Evaluation of Polymyxa betae Keskin Contaminated by Beet Necrotic Yellow Vein Virus in Soil

The fungus Polymyxa betae Keskin belongs to the family Plasmodiophoraceae and lives in the soil as an obligatory parasite of the roots of the Chenopodiaceae. When contaminated by beet necrotic yellow vein virus, this viruliferous fungus causes a serious disease of sugar beet known as rhizomania, whereas the infection by the fungus alone (aviruliferous fungus) causes only slight damage to the plant with little economic consequence. The manifestation of rhizomania in sugar beet is directly related to the concentration of infecting units of viruliferous P. betae present in the soil. (One infecting unit is a group of one or more sporosori that liberate zoospores capable of visibly infecting a plant.) By using current methods of analysis, it is possible to estimate the total quantity of P. betae present in the soil, but one cannot distinguish quantitatively the infecting units of aviruliferous from viruliferous P. betae. A new method has been developed based on the technique of the most probable number and enzyme-linked immunosorbent assay to estimate the concentration of infecting units of viruliferous P. betae in soil. The method is suitable for the routine analysis of numerous soil samples and allows one to estimate the concentration of viable forms of the fungus P. betae, whether or not contaminated by beet necrotic yellow vein virus, present in a soil affected by rhizomania or presumed healthy. The analyses performed with this method are economical and use a reagent kit and equipment in wide use.     

Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Use of the Most-Probable-Number Technique To Detect Polymyxa betae (Plasmodiophoromycetes) in Soil

The fungus Polymyxa betae is an obligate parasite of the roots of many plants of the family Chenopodiaceae. In the sugar beet, it acts as a vector of beet necrotic yellow vein virus, the agent of a serious disease known as rhizomania. With indirect methods of analysis, such as bioassay, one can establish only the presence or absence, but not the quantity, of P. betae in soil. A new method based on the technique of the most probable number (MPN) of infective units of P. betae present in the soil was developed on the basis of the biological characteristics of this microorganism. Compared with traditional bioassay methods, the MPN method is suitable for determining the contamination level of P. betae in a soil, and it appears promising for the routine analysis of many soil samples, whether they were affected by rhizomania or presumed noninfested. The instrumentation designed especially for the recovery of viable P. betae from soil with the MPN technique is made from commercially available materials, results in a saving of space during sample incubation, and permits this method to be used for any laboratory analysis.     

Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.     

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus,by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10⁶ CFU g^–1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.     

BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach

Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species.     

Genomes of the wild beets Beta patula and Beta vulgaris ssp. maritima

We present draft genome assemblies of Beta patula, a critically endangered wild beet endemic to the Madeira archipelago, and of the closely related Beta vulgaris ssp. maritima (sea beet). Evidence‐based reference gene sets for B. patula and sea beet were generated, consisting of 25 127 and 27 662 genes, respectively. The genomes and gene sets of the two wild beets were compared with their cultivated sister taxon B. vulgaris ssp. vulgaris (sugar beet). Large syntenic regions were identified, and a display tool for automatic genome‐wide synteny image generation was developed. Phylogenetic analysis based on 9861 genes showing 1:1:1 orthology supported the close relationship of B. patula to sea beet and sugar beet. A comparative analysis of the Rz2 locus, responsible for rhizomania resistance, suggested that the sequenced B. patula accession was rhizomania susceptible. Reference karyotypes for the two wild beets were established, and genomic rearrangements were detected. We consider our data as highly valuable and comprehensive resources for wild beet studies, B. patula conservation management, and sugar beet breeding research.     

Some observations on assessing Phoma betae infection of sugar-beet seed

Near-ultraviolet or ‘black’ light applied continuously from the start of incubation, facilitates tests for Phoma betae on sugar-beet seed by stimulating the production of pycnidia and restricting mycelial growth of P. betae and other fungi. Pretreatment of the seed with dilute sodium hypochlorite decreases the number of seeds with P. betae by removing superficial infection, but some of this is of significance in the field. Rubbing beet seed also decreased counts of P. betae in the laboratory and increased field emergence, primarily by removing the fungus. Griseofulvin sprayed on beet-seed plants either 2 wk or 2 days before harvest significantly decreased seed infection with P. betae, but not to a level at which further seed treatment could be omitted.     

Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains

Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67–70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep sequencing. The level of P25 variation in the populations analysed did not correlate with RB properties. Remarkably, one WT population contained P25 with RB mutations at a frequency of 11%. To demonstrate selection by Rz1 and the influence of RB mutations on relative fitness, competition experiments between strains were performed. Following a mixture of strains with four RNAs, a shift in tetrad variants was observed, suggesting that strains did not mix or transreplicate. The plant genotype exerted a clear influence on the frequency of RB tetrads. In Rz1 plants, the RB variants outcompeted the WT variants, and mostly vice versa in susceptible plants, demonstrating a relative fitness penalty of RB mutations. The strong genotype effect supports the hypothesized Rz1 RB strain selection with four RNAs, suggesting that a certain tetrad needs to become dominant in a population to influence its properties. Tetrad selection was not observed when an RB strain, with an additional P26 protein encoded by a fifth RNA, competed with a WT strain, supporting its role as a second BNYVV pathogenicity factor and suggesting the reassortment of both types.     

