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.     

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 conditions during storage of infested soil on infection of bait plants by Polymyxa betae and beet necrotic yellow vein virus

Infectivity of resting spores ofPolymyxa betae in soil stored air-dry or moist was determined by assessing infection of bait plants that were exposed to the soil. Storage of soil under air-dry conditions at room temperature resulted in a delayed onset of germination of resting spores compared to germination in soil stored under moist and cool conditions, as inferred from the infection of the bait plants. Bait plants had to be exposed for more than 12 h to flooded infested soil before germination and infection had occurred. However, when soil was prewetted for 24 h before exposing bait plants, germination, infection and transmission of beet necrotic yellow vein virus (BNYVV) were accomplished within 12 h, but only with the moistly stored soil. When resting spores isolated from roots were stored for 4 and 8 weeks under dry conditions at 22°C, germination of viruliferous spores, as measured by detection of BNYVV in bait plants exposed for 48 h to the spores, was less than that of spores stored in moist soil at 22°C. Approximately 100% of bait plants were infected after exposure to resting spores that were frozen in demineralized water or stored cool (5°C) in water or moist soil for 42 weeks. Air-dry cool storage for 42 weeks resulted in a low percentage of infection. Storage conditions of soil influence the results of bioassays for detection of rhizomania when short baiting periods are applied, whereas differences in infectivity were not detected using a bioassay with long duration.     

First Record of A and B Type Beet necrotic yellow vein virus in Sugar Beets in Sweden

Natural infections of sugar beet with Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania, have been detected for the first time in Sweden in two small areas, one on the island of Öland and one in the Southeastern part of Scania. Single strand conformation polymorphism analyses of PCR products revealed that the infections on Öland were produced by A type BNYVV, whereas those in Scania were caused by the B type. This suggests that BNYVV has been introduced into Sweden at least twice. Alternatively, the virus may have invaded sugar beet from unknown native hosts. BNYVV RNA 5 was not detected in the samples investigated.     

Partial nucleotide sequence analysis of Turkish isolates of Beet necrotic yellow vein virus (BNYVV) RNA-3

A survey was carried out to detect Beet necrotic yellow vein virus (BNYVV) in soil samples using RT-PCR and bait plant techniques from the sugar beet production area of Tokat, Turkey in 2001. More than 80% of the soil samples analyzed were found to be contaminated with the virus. The partial nucleotide sequence of cDNA corresponding to RNA-3 of BNYVV isolates were analyzed for six different regions of Tokat province. All isolates were assigned to type A strains based on RFLP analysis and DNA sequences. Sequence comparison revealed differences at amino acid positions 35, 68, 71 and 179 of the P25 coding region amongst Turkish isolates. Additionally, all Turkish isolates were compared with Japanese, French, Kazakh, Italian and Belgian isolates.     

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.     

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.     

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.     

Selection and characterization of resistance to Polymyxa betae, vector of Beet necrotic yellow vein virus, derived from wild sea beet

The plasmodiophoromycete Polymyxa betae is an obligate root parasite that transmits Beet necrotic yellow vein virus (BNYVV), the cause of sugar beet rhizomania disease. Currently, control of this disease is achieved through the use of cultivars with monogenic (Rz1) partial resistance to the virus. To improve the level and durability of this resistance, sources of resistance to the virus vector, P. betae, were sought. Over 100 accessions of the wild sea beet (Beta vulgaris ssp. maritima) from European coastal regions were evaluated for resistance in controlled environment tests. Quantification of P. betae biomass in seedling roots was achieved using recombinant antibodies raised to a glutathione‐s‐transferase expressed by the parasite in vivo. Several putative sources of resistance were identified and selected plants from these were hybridized with a male‐sterile sugar beet breeding line possessing partial virus resistance (Rz1). Evaluation of F₁ hybrid populations identified five in which P. betae resistance had been successfully transferred from accessions originating from Mediterranean, Adriatic and Baltic coasts. A resistant individual from one of these populations was backcrossed to the sugar beet parent to produce a BC₁ population segregating for P. betae resistance. This population was also tested for resistance to BNYVV. Amplified fragment length polymorphism and single‐nucleotide polymorphism markers were used to map resistance quantitative trait loci (QTL) to linkage groups representing specific chromosomes. QTL for resistance to both P. betae and BNYVV were co‐localized on chromosome IV in the BC₁ population, indicating resistance to rhizomania conditioned by vector resistance. This resistance QTL (Pb1) was shown in the F₁ population to reduce P. betae levels through interaction with a second QTL (Pb2) found on chromosome IX, a relationship confirmed by general linear model analysis. In the BC₁ population, vector‐derived resistance from wild sea beet combined additively with the Rz1 virus resistance gene from sugar beet to reduce BNYVV levels. With partial virus resistance already deployed in a number of high‐yielding sugar beet cultivars, the simple Pb1/Pb2 two‐gene system represents a valuable additional target for plant breeders.     

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.     

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.     

Evidence of a new Tomato yellow leaf curl virus in Japan and its detection using PCR

A new isolate of Tomato yellow leaf curl virus (TYLCV) has been identified from tomato plants in Kochi Prefecture in Japan and designated TYLCV-[Tosa]. The complete nucleotide sequence of the isolate was determined and found to consist of 2781 nt. In phylogenetic analyses of entire nucleotide sequences, TYLCV-[Tosa] was delineated as a single branch and was more closely related to TYLCV-[Almeria] than TYLCV isolates Ng, Sz, or Ai reported in Japan, which had spread since 1996. Isolate TYLCV-[Tosa] is suggested to be a newly introduced, novel isolate of TYLCV that dispersed into Kochi Prefecture. In addition, a rapid method using the polymerase chain reaction to separate TYLCV isolates into four genetic groups was established. This method would be useful for reliable diagnosis based on genetic differences among isolates of TYLCV.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB192965 and AB192966     

Breaking of <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis> resistance in sugar beet is independent of virus and vector inoculum densities

Beet necrotic yellow vein virus (BNYVV) is transmitted by Polymyxa betae to sugar beet, causing rhizomania disease. Resistance-breaking strains of BNYVV, overcoming single (Rz1) or double (e.g. Rz1  +  Rz2) major resistance genes in sugar beet have been observed in France and recently in the USA and Spain. To demonstrate if resistance-breaking is dependent on inoculum density, the inoculum concentration of BNYVV and P. betae in soil samples where resistance-breaking had been observed was estimated using the most probable number (MPN) method. The MPN-values obtained displayed highly significant differences with respect to the virus concentration in various soils and did not correlate with the ability to overcome resistance. Virus quantification in susceptible plants demonstrated that soils containing resistance-breaking isolates of BNYVV did not produce higher virus concentrations. The MPN assay was repeated with Rz1  +  Rz2 partially-resistant sugar beets to see if the resistance-breaking is concentration-dependent. There was no correlation between soil dilution and increased virus concentration in Rz1  +  Rz2 plants produced by BNYVV resistance-breaking strains. Determination of the absolute P. betae concentration by ELISA demonstrated that all resistance-breaking soil samples contained elevated concentrations. However, the calculation of the proportion of viruliferous P. betae did not show a positive correlation with the resistance-breaking ability. Finally resistance-breaking was studied with susceptible, Rz1 and Rz1  + Rz2 genotypes and standardised rhizomania inoculum added to sterilised soil. Results from these experiments supported the conclusion that resistance-breaking did not correlate with virus concentration or level of viruliferous P. betae in the soil.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).