Detection of beet yellows virus in sugar beet leaves and roots by enzyme-linked immunosorbent assay (ELISA)

Using the enzyme-linked immunosorbent assay (ELISA) beet yellows virus (BYV) could be detected reliably in the leaves of sugar beet andTetragonia expansa Pall. and in the roots of sugar beet. Specifio γ-globulin of BYV antiserum was coupled to horse radish peroxidase by periodate oxidation. Optimum dilutions of antigen (extract from infected leaves) were1: 50 to 1: 200 for BYV detection in sugar beet andT. expansa leaves and1: 2 to 1: 5 for detection in sugar beet roots. Extracts from beet roots are not to be purified by ultracentrifugation, however, by the described method virus can be demonstrated only in 80–90% of naturally infected sugar beet roots. The method is specific, no increase of extinction values was found in healthy or beet western yellows virus infected plants. Presence of virus can be demonstrated by visual as well as photometric evaluation. Results confirmed the suitability of peroxidase application for detection of plant viruses by ELISA.     

Reproduction of sugar beet mosaic and tobacco mosaic viruses in anthocyanized beet plants

During our studies on the interaction of anthocyanins and plant virus diseases, reproduction of sugar beet mosaic (SBMV) and tobacco mosaic viruses (TMV) was investigated. Experiments were carried out in leaves of sugar beet,Beta vulgaris cv. Dobrovicka N and its spontaneous anthocyanized mutant. SBMV induces a systemic infection while TMV is responsible for primary local symptoms in sugar beet leaves only. Our quantitative analyses onAmaranthus caudatus L. andChenopodium quinoa Wilid. showed a significant decrease in concentration of SBMV in juice extracted from anthocyanized beet plants as compared with extracts from normal green infected plants. Significant differences were also obtained when SBMV — containing juice was tested in mixtures with healthy extracts from anthocyanized and normal green plants. Also the intensity of TMV symptoms in beet leaves was considerably decreased in leaves of antho-eyanized plants.     

Utilization of agro-industrial orange peel and sugar beet pulp wastes for fungal endo- polygalacturonase production

The pectinase enzymes are involved in several industrial applications, and industrial waste is one of the largest environmental pollutants, so this study aims to Endo-polygalacturonase (endo-PG) producing using Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153 and P. variotii AUMC 4149 by using some agro-industrial wastes (dried orange peel and sugar beet pulp) as a sole raw carbon source for degradation these waste in the process of urban wastes disposal. The fermentation process was carried out as a submerged culture technique under both shaken and static culture conditions. A. niger AUMC 4156 was the most promising producer of endo-PG under static conditions while P. oxalicum AUMC 4153 was the highest producer of endo-PG under shaken conditions. Sugar beet pulp proved to be the most preferable to orange peel as the only source of carbon in both shaken and static cultures. The medium that encompassing orange peel as a single carbon source afforded the highest protein content with all tested fungal strains in stirred and static cultures in comparison with sugar beet pulp. The highest activity of endo-polygalacuronase that produced using A. niger AUMC 4156 and P. oxalicum AUMC 4153 was achieved by using sugar beet pulp at 3% concentration under static cultures, meanwhile maximal enzyme activity produced by both fungal strains required 2% sugar beet pulp under shaken cultures. Sugar beet pulp showed promised potential as a good inducer for endo-polygalacturoase production, and enzymes production depended on fungal strains, culture medium, and submerged fermentation conditions.     

Sucrose translocation and storage in the sugar beet

Several physiological processes were studied during sugar beet root development to determine the cellular events that are temporally correlated with sucrose storage. The prestorage stage was characterized by a marked increase in root fresh weight and a low sucrose to glucose ratio. Carbon derived from ¹⁴C-sucrose accumulation was partitioned into protein and structural carbohydrate fractions and their amino acid, organic acid, and hexose precursors. The immature root contained high soluble acid invertase activity (V_max 20 micromoles per hour per milligram protein; K_m 2 to 3 millimolar) which disappeared prior to sucrose storage. Sucrose storage was characterized by carbon derived from ¹⁴C-sucrose uptake being partitioned into the sucrose fraction with little evidence of further metabolism. The onset of storage was accompanied by the appearance of sucrose synthetase activity (V_max 12 micromoles per hour per milligram protein; K_m 7 millimolar). Neither sucrose phosphate synthetase nor alkaline invertase activities were detected during beet development. Intact sugar beet plants (containing a 100-gram beet) exported 70% of the translocate to the beet, greater than 90% of which was retained as sucrose with little subsequent conversions.     

