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.     

Assessment of the inoculum potential of Polymyxa betae and beet necrotic yellow vein virus (BNYVV) in soil using the most probable number method

Application of a bioassay on serial dilutions of rhizomania-infested soil provided adequate information on the level of infestation withPolymyxa betae and beet necrotic yellow vein virus (BNYVV). Different combinations of dilution ratios ratios and numbers of replicates (N) that had the same average precision were compared. A most probable number (MPN) computer programme was written to enable the comparison, because MPN tables available in literature are limited to certain dilution ratios and values of N. Most probable numbers of infective units per ml soil assessed for infested soil from the Noordoostpolder and from Tholen (the Netherlands) were 48 forP. betae with 7.1 for BNYVV and 16 forP. betae with 1.6 for BNYVV, respectively. So in these soils 10–15% of the infective population ofP. betae was viruliferous. The inoculum potential of stored soil samples was not affected by conditions during storage for 28 months (dry and warm or wet and cool).     

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.     

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.     

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.     

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...     

Inoculum potential and host range of Pofymyxa graminis1

The inoculum potential of Pofymyxa graminis was studied for 48 samples of Belgian soils and 8 samples from other European countries. After growing barley bait plants in tubes under controlled conditions on increasing dilutions of the samples in sterile sand, P. graminis was detected in 77% of the Belgian soils, the number of infection units calculated per g of soil ranging from 0.004 to 5.6. The inoculum potentials for the other origins were within these limits. The host range of P. graminis was studied by growing various Gramineae in the presence of cystosori extracted from barley roots cultivated on five soils. Barley was infected by all the isolates. Some isolates infected oat and/or rye but none wheat.     

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.     

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 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.     

Serotypes of beet soil-borne furovirus from FRG and Sweden1

Four virus isolates were obtained in FRG and Sweden from sugarbeet roots which had become infected via the soil. All isolates had rod-shaped particles of four or five different lengths. Serologically they were unrelated to beet necrotic yellow vein furovirus and several other viruses of a similar morphology including two serologically distinct sugarbeet isolates from California (US). Our isolates were, however, serologically related to beet soil-borne furovirus which has been described in England. They could be subdivided into two serotypes.     

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.     

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.     

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.     

Fluorescent staining of resting spores of Polymyxa betae as a fungal vector of rhizomania disease of sugar beet in soil

When the resting spores of Polymyxa betae were pretreated with 2% sodium dodecyl sulfate (SDS) and then stained with various fluorochromes including 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)], calcofluor, and a fluorescein isothiocyanate (FITC)-conjugated lectin, such as wheat germ lectin (WGA) or caster bean lectin, most spores fluoresced brightly. FITC-WGA mainly stained the cell surface, while DiOC6(3) stained the cytoplasm. After pretreatment with SDS and addition of FITC-WGA or DiOC6(3) to a soil suspension containing resting spores, the resting spores were distinguishable from the soil particles. This staining method is easy to use for the detection of resting spores in the soil.     

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.     

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.     

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.     

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.