Resistance against rhizomania disease via RNA silencing in sugar beet

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 (ihpRNA) 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 ihpRNA 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.     

Horizontal spread of beet necrotic yellow vein virus in soil

Horizontal dispersal of beet necrotic yellow vein virus (BNYVV) by means of viruliferous zoospores ofPolymyxa betae was studied in greenhouse experiments. BNYVV was not detected in roots of sugar beet plants grown in silver sand for 4 weeks at a root-free distance of 5 cm from eitherP. betae- and BNYVV-infected plants or BNYVV-infested soil. Spread of BNYVV from inoculum sources in the field was studied in the absence and presence of tillage practices. Active dispersal in combination with root growth from and towards point sources of inoculum contributed only little to horizontal dispersal of viruliferous inoculum and spread of disease during the season, as determined for one soil type, two different years and in the absence of tillage and tread. In the second beet crop after application of inoculum to whole field plots, more BNYVV-infected plants were detected at 2 m than at 8 m distance from the infested plots in the tillage direction. In the third year, disease incidence at 8 m was high and equivalent to that at 2 m.     

Effect of sugar beet cultivars with different levels of resistance to beet necrotic yellow vein virus on transmission of virus byPolymyxa betae

The effect of resistance of sugar beet cultivars to beet necrotic yellow vein virus (BNYVV) on virus content of resting spore clusters of the vectorPolymyxa betae was studied in controlled environments and in naturally infested fields. The total number of resting spore clusters formed in roots of a partially resistant and a susceptible cultivar did not differ when assessed 6 and 12 weeks after inoculation with viruliferous resting spores. Transmission experiments showed that in partially resistant plants, having a low virus content in the roots, the population of resting spores formed was less viruliferous than that in susceptible plants with a high virus content. Consequently, growing a resistant cultivar can be expected to delay the build-up of virus inoculum in soil.In a trial field sampled in 1991, the inoculum potential of BNYVV (most probable number of viruliferousP. betae propagules) in soil was lower after growing a partially resistant cultivar than after growing a susceptible one. On the other hand, in four sites sampled in 1990, inoculum potential in soil was hardly increased by growing sugar beet and was not significantly affected by the cultivar grown.     

Remote detection of rhizomania in sugar beets

ABSTRACT As a prelude to remote sensing of rhizomania, hyper-spectral leaf reflectance and multi-spectral canopy reflectance were used to study the physiological differences between healthy sugar beets and beets infested with Beet necrotic yellow vein virus. This study was conducted over time in the presence of declining nitrogen levels. Total leaf nitrogen was significantly lower in symptomatic beets than in healthy beets. Chlorophyll and carotenoid levels were reduced in symptomatic beets. Vegetative indices calculated from leaf spectra showed reductions in chlorophyll and carotenoids in symptomatic beets. Betacyanin levels estimated from leaf spectra were decreased at the end of the 2000 season and not in 2001. The ratio of betacyanins to chlorophyll, estimated from canopy spectra, was increased in symptomatic beets at four of seven sampling dates. Differences in betacyanin and carotenoid levels appeared to be related to disease and not nitrogen content. Vegetative indices calculated from multi-spectral canopy spectra supported results from leaf spectra. Logistic regression models that incorporate vegetative indices and reflectance correctly predicted 88.8% of the observations from leaf spectra and 87.9% of the observations for canopy reflectance into healthy or symptomatic classes. Classification was best in August with a gradual decrease in accuracy until harvest. These results indicate that remote sensing technologies can facilitate detection of rhizomania.     

Epidemiology of beet necrotic yellow vein virus in sugar beet at different initial inoculum levels in the presence or absence of irrigation

A field experiment was set up in 1988 to study the development of rhizomania disease of sugar beet at different inoculum levels of beet necrotic yellow vein virus (BNYVV) in soil. Five, tenfold different, inoculum levels were created by addition of the approximate amounts of 0, 0.5, 5, 50 and 500 kg infested soil per ha (the latter corresponding to 0.01% v/v calculated to the tillage layer). A drip irrigation treatment was applied to study the influence of soil moisture on disease. Susceptible sugar beet, cv. Regina, was grown for three consecutive years.In the first year, root symptoms were not observed, but BNYVV-infected plants were detected by ELISA in low numbers at all inoculum levels at harvest. After late drilling in 1989, high numbers of infected plants, up to 90–100% in plots with the highest inoculum level, were detected already in June. Root symptoms were also observed from June onwards. In both these years disease incidence increased in time and was significantly influenced by the initial inoculum level. In the third year, the whole field was heavily diseased, and only for the non-irrigated plots incidence differed for different initial inoculum levels. The expression of symptoms by BNYVV-infected plants was influenced by initial inoculum level, thus by the amount and timing of primary infection.Root weight at harvest was not affected, but sugar content decreased with increasing inoculum level already in 1988, leading to a reduction in sugar yield of 10% at the highest inoculum level. In 1989, both root weight and sugar content decreased progressively with increasing inoculum level, resulting in sugar yield reductions of 11–66% (down to approximately 3000 kg ha^–1) for low to high inoculum levels compared to the control. As the control plots became contaminated, all yields were low in 1990, still showing a decrease with increasing inoculum level in the non-irrigated plots, but an overall mean sugar yield of 3323 kg ha^–1 for the irrigated ones.Sodium and -amino nitrogen content of the root, additional quality parameters determining extractability of sucrose, showed an increase and decrease, respectively, with increasing initial inoculum level already in the first year. The relative differences in contents compared to those from the control were largest for Na content. A significant negative correlation was found between Na (mmol kg^–1 root) and sugar content (% of fresh weight); linear for 1988, exponential for 1989 and 1990.In spring 1989, the infestation of individual plots was assessed using a quantitative bioassay estimating most probable numbers (MPNs) of infective units of BNYVV per 100 g dry soil. The relationship between the MPns determined and root weight, sugar content and sugar yield at harvest could be described by Gompertz curves. The increase in disease incidence with increasing MPN in 1989 was adequately fitted with a logistic equation.     

