Optimierung von Saatgutbehandlungsmitteln mit Wirkung gegen Flugbrand an Gerste und Weizen (Ustilago nuda, U. tritici) unter Nutzung verbesserter Verfahren zum Nachweis der Erreger

Ziel des Projektes war die Entwicklung von Saatgutbehandlungsmitteln zur Flugbrandbekämpfung im Ökolandbau und von Techniken die geeignet sind, die Entwicklung solcher Saatgutbehandlungsmittel zu beschleunigen. In Inokulationsversuchen konnte im Feldversuch durch Ausstäuben von Sporen Saatgut mit 2-5% Flugbrandbefall erzeugt werden. Nach Einzelblüteninokulation lag der Befall oft über 60%. Anders als bei Weizen waren bei Gerste die Keimfähigkeit und das Tausendkorngewicht von solchem Saatgut deutlich reduziert. Von ca. 30 geprüften Pflanzenextrakten führten neun nach Inkorporation in PDA zu einer vollständigen Hemmung der Brandsporenkeimung. In Hemmtesten mit Mikroorganismen betrug der Anteil wirksamer Isolate bei den Trichoderma-Isolaten 43%, bei den Actinomyceten 30% und bei den übrigen Bakterien 11%. In mehrjährig durchgeführten Kleinparzellenversuchen mit flugbrandinfizierter Gerste und Weizen wurden mit einigen Behandlungsvarianten Bekämpfungserfolge erzielt. Die Effekte waren aber zu gering für die praktische Anwendung und nur bedingt reproduzierbar. Topfversuche im Gewächshaus mit hochinfizierten Saatgutchargen erbrachten ähnliche Ergebnisse. Die Wirksamkeit gegen Haferflugbrand wurde nur einmal überprüft. Wie im Falle von Gersten- und Weizenflugbrand war der Bekämpfungserfolg auch beim Haferflugbrand unbefriedigend. Eine Ausnahme bildete die Saatgutbehandlung mit Ethanol (70%). Mit ihr wurde bei Hafer eine Flugbrandwirksamkeit von ca. 80% erzielt. Nach Anfärbung mit dem Fluoreszenzfarbstoff Blankophor wurde beobachtet, dass der Pilz schon wenige Tage nach Beginn der Keimung in das Apikalmeristem und die Blattprimordien eindrang. Im 1-Knotenstadium waren die Ährenanlagen meist völlig besiedelt. Ein Protokoll für einen immunologischen (ELISA) und für einen molekularbiologischen (real-time PCR) Nachweis wurde entwickelt und zur Quantifizierung des Pilzes in Jungpflanzen angewendet. Die mit beiden Methoden erhaltenen Daten stimmten gut überein. In weiteren Versuchen konnte die Anwendbarkeit des mikroskopischen Nachweises und des ELISA für die Entwicklung von Saatgutbehandlungsmitteln und –verfahren exemplarisch gezeigt werden

Mode of Action of the Natural Product Allicin in a Plant Model:Influence on the Cytoskeleton and Subsequent Shift in Auxin Localization

Allicin is a defense substance produced by garlic cells when they are injured. It is a redox-active thiosulfinate showing redox-activity with a broad range of dose-dependent antimicrobial and biocidal activity. It is known that allicin efficiently oxidizes thiol-groups, and it has been described as a redox toxin because it alters the redox homeostasis in cells and triggers oxidative stress responses. Allicin can therefore be used as a model substance to investigate the action of thiol-specific prooxidants. In order to learn more about the effect of allicin on plants, we used pure synthetized allicin, and studied the influence of allicin on organelle movement in Tradescantia fluminensis as a cytoskeleton-dependent process. Furthermore, we investigated cytoplasmic streaming in sterile filaments of Tradescantia fluminensis, organelle movement using transgenic Arabidopsis with organelle-specifics GFP-tags, and effects on actin and tubulin in the cytoskeleton using GFP-tagged lines. Tubulin and actin were visualized by GFP-tagging in transgenic lines of Arabidopsis thaliana to visualize the influence of allicin on the cytoskeleton. Since auxin transport depends on recycling and turnover of the PIN protein involving cytoskeletal transport to and from the membrane localization sites, auxin distribution in roots was investigated using of transgenic PIN1–GFP, PIN3–GFP, DR5–GFP and DII–VENUS Arabidopsis reporter lines. Allicin inhibited cytoplasmic streaming in T. fluminensis, organelle movement of peroxi-somesperoxisomes, and the Golgi apparatus in a concentration-dependent manner. It also destroyed the correct root tip distribution of auxin, which probably contributed to the observed inhibition of root growth. These observations of the disruption of cytoskeleton-dependent transport processes in plant cells add a new facet to the mechanism of action of allicin as a redox toxin in cells

