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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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...     

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.     

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.     

禾谷多粘菌分离、培养、接种及用于大麦抗黄花叶病的鉴定

 通过禾谷多粘菌Polymyxa graminis L.休眠孢子分离接种感病大麦品种,并进行砂培养,获得13个纯化了的禾谷多粘菌分离物,且其中3个带有大麦黄花叶病毒(BaYMV)。用分别带有BaYMV和大麦温和花叶病毒(BMMV)的英国禾谷多粘菌分离物的游动孢子接种13个中国大麦品种,以及用BaMMV摩擦接种36个中外大麦品种,抗性鉴定结果游动孢子接种与摩擦接种一样,均与田间鉴定结果一致,且大麦对BaYMV的抗性与对BaMMV的抗性一致,从而这2种接种方法可用于大麦品种(系)和育种中间体对BaYMV抗性的快速鉴定和筛选。游动孢子或休眠孢子接种方法还可有效地鉴定大麦对禾谷多粘菌的抗性。     

Diversity of rDNA-ITS sequences of <Emphasis Type="Italic">Polymyxa graminis</Emphasis> from wheat and barley in Japan

Polymyxa graminis is a soil-borne obligate organism that transmits bymoviruses and furoviruses to barley and wheat. We analyzed rDNA-ITS sequences of P. graminis from roots of wheat and barley in fields in Japan and obtained five kinds of sequences; two sequences were almost the same as known ribotype Ia and IIa, respectively, and three were close to ribotype Ib. When infection of P. graminis was examined using PCR, ribotype Ia was detected only in barley, but ribotypes Ib and IIa were detected in both wheat and barley. Our analysis suggested that Japanese ribotype Ib transmits furoviruses and bymoviruses.     

四川大白菜细菌性软腐病新病原菌Pectobacterium versatile的鉴定

为明确引起四川理县大白菜软腐病的病原菌种类, 从理县梭罗沟村田间采集具有典型软腐症状的大白菜病样, 采用组织分离、致病力测定、生理生化分析、形态学和分子生物学方法进行鉴定。结果表明, 分离获得的5株细菌在NA培养基上形成的菌落呈圆形、半透明、乳白色、中心突起、边缘平滑, 在半选择性培养基CVP上培养48 h后产生凹陷。5株菌均能使大白菜、胡萝卜、马铃薯和芹菜的离体组织腐烂, 接种于白菜苗后叶片呈湿腐萎蔫症状。通过大白菜叶柄接种部位形成的病斑长径来比较菌株致病力强弱, 各菌株的致病力有显著差异。基于看家基因pgi、rpoS、mdh、proA、mtlD 和icdA(NCBI登录号为OL963562~OL963571)的多基因联合系统发育树, 分离菌株与Pectobacterium versatile聚为一簇, dnaX-leuS-recA(NCBI登录号为OL963572~OL963576)的多位点序列分析(MLSA)进一步支持以上结果, 且准确性更高。分离菌株的生理生化特征与P.versatile模式菌株一致, 综上将其鉴定为P.versatile。这是我国首次报道P.versatile引起大白菜软腐病。