Enzymes Involved in the Postharvest Degradation of Sucrose in Beta vulgaris L. Root Tissue

The reducing sugar content of sugar beet (Beta vulgaris L.) roots increased during 30 days of storage at 21 C and 160 days at 5 C as a result of an increase in acid invertase activity. Sucrose synthetase and neutral invertase activities were high at harvest but declined during storage, thus showing no relationship with postharvest reducing sugar accumulation in sugar beet roots. Acid α-glucosidase activity was detected in fresh roots but showed no activity with sucrose as a substrate.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.     

The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.     

Monosomic addition lines of Beta corolliflora Zoss in sugar beet: cytological and molecular-marker analysis

Beta corolliflora is a wild relative of sugar beet (Beta vulgaris) with 2n=4x=36 chromosomes. Monosomic addition lines (2n=19) of B. corolliflora in B. vulgaris were identified from backcross progenies between triploid hybrids (genome constitution VVC) and sugar beet. They were characterized by DNA-fingerprinting using nine different B. corolliflora-specific repetitive sequences as probes and by fluorescence in situ hybridization (FISH) using two B. corollifora specific sequences and two rDNA probes. Unique banding patterns obtained after genomic Southern hybridization enabled the classification of monosomic addition lines into 11 clusters, three of which proved to have a wild beet chromosome fragment in addition to the sugar beet chromosomes as revealed by FISH. Repetitive sequences pBC216 and pBC1416 were found to be present only on wild beet chromosomes IV and V. Chromosomes I and IV were found to carry genes for 18S and 5S rRNA, respectively. An idiogram of B. corolliflora was established in the triploid VVC hybrid on the basis of chromosome size and FISH. Eight B. corolliflora addition lines could be unequivocally identified by Southern hybridization and FISH, one addition line carrying the missing wild beet chromosome is probably not viable under greenhouse conditions. The monosomic addition lines will serve as a bridge for transferring genes from wild species to sugar beet and will help to uncover genetic relationships between species of the genus Beta.     

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

Key message

Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.

Abstract

The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R ² > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.     

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.     

Differentiation of strains of sugar beet yellows virus onTetragonia expansa Murr. and other indicator plants

Five different isolates of beet yellows virus were maintained without any changes in their properties onTetragonia expansa Murr. syn.T. tetragonoides Pall. for a long period of time. According to their characteristics and different properties especially in a diploid inbred line of sugar beet the isolates are considered to be strains of BYV and are classified into three groups: group of mild strains (the mild masked and mild strains), normal strains (the common strain) and necrotic strains (the severe necrotic and necrotic strains). The necrotic strains of BYV were relatively easily transmissible manually to sugar beet plants and other indicator species. The common strain can be transmitted to sugar beet,Chenopodium quinoa Willd. but not toC. capitatum L. Asch. Mild strains are transmissible with difficulty andC. quinoa is the only species which develops a larger number of local lesions after inoculation. In contrast to the mild masked and common strains it is manually transmissible toC. capitatum. The mild masked strain can not be transmitted to sugar beet.Nicotiana quadrivalvis Pursh. is not susceptible to mechanical inoculation with BYV. Aphid transmission withMyzus persicae (Sulz.) was positive in experiments with necrotic strains only. Mechanical transmission of BYV was successful also toC. foliosum (Moench) Asch.,C. murale L. andClaytonia perfoliata Donn. The last two species were susceptible to inoculation by aphids as well. Attempts to transmit the virus manually toT. expansa Murr. andC. giganteum Donn. failed.     