Natural history of Arabidopsis thaliana and oomycete symbioses

Molecular ecology of plant–microbe interactions has immediate significance for filling a gap in knowledge between the laboratory discipline of molecular biology and the largely theoretical discipline of evolutionary ecology. Somewhere in between lies conservation biology, aimed at protection of habitats and the diversity of species housed within them. A seemingly insignificant wildflower called Arabidopsis thaliana has an important contribution to make in this endeavour. It has already transformed botanical research with deepening understanding of molecular processes within the species and across the Plant Kingdom; and has begun to revolutionize plant breeding by providing an invaluable catalogue of gene sequences that can be used to design the most precise molecular markers attainable for marker-assisted selection of valued traits. This review describes how A. thaliana and two of its natural biotrophic parasites could be seminal as a model for exploring the biogeography and molecular ecology of plant–microbe interactions, and specifically, for testing hypotheses proposed from the geographic mosaic theory of co-evolution

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups

<p>Abstract</p> <p>Background</p> <p>Oomycetes are a large group of economically and ecologically important species. Its most notorious member is <it>Phytophthora infestans</it>, the cause of the devastating potato late blight disease. The life cycle of <it>P. infestans </it>involves hyphae which differentiate into spores used for dispersal and host infection. Protein phosphorylation likely plays crucial roles in these stages, and to help understand this we present here a genome-wide analysis of the protein kinases of <it>P. infestans </it>and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.</p> <p>Results</p> <p>Bioinformatic searches of the genomes of <it>P. infestans</it>, <it>P. ramorum</it>, and <it>P. sojae </it>reveal they have similar kinomes, which for <it>P. infestans </it>contains 354 eukaryotic protein kinases (ePKs) and 18 atypical kinases (aPKs), equaling 2% of total genes. After refining gene models, most were classifiable into families seen in other eukaryotes. Some ePK families are nevertheless unusual, especially the tyrosine kinase-like (TKL) group which includes large oomycete-specific subfamilies. Also identified were two tyrosine kinases, which are rare in non-metazoans. Several ePKs bear accessory domains not identified previously on kinases, such as cyclin-dependent kinases with integral cyclin domains. Most ePKs lack accessory domains, implying that many are regulated transcriptionally. This was confirmed by mRNA expression-profiling studies that showed that two-thirds vary significantly between hyphae, sporangia, and zoospores. Comparisons to neighboring taxa (apicomplexans, ciliates, diatoms) revealed both clade-specific and conserved features, and multiple connections to plant kinases were observed. The kinome of <it>Hyaloperonospora arabidopsidis</it>, an oomycete with a simpler life cycle than <it>P. infestans</it>, was found to be one-third smaller. Some differences may be attributable to gene clustering, which facilitates subfamily expansion (or loss) through unequal crossing-over.</p> <p>Conclusion</p> <p>The large sizes of the <it>Phytophthora </it>kinomes imply that phosphorylation plays major roles in their life cycles. Their kinomes also include many novel ePKs, some specific to oomycetes or shared with neighboring groups. Little experimentation to date has addressed the biological functions of oomycete kinases, but this should be stimulated by the structural, evolutionary, and expression data presented here. This may lead to targets for disease control.</p

Computational Prediction and Molecular Characterization of an Oomycete Effector and the Cognate Arabidopsis Resistance Gene

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses