Outbreaks of foodborne illness linked to consumptions of fresh, or partially processed, agricultural products are a growing concern in industrialized and developing countries. The incidence of human pathogens on fresh fruits and vegetables is often related to the use of recycled wastewaster in surface irrigation as well as high amounts of animal manure in agricultural management practice. Thereby the soil inhabiting fauna plays an important role in the transport and dissemination of microorganisms. The focus of the proposed project is on nematodes, well known vectors for bacteria and viruses in soil. The major goals are to: (1) survey human pathogens in soil and on/in free-living and plant parasitic nematodes in agriculture field sites irrigated with recycled wastewater or fertilized with fresh animal manure in Israel and the Palestinian Authority, (2) assess the function of nematodes as vectors in transmitting bacteria from microbial hot spots to plants, and (3) localize bacteria on and/or within the nematode and identify bacterial factors required for survival in the nematode host. Understanding the mechanisms involved in dissemination of human pathogens by nematodes will enhance the ability to develop practical means to minimize contamination of fresh produce and increase safety in food production.
Various species of pest insects cause substantial damage to agriculture every year, or transmit deadly diseases to animals and humans. A successful strategy to control pest insect populations is based on the Sterile Insect Technique (SIT), which uses the release of mass-reared, radiation sterilized male insects to cause infertile matings and thus reduce the pest population level. However, irradiation is not applicable to every insect species. Thus, new strategies based on genetic modifications of pest insects have been developed or are currently under investigation.The goal of the proposed research is to improve the development and ecological safety of genetically engineered (GE) insects created for enhanced biological control programs, including the SIT and new strategies based on conditional lethality. A major concern for GE insect release programs is transgene stability, and maintenance of their consistent expression. Transgene loss or intra-genomic movement could result in loss of strain attributes, and may ultimately lead to interspecies movement resulting in ecological risks. To address potential transgene instability, a new transposon vector that allows post-integration immobilization will be tested in the Mediterranean, Mexican and Oriental fruit fly tephritid pest species. In addition, the system will be established in the mosquito species Aedes and Anopheles - carriers of dengue and malaria.Random genomic insertion is also problematic for GE strain development due to genomic position effects that suppress transgene expression, and insertional mutations that negatively affect host fitness and viability. Diminished transgene expression could result in the unintended survival of conditional lethal individuals, or the inability to identify them. To target transgene vectors to defined genomic insertion sites having minimal negative effects on gene expression and host fitness, a recombinase-mediated cassette exchange (RMCE) strategy will be developed that. RMCE will also allow for stabilization of the target site, will be tested in tephritid and mosquito species, and will aid to the development of stabilized target-site strains for conditional lethal biocontrol. This will include a molecular and organismal evaluation of an RNAi-based lethality approach. Lethality based on an RNAi mechanism in the proposed insects would increase the species specificity and having multiple targets for lethality versus one target in existing systems. By seeking to improve transgene expressivity and stabilization of transposon-based vector systems, this proposal specifically addresses issues related to new GE insects by reducing their unintended spread after field release, and by limiting the possibilities for transgene introgression.
Cherry leaf roll virus (CLRV) is a plant pathogen of economic and ecologic importance. It is globally distributed in a wide range of forest, fruit, and ornamental trees and shrubs. In several areas of cherry and walnut production CLRV causes severe losses in yield and quality. With current reference to the rapid dissemination and strong symptom expression in Finnish birches and the Germany-wide distribution of CLRV in birches and elderberry, we continuously investigate and gradually reveal CLRV transmission pathways as by pollen, seeds or water. However, modes and interactions responsible for the wide intergeneric host transmission as well as for the exceptional CLRV epidemic in Fennoscandia still remain unknown. In this project systematic studies shall investigate biological vectors as a causal agent to finally derive control mechanisms and strategies to avoid new epidemics in different hosts and geographic regions. Detailed monitoring of the invertebrate fauna of birch stands/forests and elderberry plantations in Germany and Finland shall reveal potential vectors to subsequently study them in detail by approved virus detection methods and transmission experiments. Molecular analyses of the CLRV coat protein shall prove its role as a viral determinant for a virus/vector interaction. Consequently, this project essentially will contribute important answers on the CLRV epidemiology, and this will be a key element within the first network of research on plant viral pathogens in forest trees.
