Alternative Strategien für den Pflanzenschutz
Schwinges, Patrick; Conrath, Uwe (Thesis advisor); Schirawski, Jan (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2019
According to estimates the worldwide population will exceed the mark of nine billion people until the year 2050. Tightly connected to this development, the demand for major crops for food and feed rises. To satisfy those demands is challenging, especially as the food production is threatened by various pathogens. Since over 150 years, pesticides are an effective tool to protect plants from pathogens. However, the extensive application of fungicides not only promotes the emergence of fungal strains with reduced sensitivity to active ingredients, but also raises ecological and health concerns. Thus, a worldwide reduction of pesticide use is demanded and therefore, alternative strategies for plant protection are desired. In this study, an approach was pursued, which is based on the usage of leaf binding peptides coupled with an antimicrobial protein. By application of the amphiphilic peptides "liquid chromatography peak I" (LCI) and thanatin (THA), the coupled eGFP protein was immobilized on soybean leaves. This binding withstood not only simulated but also natural rainfall. Furthermore, the bifunctional fusion protein DS01-THA serves as protective agent and reduces Asian soybean rust (SBR) severity on inoculated soybean plants by 30 % in a rainfast manner. Another strategy that might contribute to a more sustainable agriculture is based on the identification of naturally occurring antifungal compounds that may complement current disease management strategies which widely rely on the use of chemical fungicides. Therefore, in the second part of this study the focus was to identify antifungal molecules that are secreted to plant leaf surface. To disclose such plant protectants a variety of plant species were inoculated to identify plants on whose leaf surface the germination of P. pachyrhizi was impaired. Only sunflower plants were found to suppress the germination of fungal spores. This effect is likely associated with the presence of the fluorescent coumarins, scopoletin and ayapin. By investigating the biochemical function of enzymes from the phenylpropanoid pathway genes were identified, which are likely involved in coumarin biosynthesis in sunflower plants. Heterologous expression of candidate genes in E. coli facilitated the production of the coumarins esculetin, scopoletin, isoscopoletin and scoparone in precursor feeding experiments. When co-expressed in Nicotiana benthamiana HaF6’H1 and HaOMT3 not only enabled biosynthesis of antifungal scopoletin but also scoparone, which is not native to this plant. Because foliar applied scoparone provided protection of soybean from Asian soybean rust, engineering of this metabolic pathway in soybean may provide a possibility to reduce susceptibility to this devastating disease and decrease fungicide usage.In addition, whole transcriptome analysis of sunflower leaves identified CYP genes which expression correlated with the accumulation of ayapin upon treatment with CuCl2. This data may serve as a basis to elucidate the so far unknown biosynthetic route of this antifungal compound and enable engineering of crops that are capable of synthesizing ayapin. Generating plants that are capable of producing blends of different coumarins may hinder adaptation of pathogens to these antifungal substances and may provide more durable protection.