Use of zoospores of Polymyxa betae in screening beet seedlings for resistance to beet necrotic yellow vein virus

A system to culture viruliferousPolymyxa betae and to produce zoospores is described. The zoo spores were used for inoculation of beet seedlings, grown in nutrient solution, in tests for resistance to beet necrotic yellow vein virus (BNYVV). On most occasions in a time course experiment, and with various zoospore cultures, the partially resistant cultivar Rima and the accession Holly-1–4 had virus concentrations similar to the susceptible cultivar Regina, but the virus concentration inBeta vulgaris ssp.maritima accession WB42 was significantly lower (P<0.05). ‘Regina’ could be distinguished from various resistant accessions by a significantly higher virus concentration (P<0.05) shortly after inoculation, or after transplanting the seedlings from the nutrient solution into sand. Results of screening for resistance to BNYVV, using zoospores for inoculation, did not correspond with results of a test in which infested soil was used.Tests in which seedlings are grown in nutrient solution and inoculated with zoospores are suitable for the detection of accessions with a high level of resistance to BNYVV. To obtain virus infection in all plants, the optimal density of the zoospore suspension should first be determined and plants should be assayed shortly after inoculation.     

Epidemiology of beet necrotic yellow vein virus in sugar beet at different initial inoculum levels in the presence or absence of irrigation: Dynamics of inoculum

Using field plots where rhizomania had not previously been detected, different inoculum levels of beet necrotic yellow vein virus (BNYVV) were created by application of infested soil. A susceptible sugar beet cultivar (cv. Regina) was grown for two consecutive years (1988 and 1989), in the presence or absence of drip irrigation. In soil samples taken in spring 1989, the different initial inoculum levels of 1988 could be distinguished using a quantitative bioassay estimating most probable numbers (MPNs) of infective units per 100 g dry soil. The first sugar beet crop resulted in a tenthousandfold multiplication of inoculum of BNYVV (viruliferousPolymyxa betae). Mean MPNs of BNYVV ranged from 0.6 and 7 per 100 g soil for the lowest inoculum level to 630 and 1100 per 100 g for the highest level, in plots without and with irrigation, respectively. In spring 1990, MPNs had again increased. In both years, the initial inoculum level of 1988 had a significant linear effect on log-transformed MPNs of BNYVV determined. Log-transformed MPNs for 1990 and 1989 showed a positive linear correlation, despite a decreasing multiplication ratio at higher inoculum levels. Drip irrigation during one or two years enhanced the increase in MPN of BNYVV, which was reflected by the enhancement of multiplication ratios at all inoculum levels. The totalP. betae population was also higher after growing two irrigated crops than after growing two non-irrigated ones.     

Pathogenetic roles of beet necrotic yellow vein virus RNA5 in the exacerbation of symptoms and yield reduction,development of scab-like symptoms,and Rz1-resistance breaking in sugar beet

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.     

Antagonistic effects of natural Pseudomonas putida biotypes on Polymyxa betae Keskin,the vector of Beet necrotic yellow vein virus in sugar beet

Journal of Plant Diseases and Protection - This in vivo study investigated the ability of fluorescent Pseudomonas spp. to suppress Polymyxa betae, a vector of Beet necrotic yellow vein virus...     

Polymyxa betae zoospores as vectors of beet necrotic yellow vein furovirus1

When beet seedlings exposed as bait plants in infested soil were placed in a nutrient solution, they released Polymyxa betae zoospores, infected with beet necrotic yellow vein furovirus. The roots produced the first zoospores 5 days after the start of soil baiting. When seedlings were inoculated with zoospore suspensions, infection occurred within 5 min and reached a maximum in 30 min. The suspensions remained infectious for at least 2 h after removal of the bait plants from which the zoospores were released. So many spores were released into the suspension that disease transmission could be obtained within half an hour from an infected plant to a healthy plant, placed together into fresh medium. Suspensions could be diluted 1/16 with nutrient solution without any loss of infectivity, whereas 1/4 dilution with tap water resulted in a complete loss of infectivity.     

