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.     

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.     

甜菜多粘菌传带甜菜坏死黄脉病毒的细胞定位研究

 利用砂培体系继代培养不同病区甜菜多粘菌(Polymyxa betae),经酶联检测,分离得到2个带毒率高的分离株N,HR₁₂。应用甜菜坏死黄脉病毒(BNYVV)抗血清和免疫金标记技术分析了甜菜根中P.betae不同发育阶段与病毒的关系。在初生原质体、游动孢子囊以及未成熟的游动孢子中观察到被金颗粒标记的病毒粒子,在休眠孢子外围也观察到金标记的病毒粒子,但在休眠孢子内未直接观察到病毒粒子,只是在其内壁及液泡中常见有标记上的金颗粒。     

The effect of methyl bromide fumigation on rhizomania inoculum in the field

The first outbreak of rhizomania disease in the UK occurred in 1987 and was limited to a single sugar-beet crop in Suffolk. In an attempt to prevent the spread of this disease, the crop was first destroyed by herbicide. In 1988, to reduce the level of rhizomania still present in the soil, the field was treated with methyl bromide at a rate of 900 kg/ha prior to seeding for permanent pasture. Levels of methyl bromide were monitored during the fumigation. A mean concentration time product of 5500 mgh/1 was achieved after 72 h at the soil surface and of 3300-4100 mg-h/1 at a soil depth of 0.3 m after 24 h. Soil samples were taken from five plots across the field before and after fumigation. In the plot with the highest initial inoculum levels, further samples were taken at three depths down to 0.61 m. Sugar-beet seedlings were grown in all soil samples as a bait test for rhizomania inoculum. The presence or absence of Polymyxa betae was observed by microscopical examination, and an enzyme-linked immunoassay was used for the detection of beet necrotic yellow vein virus (BNYVV). The results showed that the methyl bromide treatment had reduced rhizomania inoculum and BNYVV in the soil to levels that were undetectable by the procedures used.     

Identification and characterization of resistance to rhizomania in an ecotype of Beta vulgaris subsp. maritima

An ecotype (S023) of Beta vulgaris subsp. maritima was identified which was highly resistant to beet necrotic yellow vein virus (BNYVV) but susceptible to its fungal vector, Polymyxa betae . A comparative study of viral development in the roots showed that the resistance was effective from the early stages of infection. Mechanical inoculation experiments suggested that the resistance was not due to a lack of transmission of BNYVV by its fungal vector. Immunogold-silver labelling of the viral coat protein in root sections confirmed the existence of BNYVV transmission in the resistant ecotype. Virus replicated in isolated root protoplasts of both susceptible and resistant genotypes. Viral particles localized by immunogold-silver labelling diffused over short distances from the primary infected root cells in both resistant and susceptible plants. Evidence for inhibition of long-distance movement in the resistant ecotype requires further investigation.     

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.     

Modelling the effect of temperature on the development of Polymyxa betae

Polymyxa betae is the fungal vector of beet necrotic yellow vein virus (BNYVV), which is the causal agent of the sugar beet disease rhizomania. The within-season dynamics of the fungus are a crucial factor in the occurrence and severity of rhizomania. Late infection of the host by viruliferous fungi enables host resistance to the virus to develop and hence limits crop damage. A previously published mechanistic model for the dynamics of Polymyxa betae is extended in this paper to incorporate the effect of temperature on the germination of resting spores, and on the latent periods between infection and the production of secondary zoospores and new resting spores. It is shown that, for UK temperature conditions, the effect of sowing date on infection is greater than that of year-to-year variations in temperature associated with a single representative sowing date. The variation in inoculum build-up predicted when temperature data from a range of soil types were used in the model agreed with field observations, where higher levels of infection are observed on sandy soils than on black fen peat soils. The difference was most distinct when daily maximum soil temperature values were used to drive the model rather than rolling 24-hour average values.     

