dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Progress towards the understanding and control of sugar beet rhizomania disease

Rhizomania is a soil-borne disease that occurs throughout the major sugar beet growing regions of the world, causing severe yield losses in the absence of effective control measures. It is caused by Beet necrotic yellow vein virus (BNYVV), which is transmitted by the obligate root-infecting parasite Polymyxa betae . BNYVV has a multipartite RNA genome with all natural isolates containing four RNA species, although some isolates have a fifth RNA. The larger RNA1 and RNA2 contain the housekeeping genes of the virus and are always required for infection, whereas the smaller RNAs are involved in pathogenicity and vector transmission. RNA5-containing isolates are restricted to Asia and some parts of Europe, and these isolates tend to be more aggressive. With no acceptable pesticides available to restrict the vector, the control of rhizomania is now achieved almost exclusively through the use of resistant cultivars. A single dominant resistance gene, Rz1 , has been used to manage the disease worldwide in recent years, although this gene confers only partial resistance. More recently, new variants of BNYVV have evolved (both with and without RNA5) that are able to cause significant yield penalties on resistant cultivars. These isolates are not yet widespread, but their appearance has resulted in accelerated searches for new sources of resistance to both the virus and the vector. Combined virus and vector resistance, achieved either by conventional or transgenic breeding, offers the sugar beet industry a new approach in its continuing struggle against rhizomania.     

Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

Gehalt an Beet Necrotic Yellow Vein Virus (BNYVV) in der Hauptwurzel von Zuckerrübenpflanzen verschiedener Sorten und deren Leistung unter Rizomaniabefall im Feld

BNYVV concentration in the tap roots of sugar beet varieties grown in rhizomania-infested fields During plant development, the BNYVV concentration in several commercially available rhizomania-tolerant sugar beet varieties and one susceptible variety was examined as an index of the intensity of infection. The root weight, sugar content and sugar yield of the same varieties in fields naturally infested with rhizomania were also measured. Significant negative correlations were found between the average virus concentration in the tap root and yield parameters in infested fields. These were largely independent of the growth stage of beet plants used for virological investigations. However, the negative correlations between virus concentration and yield were not significant if rhizomania-tolerant varieties only were compared. The possibility that virus concentration might be used as a criterion for selection in addition to yield performance is discussed. This may lead to selection that is targeted more directly at rhizomania resistance and thereby accelerate breeding work.     

Control of chestnut blight by the use of hypovirulent strains of the fungus Cryphonectria parasitica in northwestern Spain

Chestnut blight is controlled in Europe by using Cryphonectria hypovirus CHV1, a non-encapsulated RNA virus. The chestnut blight fungus, Cryphonectria parasitica, is weakened by the virus, and healing tissue growth occurs in the host tree. Transmission of this cytoplasmic hypovirus is restricted by the incompatibility system of the fungus, so that the hypovirus can be transmitted only between isolates of the same or closely related vegetative compatibility (vc) types. Hypovirulent isolates of C. parasitica (all of the French subtype CHV1-F1) from Castilla y León (NW Spain) were compared with virulent isolates in both laboratory (cut stems) and field inoculations (in two orchards in the province of León and one orchard in the province of Zamora). The tests were performed with the most common vc types in the region, EU1 and EU11. The cut stem assay revealed that the hypovirulent isolates of vc type EU1 did not reduce the growth of virulent cankers. By contrast, four hypovirulent strains H1, H4, H5 and H6 (all vc type EU11) reduced the growth of virulent isolates in the cut stem assay. Field tests showed that hypovirulent isolates of EU1 and EU11 were effective in reducing canker in both orchards in León with all treatments tested; however, in Zamora, where only EU11 was tested, all the treatments failed except H1, which was able to reduce growth of the canker eighteen months after the inoculation. The development of hypovirulence suggests that hypovirus subtype F1 is well adapted in the province of León. Both naturally extended and inoculated hypoviruses appear to have reduced the incidence of the canker, thus improving chestnut stands. However, the inoculations were not as effective in the orchards in Zamora. This indicates that the disease could be controlled in Castilla y León by inoculation of trees with hypovirulent strains, but that more tests should be done in provinces where the hypovirus is still not present.     

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.     

