Licorice, cucumber, downy mildew: tracing the secret Interactions between the plant extract, the host and the pathogen

In ancient times, right after they started to domesticate plants, humans had to become plant protectors. The first protection strategies were mainly against herbivorous animals of any kind, arthropods as well as vertebrates. Thousands of years later humans discovered microorganisms like bacteria and fungi and soon correlated them with the disease they, their livestock and their plants had to suffer. The control agents they used against plant pests were mainly inorganic substances such as sulfur, arsenic or mercury. Their own diseases, in contrast, were fought with medicinal plants for thousands of years, more or less successfully. After problems caused by chemical pesticides, like DDT, became obvious (enrichment in the food chain and with that in the human tissues as well), the search for alternatives started. In more recent times more and more scientific studies investigate the potential of medicinal plants as plant protection agents. The aim of this thesis was to investigate the potential of ethanolic extract of leaves of the medicinal plant Glycyrrhiza glabra (licorice) as a control agent against one of the main pests in cucumbers, the Oomycete Pseudoperonospora cubensis (downy mildew). The first question addressed was: Has the licorice extract a potential as alternative control agent, not only in the laboratory, but also under commercial conditions in greenhouses? The second question addressed was: If it shows a potential, what is the active ingredient? And the final question addressed was: What is the mode of action? Under semi-commercial conditions the licorice extract has a high potential as control agent against cucumber downy mildew (efficacy up to 83.0%, application interval 7 -11 days, 3% extract concentration). Besides this, in semi-commercial trials a dark green color of licorice extract treated plants was observed. The crude licorice extract was fractionated by shake-out procedure in 6 fractions. The active ingredient was found to be part of extract fraction F6, which contains acidic substances. This fraction reached efficacies up to 97.6% in bioassays on treated cumber plants. Three flavanoids were detected in sub-fractions of fraction F6 and identified as glabranin, licoflavanon and pinocembrin. All three are known for their antimicrobial effect and were effective against another Oomycete, Phytophthora infestans in in vitro assays. Also, it is known that G. glabra extract kills zoospores of P. cubenis in vitro (Schuster et al. 2010) which may be due to the detected flavanoids. However, the effect of fraction F6 is not explainable by the action of the antimicrobials glabranin, licoflavanon and pinocembrin alone. Sub-fractions not containing those three substances showed efficacies up to 89.2%. This together with the observed dark green color of treated plants in semi-commercial trials led to the assumption that licorice leaf extract has not only a direct effect on the pathogen but influences the plant itself. This assumption was supported by the finding of elevated chlorophyll and anthocyanin content in treated plants and a positive effect on the stress indicator chlorophyll fluorescence. Even in highly infected fraction F6 treated plants (disease severity 92.5%) the Fv/Fm value (0.80) was stable during the whole measuring period, whereas the Fv/Fm value of also highly infected water treated plants decreased (disease severity 100.0%) from 0.80 to 0.69 in the same time. Furthermore, an elevated H2O2 level in the leaf tissues of licorice extract treated uninfected cucumber plants and an up-regulation of PR-1 mRNA could be found. The level of PR-1 mRNA was approximately 4 times higher than in water treated plants. Both, an elevated H2O2 level and an up-regulation of Pr-1mRNA expression are strong indicators for induced resistance. Since PR-1 has antifungal activity, this high amount of PR-1 is a possible explanation for inhibition of germ tube growth of P. cubensis zoospores on licorice treated leaf discs found in this thesis. The germ tube length on water treated leaf discs was 17.3-26.6 µm, whereas the germ tube length on licorice treated leaf disc was only 1.6-6.4 µm. Based on these results it was concluded that the ethanolic leaf extract of G. glabra is a highly potent control agent against cucumber downy mildew (P. cubensis) and its mode of action is a combination of direct effects on the pathogen development and induced resistance