P type than in A or B type infections indicating that the P type moves more rapidly in the plants than the two other BNYVV types. The percentage of plants in which the virus reaches only a low concentration (less than 56 ng/ml of sap) is much lower in P type than in A or B type infections. Frequency distribution diagrams of individual plants showing different BNYVV levels reveal considerable differences between various cultivars.

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets (Beta vulgaris ssp. maritima) has to be considered. In this study the transfer of BNYVV‐ (beet necrotic yellow vein virus) resistance and herbicide‐tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.

SALINE SOIL CONDITION DECREASES RHIZOMANIA INFECTION OF BETA VULGARIS

Rhizomania, caused by the beet necrotic yellow vein virus (BNYVV), is a world-wide problem for sugar beet production. Although rhizomania was first detected in sugar beet in the northern Italian Po Valley, wild beet on the Adriatic coast near the Po estuary show no virus infection under natural conditions. Our aim was to examine whether this absence of virus infection is caused by endogenous wild beet resistance or by the saline soil conditions of coastal habitats. Greenhouse experiments were carried out with pot plants grown from coastal seed and with a BNYVV-susceptible sugar beet variety as control. The plants were grown in highly BNYVV infested soil. To simulate saline conditions, the pots were periodically irrigated with sea salt solutions of two different concentrations or with tap water as a control. The BNYVV levels in the plant were measured by ELISA. Without salt, a range of tolerances to BNYVV was observed in plants derived from different coastal populations. One population was highly virus resistant, two were more heterogeneous and three populations were highly susceptible. In the presence of salt the data showed a significant reduction of the number of infected plants. These results indicate that the absence of virus in wild beet populations is mainly due to the saline soil conditions in coastal habitats. This phenomenon will be important for the biosafety assessment of gene flow of BNYVV resistance genes from transgenic sugar beet to wild beet populations in coastal habitats.

Cell-to-cell movement of beet necrotic yellow vein virus: I. Heterologous complementation experiments provide evidence for specific interactions among the triple gene block proteins.

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.

The genome organization of the broad bean necrosis virus (BBNV).

The genome of the broad bean necrosis virus Oita-isolate (BBNV-O) [RNA1 (6.0 kb), RNA2 (2.8 kb) and RNA3 (2.4 kb)] was cloned and sequenced. Computer analysis indicates that methyltransferase, helicase and RNA-dependent RNA polymerase (RdRp) motifs are present in RNA1. The viral capsid protein (CP) cistron is located at the 5' terminal end of RNA2 and the Mr of CP (20 K) is close to that determined by SDS-PAGE analysis. An ochre codon (UAA) in the CP cistron is thought to be partially suppressed to produce a large readthrough protein. RNA3 possesses typical motifs of triple gene block proteins, which are also reported in several other plant viruses. The furovirus genome organization and phylogenetic analysis using RdRp and CP amino acid sequences suggest that BBNV is closely related to potato mop-top virus (PMTV), but is relatively distantly related to other furoviruses. The data also suggest that the genus Furovirus should be separated into several genera: the prototypical genus Furovirus, which excludes the following viruses: the PMTV group including BBNV; the beet necrotic yellow vein virus (BNYVV) group; and the peanut clump virus (PCV) group.

Vascular movement of beet necrotic yellow vein virus in Beta macrocarpa is probably dependent on an RNA 3 sequence domain rather than a gene product.

RNAs 1 and 2 of beet necrotic yellow vein virus (BNYVV) carry the functions enabling viral RNA replication, cell-to-cell movement, virus assembly and vascular movement of the virus in the systemic host Spinacea oleracea. In Beta macrocarpa, on the other hand, BNYVV RNA 3 is required for vascular movement. Replication-competent RNA 3 transcripts carrying various point mutations and deletions were coinnoculated with RNAs 1 and 2 to young leaves of B. macrocarpa and the ability of the virus to multiply on the inoculated leaves and to invade the plant systemically was examined. None of the RNA 3 mutants tested interfered with virus multiplication in the inoculated leaves. Point mutations designed to specifically block or truncate translation of the ORFs of the two known RNA 3 gene products, P25 and N, did not interfere with vascular movement. Vascular movement was not inhibited by deletions eliminating the short 5'-proximal ORF on RNA 3 (ORF A) or by point mutations blocking putative translation of the short 5'-proximal ORF (ORF S) on RNA 3sub, a subgenomic RNA derived from RNA 3. On the other hand, deletions in a 'core region' encompassing nucleotides 1033-1257 of RNA 3 completely blocked vascular movement of the virus while removal of sequences flanking the core region lowered its efficiency. The observations suggest that some feature of the RNA 3 sequence rather than an RNA-3 coded protein is important for vascular movement of BNYVV in B. macrocarpa.