Widespread distribution of Beet necrotic yellow vein virus in Iranian sugar beet industry

Beet necrotic yellow vein virus (BNYVV) is the most devastating pathogen of sugar beet worldwide. This virus has been reported in the majority of sugar beet growing regions of Iran as well. For the present study, we collected samples from different sugar beet varieties with suspected symptoms of BNYVV from the main important sugar beet growing regions in eight provinces of Iran. Infection of collected samples to BNYVV was tested by ELISA and RT-PCR. Upon testing of 167 collected samples of BNYVV suspected through ELISA and RT-PCR, 115 (68.9%) were infected. Different incidences of BNYVV through surveyed provinces may represent the presence of diverse infective viral sources or resistance genes in tested sugar beet varieties which need further attempts to develop control strategies. Results also showed that BNYVV has been recently distributed throughout some surveyed regions. Otherwise, trace infection or resistance to BNYVV infection in some varieties of distinct regions may represent proper sources of resistance to BNYVV.     

Suppression of Fusarium oxysporum with recombinant polygalacturonase inhibiting proteins (BvPGIPs) extracted from sugar beet roots

Soil-borne fungus Fusarium oxysporum f. sp. betae (Fob) is the causative agent of Fusarium yellows in sugar beet. Leaf interveinal yellowing and root vascular discoloration significantly reduce root yield as well as sucrose content and juice purity. Fob, like other fungal pathogens, initiates disease development by secreting polygalacturonase (PG) enzymes to break down plant cell walls during early stages of infection. To protect themselves, plants produce polygalacturonase-inhibiting proteins (PGIPs). In our study of sugar beet root defense responses, several PGIP genes (BvPGIPs) were identified. To determine if BvPGIPs inhibit Fob PGs, genes BvPGIP1, BvPGIP2 and Bv(FC607)PGIP1 were fused with the CaMV 35S promoter and each was expressed individually in sugar beet hairy roots. We demonstrate that all three recombinant BvPGIP proteins inhibited Fob and F. oxysporum f. sp. gladioli (Fog) PGs. A comparable level of BvPGIP activity was observed against Fob PGs, while BvPGIP2 showed higher activity against Fog PGs. Similar results were obtained when recombinant PGIPs were used to bioassay effects on Fob and Fog spore germination and hyphal growth. This is a first report that documents F. oxysporum inhibition by overexpressing BvPGIPs that may lead to improved Fusarium yellows resistance in sugar beet.

     

Eradication of Polymyxa betae by Thermal and Anaerobic Conditions and in the Presence of Compost Leachate

The abiotic conditions required for eradication of Polymyxa betae, the vector of Beet necrotic yellow vein virus in sugar beet, were investigated. Survival of resting spores of P. betae was determined under aerobic (30 min, 4 days and 21 days) and anaerobic (4 days) conditions under several temperature regimes in a water suspension and in leachate extracted from an aerobic compost heap. In water under aerobic conditions the lethal temperature was 60, 55 and 40°C for exposure times of 30 min, 4 days and 21 days, respectively. The effect of compost leachate and/or anaerobic conditions on survival of P. betae depended on temperature. After incubation for 4 days at 20°C, no significant effects of anaerobic conditions or leachate on the survival of P. betae were found. However, at 40°C for 4 days under anaerobic conditions, survival of P. betae was significantly lower than survival under aerobic conditions in water as well as in leachate. In leachate taken from an aerobic compost heap, aerobically incubated at 40°C for 4 days, survival of P. betae was significantly lower than survival in water at the same temperature. As anaerobic spots are prevalent in aerobic compost heaps, especially during the thermophilic phase, actual inactivation temperatures under composting conditions are likely to be lower than the temperatures we found for eradication in water under aerobic conditions.     

Virus Content and Virulence of Polymyxa betae Keskin Isolates Obtained from Different Regions in Europe

Polymyxa betae isolates were obtained by means of bait plants from a large number of soil samples collected in eastern Germany. Additional P. betae isolates were received from several institutions in western Germany and abroad. Isolates were grown on sugarbeet seedlings and tested for the presence of beet necrotic yellow vein virus (BNYVV) and beet soilborne virus (BSBV). BNYVV was only present in isolates from western Germany and abroad but absent in all isolates from eastern Germany., In contrast, BSBV was detected in more uniform geographic distribution in 14 out of 33 P. betae isolates tested. The virulence of P. betae isolates was estimated on the basis of the extent of resting spore formation in the root system of sugarbeet seedlings. Differences in virulence were found among virus-free as well as virus-carrying P. betae isolates. The mean value of virulence ratings was distinctly lower with BNYVV-carrying isolates and slightly lower with BSBV-carrying isolates as compared to virus-free isolates.