The effect of sowing date on infection of sugar beet by Polymyxa betae

The effect of sowing date on the infection of sugar-beet seedlings by Polymyxa betae was examined in a small-plot experiment on a naturally infested site. Seed was sown on seven occasions at weekly intervals from late March to early May. From each sowing, plant samples were taken at approximately weekly intervals over a period of 7 weeks. The extent of root infection by P. betae and the dry weight of plants was determined at each sampling date, and the progress of infection and rate of plant growth were examined against time and thermal time. Infection occurred sooner after sowing and the subsequent rate of fungal development was more rapid in late-sown than in early-sown plants. Early sowing allowed germination and growth of sugar beet at temperatures too low for fungal infection. The growth of late-sown plants appeared to be reduced by P. betae infection. The implications of these findings for the development of rhizomania disease are 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.     

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.     

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.     

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.     

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.     

Survival of Polymyxa betae during processing of vegetables and sugarbeet

Samples of effluent were taken at various stages in a range of waste-water treatment systems from seven sugarbeet factories and 14 vegetable processors and tested by a seedling-baiting method. None of the systems examined appeared completely to remove Polymyxa betae , the fungal vector of beet necrotic yellow vein furovirus, the cause of rhizomania disease of sugarbeet. In laboratory experiments, neither anaerobic conditions, raising the pH to 12 nor treating with peracetic acid had any discernible effect on P. betae viability. It is concluded that there is a risk that rhizomania disease could be spread by waste water from processing infected sugarbeet or vegetables from infested land, although there is some evidence that this risk is reduced where systems involving extensive settlement are used.     

Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae , the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae . A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae -resistant accessions of the wild sea beet ( Beta vulgaris ssp. maritima ), which might be used to restrict the transmission of BNYVV, were identified.     

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.     

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.     

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.     

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.     

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。     

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.     

The incidence of Polymyxa betae and other fungal root parasites of sugar-beet in Britain

A 2-year survey of soils from a total of 208 fields, using sugar-beet seedlings as bait plants, showed Polymyxa betae to be ubiquitous in the sugar-beet-growing areas of Britain. It was detected in some soils which had not grown a host crop for up to 17 years. However, infection of roots was relatively infrequent in plant samples taken from 134 survey fields in early summer and the density of colonization always low. Three other non-mycelial fungi, Olpidium brassicae, Lagena radicicola and Rhizophydium graminis were also common parasites of sugar-beet roots detected in soil bioassays. Infection of plant samples by O. brassicae was particularly severe.     

Resistance to Polymyxa betae in wild Beta species

Resistance to Polymyxa betae , the fungal vector of beet necrotic yellow vein virus, was studied in two wild beet species. Beta patellaris and B. procumbens. Plants grown in naturally infested soil or exposed to zoospore suspensions were examined in order to determine the stage in the life cycle of the fungus at which resistance was operating. Resting spores were never observed in the resistant species. Microscopic examination of stained transverse sections of fibrous roots taken at intervals after inoculation showed no evidence of even the earliest infection structures, the plasmodia; these were detected frequently in the epidermal cells of the susceptible Beta vulgaris. Use of the fluorescent stain DiOC₆(3) to label zoospores showed that these were attracted to and attached themselves to the roots and root hairs of resistant species in the same way as to susceptible species. Maximum zoospore attachment was observed 1–6h after roots were exposed to zoospore suspensions. There was no obvious difference in the numbers attracted to resistant and susceptible hosts. Apparent infection of root hairs by encysted zoospores was observed in all three species. The resistance mechanism in the wild species must operate soon after this initial infection, possibly involving a hypersensitive response that prevents the subsequent development of plasmodia in epidermal cells.     

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.     

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.     

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.     

Sugarbeet rhizomania in England1

Concern about the introduction of rhizomania to UK heightened when the disease was confirmed in The Netherlands in 1983. A series of precautionary measures supported by legislation was quickly enacted to reduce this risk. Extensive surveys of harvested beet and of growing crops failed to reveal any infection until 1987 when a single isolated outbreak was found near Bury St Edmunds in Suffolk. Plants in the affected field showed typical rhizomania symptoms and laboratory tests confirmed the presence of beet necrotic yellow vein furovirus (BNYVV). Further investigation of isolates from the outbreak field revealed the presence of beet soil-borne furovirus in addition to BNYVV and sometimes both were present in mixed infections. Intensive surveys in the immediate area of the outbreak, both by field inspections of growing crops and by soil bait testing, confirmed infection only in the one field and the margin of the adjacent field. Immediate containment measures were followed by soil sterilization with methyl bromide to minimize the movement of infection from the area. Surveys of beet will continue, but results to date suggest that the distribution of the disease is limited in extent.