Ziel des Projekts ist die Entwicklung von speziellen Limiter-Techniken für Strömungs- und Transportprobleme. Im Mittelpunkt der geplanten Arbeiten stehen hochauflösende Finite-Elemente-Verfahren zur Simulation von Ozeanströmungen auf unstrukturierten Gittern. In erster Linie sollen die von den Antragstellern entwickelten und analysierten Ansätze an problemspezifische Anisotropien angepasst werden, die in geophysikalischen Anwendungen auftreten. Die neuen Limiter werden in der Lage sein, die horizontalen und vertikalen Komponenten separat zu beschränken, um eine übermäßige Verschmierung durch die numerische Diffusion zu verhindern. Darüber hinaus sollen die bisherigen Limiter-Konzepte auf Vektorfelder - insbesondere Geschwindigkeiten und diffuse Flüsse - erweitert werden. Um die Positivitätserhaltung im Rahmen eines vertikalen Wirbelviskositätsmodells zu garantieren, ist eine Sonderbehandlung für heterogene Diffusionskoeffizienten und nichtlineare Quellterme vorgesehen. Die anisotropen Flusskorrektur-Schemata und Ableitungslimiter sollen in die Software-Pakete UTBEST3D und FESOM integriert werden. Die erwartete Reduktion der unphysikalischen Vermischung soll durch detaillierte numerische Studien quantifiziert werden.
Cassava is a crop used by subsistence farmers in tropical and subtropical regions. It is extensively cultivated in certain regions of India and also in other Asian and African countries, and its cultivation is expected to further increase due to climate changes and increased water shortage, since cassava is drought-adaptive. Viruses, especially the begomoviruses Indian cassava mosaic-, Sri Lankan cassava mosaic- and African Cassava mosaic viruses (ICMV,SLCNV,ACMV here commonly I/SL/ACMV) cause dramatic losses in cassava cultivation. The plant recognizes viral RNA in the form of double-strand (ds)RNA, a by-product of virus replication. Long dsRNA is chopped by dicer enzymes into small interfering (si)RNAs, 21 to 24 base pair duplexes, consisting of a guide and a passenger-strand. The guide-strand forms together with the Slicer protein AGO a 'RISC'-complex which recognizes and cleaves cognate, i.e. in our case viral RNA. A remarkable feature of RNA interference (RNAi) is that, once started, it initiates a positive feedback loop, whereby the sliced RNA fragments are converted to more dsRNA by host RNA-dependent RNA polymerase, which give rise to more siRNAs. Furthermore, siRNAs or other components of the silencing pathway invade the plant systemically and protect from virus proliferation. Our antiviral strategy is to shift this equilibrium in favour of the plant by providing dsRNA cognate to the virus sequences. As a result, more siRNAs are produced, more virus RNA is destroyed and more systemic silencing signal reaches the growing tissue. Finally plants recover fast from initial infection or even become immune to incoming virus. Our project involves isolation, sequencing and cloning of the I/SL/ACV virus populations and their satellites occurring in the states of Andhra Pradesh, Maharashtra, Kerala, and Tamil Nadu. From these sequences a series of constructs will be derived that express hairpins, i.e. dsRNA with a loop between the RNA arms. We will construct variants that target all these viruses, including those that mimic natural micro (mi)RNAs or transacting (ta)siRNAs down-regulating host genes, and those provided with constitutive or virus-inducible promoters. We will optimize these constructs in transient assays to select those most efficient in initiating siRNA production. Selected constructs will then be cloned in Agrobacterium Ti-plasmid vectors and used for transformation of host plants. Although virus-resistant cassava is our goal, preliminary tests of the constructs could also be performed with the easily transformable Nicotiana benthamiana in one of our labs, as has been done by the applicants for African cassava mosaic begomovirus or with a more easily transformable model cassava cultivar with the help of Dr. Peng Zhang, Chinese academy of Science, Shanghai, thus extending a former collaboration of the Basel group with him.