The use of monoclonal antibodies to detect beet mild yellowing virus and beet western yellows virus in aphids

Information on infectivity of the aphids which invade sugar beet root crops each Spring is required for forecasting incidence and providing advice on control of virus yellows. Monoclonal antibodies, produced in the USA to barley yellow dwarf virus (BYDV) and in Canada to beet western yellows virus (BWYV), were used to distinguish between sugar-beet-infecting strains of the luteovirus beet mild yellowing virus (BMYV), and the non-beet-infecting strains of the closely-related BWYV in plant and aphid tissue. Totals of 773 immigrant winged Myzuspersicae and 124 Macrosiphum euphorbiae were caught in water traps in a crop of sugar beet between 25 April and 5 August 1990. Using the monoclonal antibodies and an amplified ELISA, 67%M. persicae and 19%M. euphorbiae were shown to contain BWYV; 8%M. persicae and 7%M. euphorbiae contained BMYV. In studies with live winged aphids collected from the same sugar beet field during May, 25 of 60 M. persicae and two of 13 M. euphorbiae transmitted BWYV to the indicator host plant Montia perfoliata; two M. persicae and two M. euphorbiae transmitted BMYV. In another study three of 65 M. persicae and one of three M. euphorbiae in which only BWYV was detected, transmitted this virus to sugar beet.     

Characteristics of Serially Produced Zoospore Suspensions of Polymyxa betae for Transmission of Beet Necrotic Yellow Vein Virus

Zoospore suspensions of Polymyxa betae were analysed for their potential as inocula to infect sugar beet plants with beet necrotic yellow vein furovirus. The infectivity could be maintained when zoospore suspensions were serially transferred. When zoospore-producing seedlings were individually transferred some of these seedlings lost their infectivity after several passages. Infectivity was first detected in suspensions within I day after inoculation of the plant by zoospores. The suspensions remained infectious for at least 10 h after removal of the plants producing viruliferous zoospores. Both the number of test plants infected and the concentration of virus that developed were greater at 25 C than at 20 C.     

SUGAR BEET YELLOWS: A PRELIMINARY STUDY OF THE DISTRIBUTION AND INTERRELATIONSHIPS OF VIRUSES AND VIRUS STRAINS FOUND IN EAST ANGLIA, 1955-57

Aphid transmissions to sugar beet seedlings from yellowed sugar beet leaves collected from commercial fields in East Anglia during the summers of 1955, 1956 and 1957, showed the occurrence of two yellowing viruses. One was sugar beet yellows virus (SBYV) and produced vein-etch and yellowing symptoms on beet seedlings in the glasshouse; the other produced yellowing but no etch. These two viruses were apparently unrelated, so that sugar beet tolerant to one of them would not necessarily be tolerant to the other. The second virus, called 'sugar beet mild yellowing virus' (SBMYV), decreased the root yield of sugar beet plants grown under glass, by as much as did the milder SBYV strains, but less than did the severe SBYV strains. The proportions of the two viruses in the samples differed from year to year and from place to place.     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

Sucrose phosphatase associated with vacuole preparations from red beet, sugar beet, and immature sugarcane stem

The specific phosphatase, sucrose phosphate phosphohydrolase (sucrose phosphatase, EC 3.1.3.24) was present in vacuole preparations from storage tissue of red beet (Beta vulgaris L.), sugar beet (Beta vulgaris L. cultivar Kawemono), and immature sugarcane (Saccharum spp. hybrid, cultivar NCO 310). In red beet vacuole preparations the specific activity of sucrose phosphatase, using the naturally occurring vacuole marker, betanin, as reference, was higher than the specific activity of cytoplasmic markers, phosphoenolpyruvate carboxylase and glucose 6-phosphate dehydrogenase, suggesting that sucrose phosphatase is associated with the vacuoles. High speed centrifugation of lysed vacuoles did not result in precipitation of the enzyme indicating that the enzyme is not tightly bound to the tonoplast. Sucrose phosphatase was more sensitive to inhibition by sodium vanadate and less sensitive to ammonium molybdate than was the nonspecific phosphatase which was also present in the extracts. Sucrose phosphatase might be part of the group translocator proposed recently to operate in the tonoplast of sugarcane and red beet.