Furovirus isolation and RNA extraction.

The Furovirus (fungus-transmitted rod-shaped) group, whose type member is soil-borne wheat mosaic virus (SBWMV) (), is a very heterogeneous group infecting a wide variety of hosts. The group consists of viruses that are naturally transmitted by soil-borne fungal vectors, generally of the family Plasmodiophorales. Virions are rigid rods and contain two or more genome components consisting of positive-sense RNA. Furoviruses typically infect roots, but can also invade, or be inoculated to, leaves. As sequence data for different furoviruses has become available, it has become clear that there are significant differences in genetic organization within the group (,). We find it convenient to divide the viruses into two major subgroups (Fig. 1), which are distinguished by the nature of the protein(s) putatively involved in cell-to-cell movement. Subgroup 1 consists of SBWMV, which appears to have a movement protein (MP) of the type characterized by the 30-kDa protein of tobacco mosaic virus (TMV) (). Subgroup 2 furoviruses (beet necrotic yellow vein virus [BNYVV], peanut clump virus [PCV], and potato moptop virus [PMTV]) do not have a TMV type MP but instead possess a cluster of three slightly overlapping genes known as the triple gene block (TGB), which has been shown for BNYVV (), and is presumed for the other viruses (, , ) to mediate viral cell-to-cell movement. Open image in new window Fig 1. Genetic organization of characterized members of the furovirus group. RNA1 of PMTV has not yet been sequenced, but presumably encodes the viral replicase. ORFs are represented as hollow rectangles. Asterisks indicate suppressible termination codons. TGB, tripe gene block; Cys-rich, cysteine rich protein; C P, coat protein; mvt, putative movement protein of SBWMV; AA, poly(A) tail; black rectangle, consensus core polymerase motif ().

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

AbstractRhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants.The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA4111800 and AS71100 at 9.5 and 18.5cM, respectively. Moreover, in our I2 population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’‐type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’‐based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.

Characteristics of Beet Soilborne Mosaic Virus, a Furo-like Virus Infecting Sugar Beet.

Beet soilborne mosaic virus (BSBMV) is a rigid rod-shaped virus transmitted by Polymyxa betae. Particles were 19 nm wide and ranged from 50 to over 400 nm, but no consistent modal lengths could be determined. Nucleic acids extracted from virions were polyadenylated and typically separated into three or four discrete bands of variable size by agarose-formaldehyde gel electrophoresis. RNA 1 and 2, the largest of the RNAs, consistently averaged 6.7 and 4.6 kb, respectively. The sizes and number of smaller RNA species were variable. The molecular mass of the capsid protein of BSBMV was estimated to be 22.5 kDa. In Northern blots, probes specific to the 3' end of individual beet necrotic yellow vein virus (BNYVV) RNAs 1-4 hybridized strongly with the corresponding BNYVV RNA species and weakly with BSBMV RNAs 1, 2, and 4. Probes specific to the 5' end of BNYVV RNAs 1-4 hybridized with BNYVV but not with BSBMV. No cross-reaction between BNYVV and BSBMV was detected in Western blots. In greenhouse studies, root weights of BSBMV-infected plants were significantly lower than mock-inoculated controls but greater than root weights from plants infected with BNYVV. Results of serological, hybridization, and virulence experiments indicate that BSBMV is distinct from BNYVV. However, host range, capsid size, and the number, size, and polyadenylation of its RNAs indicate that BSBMV more closely resembles BNYVV than it does other members of the genus Furovirus.

Nicotiana benthamiana plants expressing beet necrotic yellow vein virus (BNYVV) coat protein-specific scFv are partially protected against the establishment of the virus in the early stages of infection and its pathogenic effects in the late stages of infection.

Transgenic plants of Nicotiana benthamiana expressing single chain antibody fragments (scFv) specific for the coat protein of beet necrotic yellow vein virus (BNYVV) and non-expressing control plants were inoculated with BNYVV mechanically and by means of the vector Polymyxa betae. The scFv were presumably expressed in the endoplasmic reticulum (ER). The average time needed for infections to become detectable was longer in the scFv-expressing plants than in the non-expressing control plants. In addition, the scFv-expressing plants were partially protected against the pathogenic effects exerted by the virus on N. benthamiana plants in the late stages of infection.

Detection and Characterization of a Distinct Type of Beet Necrotic Yellow Vein Virus RNA 5 in a Sugarbeet Growing Area in Europe

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.