Investigations concerning soil-borne viruses in sugarbeet in Sweden1

Some experiments with soil-borne beet viruses in cement tubes in a wire netting enclosure are described. It is confirmed that rhizomania (virus + vector) originating from German soil can survive and cause rhizomania in Sweden. Antisera produced in 1987 to one German BNYVV isolate and to one Swedish soil-borne beet virus isolate, 86-109, which is distinct from BNYVV, were used to check ELISA reactions in the tube beets. Positive ELISA was obtained not only for BNYVV but also for the 86-109 virus from tubes with German inoculum. Beets from tubes with Swedish inoculum reacted only against 86-109 antiserum. In 1988-09, ELISA of 118 sugarbeet plants from Öland and 73 from Skåne, collected in 42 different fields with spots resembling rhizomania, showed no or weak reactions against 86-109 antiserum, in contrast to plants collected in 1987. However, after transplanting the field plants into a warm glasshouse and using bait plants it was shown in ELISA and in transmission to Chenopodium quinoa that many of the bait plants became infected with the 86-109 and ‘related viruses’ but not with BNYVV. Viruses of the 86-109 type seem to be common both in Sweden and elsewhere but may escape detection, especially in mixed infections with BNYVV.     

Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.     

The host range of Polymyxa betae in Britain

The host range of Polymyxa betae on common arable weed species in Britain was determined by growing plants in naturally infested soil and examining their root systems for the presence of resting spores (cystosori). Of the 24 species tested, only Atriplex patula and Chenopodium album of the Chenopodiaceae, and Silene alba of the Caryophyllaceae, were found to be heavily infected. S. alba is a newly recorded host species for Polymyxa. The host specificity of isolates of P. betae from Beta vulgaris, C. album and A. patula was investigated by observing which of 11 test plants could be infected by the isolates obtained from this soil. Three main biotypes of P. betae appeared to be distinguishable: one which was able to infect all chenopodiaceous species; one which had a narrower host range; and one which was able to infect S. alba. The role of weed species in the epidemiology of rhizomania is discussed.     

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.     

Identification of rhizomania-infected soil in Europe able to overcome <Emphasis Type="Italic">Rz1</Emphasis> resistance in sugar beet and comparison with other resistance-breaking soils from different geographic origins

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is vectored by Polymyxa betae. The disease can only be controlled by growing partially resistant sugar beets, which quantitatively reduce virus replication and spread. None of the known major resistance genes (Rz1, Rz2, Rz3), alone or in combination, are able to prevent BNYVV infection entirely. Here we report for the first time the identification of a Spanish soil, containing an A-type BNYVV with RNA 1-4, displaying Rz1 resistance-breaking abilities comparable to soils from the USA and to those from France containing the French (Pithiviers) P-type BNYVV with RNA 5. A resistance test with several soil samples vs. different sugar beet cultivars was conducted under standardised conditions. Sugar beets were analysed after 12 weeks of greenhouse cultivation for taproot weight, BNYVV and relative P. betae content. The soil samples from Spain, France and the USA produced high virus contents and strong rhizomania symptoms in Rz1 plants, indicative of resistance-breaking abilities. In addition, all resistance-breaking soil samples produced detectable virus concentrations in plant lateral roots of the Rz1 + Rz2 cultivar, and plants grown in the Spanish soil sample also had reduced taproot weight and displayed severe rhizomania disease symptoms. Additionally, the main pathogenicity factor P25, responsible for the formation of BNYVV symptoms, showed high sequence variability in the amino acid tetrad at position 67–70. The results suggest the geographically independent selection of BNYVV resistance-breaking isolates following the uniform cultivation of Rz1-containing sugar beet cultivars.     