Although the use of genetically modified plants for bioremediation, or the in situ cleaning of contaminated sites, has been known for quite some time, little attention has so far been paid to the production of antibodies in plants and their ex vivo application in selective depletion. Therefore, highly affine and specific antibodies against algal toxins using microcystin as an example will be produced in plants at low cost within this research project. The basis is a monoclonal antibody (Mab 10E7, species: mouse) generated in a former research project. The sequence of the variable domains will be determined, optimized for plants and sub cloned into suitable plant transformation vectors, which already contain constant antibody sequences. In addition, a scFv fragment containing different tag sequences and fusion proteins will be constructed. Leaf-based (tobacco) as well as seed-based (barley) systems will be used.Affinity-purified plant-produced antibodies (plantibodies) will be characterized in detail for their binding properties using microtitre plate-ELISA and surface plasmon resonance (SPR). The monoclonal mouse antibody will be used as reference. To assure cost-efficiency for future applications, roughly purified fractions (sequential pH and temperature treatment followed by filtration) will be tested for the upscaling. Following immobilization of the plantibody fractions on suitable substrates, for instance membranes, porous polymer monoliths or in porous glasses, their application for depletion will be defined using model water samples spiked fortified with microcystins.
Nuklidvektoren wurden ermittelt, um die Messungen für die Freigabe nach § 29 der Strahlenschutzverordnung zu erleichtern. Dafür müssen die Nuklidvektoren aber repräsentativ und ausreichend konservativ sein. Im Vorhaben wurde die bisherige Praxis bei der Ermittlung von Nuklidvektoren dargestellt. Ausgehend von der Definition des Begriffs 'Repräsentativität' sind die möglichen Vorgehensweisen bei der Ermittlung repräsentativer Nuklidvektoren beschrieben. Die konkrete Gestaltung des Untersuchungsprogramms ist von der Zielstellung und der radiologischen Situation des zu untersuchenden Objekts abhängig. Für die einzelnen Schritte lassen sich aber verschiedene Möglichkeiten und Regeln für ein sachgerechtes Vorgehen beschreiben. Bei der Erstellung der Probenahmestrategie ist die Kompetenz des Bearbeiters der entscheidende Faktor für die Richtigkeit.
Ziel des Projektes ist die Erstellung einer Literaturübersicht zum Stand der Entwicklung transgener Gehölze und zu den Möglichkeiten (Risikopotential) eines horizontalen Gentransfers in Forstgehölzen. Bei der Erzeugung transgener Gehölze werden binäre Vektoren verwendet, die aus den natürlichen Ti-Plasmiden von Agrobakterien entwickelt wurden. Mit Hilfe dieser Vektorsysteme wird die zu übertragende rekombinante DNA in die Pflanzenzellen eingeschleust. Daneben kann die DNA über bakteriellen Gentransfer auch in andere Bakterien übertragen werden. Durch das Vorhandensein endophytischer Bakterien in Bäumen und die relativ lange Persistenz der Agrobakterien in transformierten Gehölzen besteht somit ein Risiko des horizontalen Gentransfers, das bisher kaum beschrieben wurde. In der Studie wird der Kenntnisstand zum Vorkommen von Endophyten in Forstgehölzen, zur Persistenz von Agrobakterien sowie zu den Mechanismen des bakteriellen Gentransfers dokumentiert. In Verbindung mit den Eigenschaften der verwendeten Vektoren werden hieraus prinzipielle Möglichkeiten für den Gentransfer in die Endophytenflora beschrieben und Schlussfolgerungen für den Forschungsbedarf abgeleitet.
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