The evolutionary history of Beet necrotic yellow vein virus deduced from genetic variation, geographical origin and spread, and the breaking of host resistance

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet and has been found worldwide, probably as the result of recent worldwide spread. The BNYVV genome consists of four or five RNA components. Here, we report analysis of sequence variation in the RNA3-p25, RNA4-p31, RNA2-CP, and RNA5-p26 genes of 73 worldwide isolates. The RNA3-p25 gene encodes virulence and avirulence factors. These four sets of gene sequences each fell into two to four groups, of which the three groups of p25 formed eight subgroups with different geographical distributions. Each of these subgroup isolates (strains) could have arisen from four original BNYVV population and their mixed infections. The genetic diversity for BNYVV was relatively small. Selection pressure varied greatly depending on the BNYVV gene and geographical location. Isolates of the Italy strain, in which p25 was subject to the strongest positive selection, were able to overcome the Rz1-host resistance gene to differing degrees, whereas other geographically limited strains could not. Resistance-breaking variants were generated by p25 amino acid changes at positions 67 and 68. Our studies suggest that BNYVV originally evolved in East Asia and has recently become a pathogen of cultivated sugar beet followed by the emergence of new resistance-breaking variants.     

Serologic evidence of human infection by Francisella tularensis in the population of Castilla y León (Spain) prior to 1997

Prior to an outbreak in Castilla y León in December 1997, tularaemia was practically non-existent in Spain. In this paper we studied the prevalence of antibodies against Francisella tularensis in a representative sample of the population (4825 people) from Castilla y León (Spain) in samples collected before this outbreak. Antibodies against F. tularensis were detected in nine (0.19%) of the 4825 sera, with antibody titres ranging from 1/20 to 1/160. Of these nine sera, one was positive in seroagglutination against Brucella. Seroagglutination against other bacteria (Yersinia enterocolitica O:9 and O:3 and Proteus OX19) was negative in all sera. Seroprevalence of antibodies in females was 0.20% and 0.17% in males; no statistically significant differences were found in prevalence in terms of sex, age or province.     

Widespread distribution of Beet necrotic yellow vein virus in Iranian sugar beet industry

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.     

Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains

Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67–70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep sequencing. The level of P25 variation in the populations analysed did not correlate with RB properties. Remarkably, one WT population contained P25 with RB mutations at a frequency of 11%. To demonstrate selection by Rz1 and the influence of RB mutations on relative fitness, competition experiments between strains were performed. Following a mixture of strains with four RNAs, a shift in tetrad variants was observed, suggesting that strains did not mix or transreplicate. The plant genotype exerted a clear influence on the frequency of RB tetrads. In Rz1 plants, the RB variants outcompeted the WT variants, and mostly vice versa in susceptible plants, demonstrating a relative fitness penalty of RB mutations. The strong genotype effect supports the hypothesized Rz1 RB strain selection with four RNAs, suggesting that a certain tetrad needs to become dominant in a population to influence its properties. Tetrad selection was not observed when an RB strain, with an additional P26 protein encoded by a fifth RNA, competed with a WT strain, supporting its role as a second BNYVV pathogenicity factor and suggesting the reassortment of both types.     

Development of coupling-repulsion-phase SCAR markers diagnostic for the sugar beet Rr1 allele conferring resistance to rhizomania

In sugar beet genotypes with the Holly type of resistance to rhizomania, a disease due to infection of the beet necrotic yellow vein virus (BNYVV), the major gene rrl is responsible for resistance. Twelve RAPD markers linked to rrl were selected by BSA and mapped on linkage group IV using a segregating population previously analysed by the same group. Markers F61050 and N9600 were tightly linked, respectively in coupling and repulsion, to the Rrl allele (recombination values of 1.4 cM for both markers). After sequencing the products amplified by F61050 and N9600, new PCR primers were used to generate the two SCAR markers F6 and N9. The simultaneous use of these markers in a PCR reaction allows the correct fingerprinting of rrl rrl, Rrl rrl and Rrl Rrl sugar beet plants in populations segregating for the Holly resistance. In a group of sugar beet elite lines containing the Holly type of rhizomania resistance, SCAR F6 is always present whereas the SCAR N9 fragment is absent. Thus, in marker-assisted selection with coupling-repulsion-phase markers, SCAR F6 can be used in combination with N9, or together with any other RAPD marker linked in repulsion to the Rrl allele.     

Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.