Publikationsserver der RWTH Aachen University
Isolation von Ascosporen des pflanzenpathogenen Pilzes Venturia inaequalis und Charakterisierung von Antikörperinteraktionen zur Entwicklung eines immunogravimetrischen Sensors
Abstract
dc:descriptionThe work presented here was performed in the frame of an interdisciplinary project. The aim of this project was the development of a biosensor detecting ascospores of Venturia inaequalis, the causual agent of apple scap. It was shown, that such a biosensor could be a useful addition to present-day scab-prediction models. In the examined years 2002 and 2003 the weather-based forecast-models had misjudgements that caused on the one hand unnecessary environmental pollution with fungicides and on the other hand did not prevent the control of infections. Our data clearly showed, that the common models to forecast the release of ascospores are not sufficient to assure non-polluting and safe control of apple scab. Isolation of ascospores: The biological part of the sensor was intended to consist of antibodies specifically recognizing the ascospores of Venturia inaequalis. To generate these antibodies it was necessary to isolate a large number of highly purified ascospores. At first the spores were harvested in a batch-procedure. These spores were further purified by physical methods: sieving, density-gradient centrifugation and anion-exchange-chromatography. During these procedures about 80% of the initially purified spores were lost. Also a change of antigen structures during processing could not be excluded. Therefore, other methods were established. Ultimately, the spores used for the project were harvested using the natural discharge of the spores from infected leaves. The spores were collected from lids of petridishes and washed. It was unlikely that surface-antigens were changed during the isolation. Sporetrap: To use the sensor in the orchards it was necessary to develop a sporetrap which was able to collect the spores from the air and could transfer them into a aqueous environment. Such a sportrap was developed and successfully tested in an orchard. Analysis of the surface of the spores: A spore-surface and antigen analysis was necessary for optimising the antibodies towards the requirements of the sensor. To this end, a mouse polyclonal antiserum was available containing antibodies directed against Venturia spores. Treatment of spores with various enzymes showed that the antigens and the spore-surface had different characteristics. One set of the antibodies in the serum was reactive against N-linked sugar-chains of glycoproteins. The remaining antibodies recognized a protein. Both antigens were found to be securely anchored in the cell wall of the spore. Successive degradation of the outer layers of the cell wall showed four biochemically distinct layers: the spores are covered with a detergence soluble coat which is fixed to a glucan layer. Underneath the glucan a cellulose layer was found which is interconnected with a chitin lattice. The morphology of the spores by all these treatments was not altered. cDNA-screening: Using the mouse serum, we isolated a gene from a cDNA expression library, derived from mycelia of Venturia inaequalis. The sequence of the cDNA was determined. By means of PCR, the gene was amplified from the genome and also sequenced. No introns were found. The gene encodes a protein of 31 kDa showing similarity to esterases. Esterase activity was found in bacteria expressing the cDNAhowever, no cutinase activity could be detected.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2004
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Wolters, Andreas Helmut
- Contributors dc:contributor
-
- Slusarenko, Alan
Subjects
dc:subject × 11Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:61983