Differences in temperature requirements between Polymyxa sp. of Indian origin and Polymyxa graminis and Polymyxa betae from temperate areas

The temperature requirements of three single cystosorus strains of Polymyxa sp. from India were studied at 15–18, 19–22, 23–26 and 27–30 °C (night-day temperature), and compared with the temperature requirements of three strains of P. graminis from Belgium, Canada and France and two strains of P. betae from Belgium and Turkey. Sorghum was used as the host-plant for the Indian strains; the strains of P. graminis and P. betae from temperate areas were cultivated on barley and sugar beet, respectively. The cystosori germination and the development of plasmodia, zoosporangia and cystosori of Polymyxa sp. from India were optimal at 27–30 °C. Infection progression was slower at 23–26 °C than at 27–30 °C. At 19–22 °C, infection was insignificant. No infection occurred below 19 °C. In contrast, the infection of barley with P. graminis strains from temperate areas was optimal at 15–18 °C, but at 19–22 °C the progression appeared inconsistent and infection stayed low. Above 22 °C, infection was insignificant. P. betae strains showed consistent infection in the range of 15–18 °C to 27–30 °C. Plasmodia formation and cystosori detection of the Belgian strain were slightly advanced at 23–26 °C compared to 19–22 °C but clearly restrained at 27–30 °C. Fungus development of the P. betae strain from Turkey was almost as high at 27–30 °C as at the lower temperatures. These results strengthen the case for distinguishing between Polymyxa sp. from India and P. graminis or P. betae from temperate areas.     

A rapid method for direct detection of Polymyxa DNA in soil

Polymyxa spp. are vectors for a number of economically important soilborne plant viruses. The development of a technique to detect virus and vectors directly in soil would be useful for epidemiological studies and assessment of disease risk prior to planting. A rapid method was developed to extract and quantify Polymyxa spp. DNA from soils. DNA was extracted from three soils infested with Polymyxa betae and three infested with P. graminis using an EDTA lysis buffer in combination with a MagneSil™ DNA extraction kit and Kingfisher™ magnetic particle processor. Primers and probes designed to correspond to sequences within the internal transcribed spacer region 2 (ITS2) of ribosomal DNA enabled recovery and amplification of P. betae and P. graminis DNA using real-time PCR and TaqMan chemistry. For the P. graminis- infested soils, the purity of DNA obtained was sufficient to allow Polymyxa DNA to be amplified without dilution to remove inhibitors, but with P. betae- infested soils, amplification was only achieved if the DNA was diluted 1:10. Using TaqMan PCR, a standard curve was constructed from uninfested soil spiked with known numbers of P. betae cystosori; the quantity of P. betae inoculum from naturally infested soil was then extrapolated from the curve. This technique offers a sensitive method of extracting, detecting and quantifying Polymyxa spp. DNA in soil.     

Infection of sugar beet by Polymyxa betae in relation to soil temperature

The effects of soil temperature on infection of sugar-beet roots by the soil-borne fungus Polymyxa betae were investigated in controlled environments. Pre-germinated seeds were sown in pots of naturally infested soil and seedlings sampled at frequent intervals over a period of several weeks. Within the range 10-30°C, the optimum soil temperature for infection was c. 25°C; the time between sowing and the first detectable infection was shortest and the subsequent rate of infection most rapid at this temperature. No infection was observed over 80 days at 10°C.
Both root and shoot dry weight were reduced on plants growing in infested soil at 15, 20 and 25 C compared with those growing in uninfested soil. In general, root growth was more severely affected than shoot growth and the effects were most pronounced at 20°C. These results were confirmed in a subsequent experiment in which P. betae -infected root material was used as the inoculum. In addition to its role as the vector of beet necrotic yellow vein virus (the cause of Rhizomania disease), the significance of P. betae as a plant pathogen in its own right is discussed.     

Characterization of Polymyxa species by restriction analysis of PCR-amplified ribosomal DNA

A molecular method is described to aid identification of the obligate parasite Polymyxa and discriminate between species ( P. betae and P. graminis ) and isolates. DNA was extracted from zoospores, resting spores and roots infected with P. betae and P. graminis and compared with that from negative control plants that were not inoculated with Polymyxa but were grown at the same time under the same conditions. The ribosomal internal transcribed spacers and 5.8S rDNAs were amplified by the polymerase chain reaction and digested with restriction enzymes to detect molecular differences between the species and isolates. There were differences between P. betae and P. graminis and two subgroups within P. graminis but so far this has not been correlated with any other biological property.     

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.     

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.     

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 56ng/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.     

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.