Differential abundance of proteins in response to Beet necrotic yellow vein virus during compatible and incompatible interactions in sugar beet containing Rz1 or Rz2

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.     

Epidemic progress of beet necrotic yellow vein virus: Evidence from an investigation in Japan spanning half a century

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.     

The role of alternative hosts of Polymyxa betae in transmission of beet necrotic yellow vein virus (BNYVV) in England

The host range of beet necrotic yellow vein virus (BNYVV) and Polymyxa betae was determined by growing plants in naturally infested soils from rhizomania outbreaks in England. Apart from Beta vulgaris , plant species infected by BNYVV were included in the families Chenopodiaceae ( Atriplex patula, Chenopodium bonus-henricus, C. hybridum, C. polyspermum and Spinacia oleracea ), Amaranthaceae ( Amaranthus retroflexus ) and Caryophyllaceae ( Silene alba, S. vulgaris, S. noctiflora and Stellaria graminea ). Only P. betae isolates from B. vulgaris, C. polyspermum and S. oleracea were found to be able to transmit BNYVV back to sugar beet. When a range of weed plants from infected fields were tested, none were found to be infected by BNYVV. Therefore, it seems likely that the weed hosts play only a minor role in the spread of rhizomania disease compared to that of sugar beet, other Beta vulgaris crop types or spinach.     

A method for detecting Polymyxa betae and beet necrotic yellow vein virus in soil using sugar-beet as a bait plant

Samenvatting Door in Petrischalen met grond bietezaailingen als lokplant voor de obligaatparasitaire schimmelPolymyxa betae, een vector van het rhizomanievirus, te laten fungeren, kunnen zowel kwalitatieve als kwantitatieve gegevens over de besmetting van de grond met de schimmel en het virus worden verkregen. De methode en enkele ermee bereikte resultaten worden beschreven.     

Inoculum potential and host range of Polymyxa betae and beet necrotic yellow vein furovirus1

The inoculum potential of Polymyxa betae and BNYVV was studied from 52 random samples of Belgian soils and 10 samples from other European countries, by culture of bait plants in tubes under controlled conditions on serial dilutions of the soils in sterile sand. P. betae was detected in all samples within the range of 0.01 to 27.1 infection units per g of soil. BNYVV was detected by ELISA on root extracts of bait plants grown on three Belgian soil samples. All the tested samples from rhizomania-infested areas in France, FRG, the Netherlands, Switzerland and Austria, were found to be infested by BNYVV by this technique. For BNYVV survey, the plant bait technique appears more reliable than the analysis of rootlets collected in the field and observation of external symptoms in case of low BNYVV infestations or non-expression because of unfavourable environmental conditions. P. betae isolates from various origins heavily infected Beta spp. but only moderately spinach. Chenopodium album was slightly infected by 2 of the 7 isolates, C. murale by 4 of them.     

Influence of RNA composition of beet necrotic yellow vein furovirus isolates and of the sugarbeet cultivar on the translocation of the virus in mechanically inoculated sugarbeet roots1

The roots of young sugarbeet seedlings (5-12 days old) were inoculated mechanically with beet necrotic yellow vein furovirus (BNYVV). Vortexing of the seedlings in virus-containing suspensions proved to be especially effective. Higher percentages of infected tap roots were observed with a highly susceptible than with a tolerant cultivar. In the susceptible cultivar, the virus spread more readily from the tap root to the newly formed lateral roots than in the tolerant cultivar, indicating that the latter had a partial resistance to the virus. The translocation of the virus also depended on its RNA composition. An isolate with intact RNA3 and RNA4 was much more efficiently translocated to the lateral roots than an isolate which lacked these two RNAs. So far we have been unable to infect hairy roots of sugarbeet with BNYVV. These hairy roots had been induced by infection with Agrobacterium rhizogenes and were propagated in liquid culture.     

Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil

The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.     

A greenhouse test for screening sugar-beet (Beta vulgaris) for resistance to beet necrotic yellow vein virus (BNYVV)

Small differences in activity between batches of purified beet necrotic yellow vein virus (BNYVV) were observed in ELISA. A four-parameter modelled dose-response curve of purified BNYVV was used for the conversion of ELISA values to virus concentrations. Seedlings of the susceptible cultivar Regina and the partially resistant cultivars Nymphe and Rima were tested for resistance to BNYVV in a mixture of sand and infested soil. Plants were grown in a green-house with low nutrient supply and at temperatures below the optimum of both the vectorPolymyxa betae and BNYVV. Root systems were small and consisted mainly of lateral roots. Significant differences in average virus concentrations were found between cultivars, both when using the complete root systems and when using either the top or the bottom part of the root systems. Average virus concentrations in Regina were always significantly higher than in Rima and higher than in Nymphe on all occasions except one (P<0.05). Differences between Nymphe and Rima were less evident. Variation between plants was greatest within Rima. The test described in this paper can be used for the discrimination of different cultivars and for the identification of individual plants with resistance to BNYVV.