Browsing by Subject "ELISA"

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    Analyzing resistance to ergot caused by Claviceps purpurea [Fr.] Tul. and alkaloid contamination in winter rye (Secale cereale L.)
    (2022) Kodisch, Anna; Miedaner, Thomas
    Ergot caused by Claviceps purpurea [Fr.] Tul. is one of the oldest well-known plant diseases leading already in medieval times to severe epidemic outbreaks. After the occurrence of honeydew, the characteristic ergot bodies called sclerotia are formed on the ear. These are containing toxic ergot alkaloids (EAs). Strict limits are set within the European Union. Rye (Secale cereale L.) as cross-pollinating crop is particularly vulnerable to ergot since the competitive situation of fungal spores and pollen during flowering. Nevertheless, even today the threat is real as agricultural practice is changing and screening studies revealed EAs in samples of the whole cereal value chain frequently. The aims were to establish a harmonized method to test ergot resistance and EA contamination in winter rye, to clarify major significant factors and their relevance and to reveal the suitability of one commercial ELISA test. Further, effort was paid to examine the covariation of ergot amount and EA content considering different factors because of prospective legislative changes. Genotypes showed significant variation for ergot severity and pollen-fertility restoration after natural infection as well as artificial inoculation whereas a high positive correlation could be found between both treatments. Additionally, variances of environment, general combining ability (GCA), specific combining ability (SCA), and interactions were significant. Although male pollen-fertility restoration was of utmost importance, the female component was also significant. This illustrates that apart from promising selection of high restoration ability the maternal restorability could be exploited in future breeding programs especially when a high pollen amount is already reached. A large-scale calibration study was performed to clarify the covariation of ergot severity, EA content (HPLC, ELISA) considering genotypes, locations, countries, years, and isolates. EA profile was rather stable across country-specific isolates although large differences regarding the EA content were detected. The moderate covariation between ergot severity and EA content (HPLC) indicates that a reliable prediction of the EA content based on ergot severity is not possible what can also not be explained by grouping effects of the factors. Further, EAs seem not to act as virulence factor in the infection process since EA content showed no relationship to disease severity. Additionally, the missing correlation of ELISA and HPLC leads to the conclusion that the ELISA is not an appropriate tool what can be used safely to screen samples regarding ergot in the daily life. The genetic variation of male-sterile CMS-single crosses was analysed in a special design without pollen in field and greenhouse to identify resistance mechanisms and to clarify whether ergot can be reduced in the female flower. At this, comparison of needle and spray inoculation revealed medium to high correlations illustrating that both methods were suitable for this research. Significant environment and genotype by environment interaction variances were detected. So, testing across several environments is necessary also without pollen. Further, small but significant genotypic variation and identification of one more ergot-resilient candidate revealed that selection of female lines could be promising to further reduce ergot. The EA content was lower for less susceptible genotypes. Thus, EA content can be considerably reduced by breeding. A strong positive correlation could be found for ergot severity and EA content when analysing 15 factorial single crosses. The male pollen-fertility restoration was also here the most relevant component but the female component contributed an obviously higher proportion for the EA content than for ergot severity. In conclusion, this thesis demonstrate that implementing of a high and environmental stable male fertility restoration ability via exotic Rf genes can effectively reduce ergot although also the female restorability enables great opportunities. The unpredictable covariation between ergot amount and EA content illustrates that both traits have to be assessed, in particular the EA content by a valid HPLC approach to guarantee food and feed safety.
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    Entwicklung von spezifischen PCR-ELISA Nachweissystemen für Coxiella burnetii, Francisella tularensis und Orthopockenviren
    (2001) Kohlhaußen, Simone; Böhm, Reinhard
    In the present work specific systems on the basis of the PCR were developed for the detection of Coxiella burnetii, Francisella tularensis and Orthopoxviruses. The detection of the pathogens is possible without additional cultivation. In order to exclude false-negative PCR results the amplification reaction was established as a competitive PCR in which an internal control DNA is included as an amplification check. With regard to a possible automation the detection-systems were constituted in form of a 'PCR ELISA'. The colorimetric detection of the amplicons takes place thereby in streptavidin coated microtiter plates. For each system biotinylated capture probes for both the internal control DNA (ST) and the pathogen specific DNA (WT) have been developed. For the use of these proof systems in the clinical laboratory, the routine application of the tested decontamination procedure by means of UNG during the execution of the PCR ELISA is recommended. For all three systems a detection of the pathogens is possible within one working day. For the species specific detection of F. tularensis and the genus specific detection of Orthopoxviruses the PCR ELISA systems established here represent the first developments of this type with integrated amplification control and potential for quantification.