RT Dissertation/Thesis
T1 Development of alternative strategies for the control of the important phytopathogens Phytophthora infestans (Mont.) and Erwinia amylovora (Burrill)
A1 Swaidat,Ihsan
WP 2008/02/13
AB Within the framework of two projects, financed by DBU (Deutsche Bundesstiftung Umwelt) and ProInno ("Förderung der Erhöhung der Innovationskompetenz mittelständischer Unternehmen"), respectively, in co-operation with an industrial partner, alternative phytosanitary compounds from natural sources have been screened. High throughput screening systems were developed and used for testing of large numbers of extracts of Actinomycetes in 96-well multiplates against Phytophthora infestans and Erwinia amylovora, the causal agents of tomato late blight and apple fire blight, respectively. These important phytopathogens were chosen as models. According to the Pflanzenschutzgesetz (PflSchG) §2 Nr. 10a, plant strengthening compounds (Pflanzenstärkungsmittel) should not act directly against the pathogen, but via an induction of plant resistance mechanisms. Therefore, one of the projects (DBU) aimed to exclude direct action on P. infestans. Based on the GFP-fluorescence of the P. infestans transformant 208m2, a fluorescence optical measurement of mycelium growth respectively growth inhibition was developed, to test the influence of the extracts and extract fractions. Only 52 out of 8335 extracts significantly inhibited the mycelium growth (Pi+) in this test, and thus had to be excluded according to §2 Nr.10a PflSchG for a potential commercial application. Searching for resistance inducing activity, all extracts were re-tested in parsley cell culture as a model for putative resistance induction characterised by formation of furanocoumarin phytoalexins. Only 42 out of the whole set of extracts tested, induced furanocoumarins (Pc+) significantly. In order to test if the induction correlates with a successful defence of host tissue against P. infestans, detached tomato leaves were treated with these Pc+ extracts. Only one extract resulted in formation of small sharp necrosis symptoms after pathogen inoculation, leading to strongly reduced infection and inhibited sporulation. The identification of the active ingredient is currently performed. When comparing the 52 direct acting substances (Pi+) with the potentially resistance inducing compounds (Pc+), three were also found actively inhibiting P. infestans in the host tissue. These three extracts also induced limited dark brown necrosis, suggesting an induction of hypersensitive reaction (HR). Infection area and sporulation level were reduced to levels below 25% of total leaf area. Microscopic investigations showed non-germinated or abnormally shaped germinated sporangia. Promising extracts were fractionated by the cooperation partner. Fraction 1 of one Pi+-extract (014 008-2) reduced the sporulation level and the size of the infection area to 5 and 25%, respectively, compared to the control. Application of fraction 2, although less effective than fraction 1, produced sporangiophores that were morphologically abnormal carrying no sporangia, indicating a possible highly specific action on a certain developmental step of the pathogen. In case of the extracts tested in vitro against E. amylovora, only 60 out of 5236 extracts inhibited bacterial growth. Only extracts showing a similar effect as the streptomycin positive control (12 extracts), were also tested in the cell culture of parsley for a potential resistance induction. The effect of such promising extract (000 391 CF) was compared in in vitro apple plantlets with two purified substances identified in the in vitro growth inhibition screening (tubercidin and streptothricin) and two commercially available resistance inducers (Prohexadion-Calcium, Bion®) in addition to streptomycin as control. Streptothricin was found nearly as effective as the streptomycin control (2 and 0% diseased shoots, respectively). 000 391 CF with chuanghsinmycin as active ingredient in unknown concentration, however, was less effective together with tubercidin; both compounds were originally classified effective in the in vitro screening (approximately 20 and 30%, respectively). The resistance inducers showed a maximal effectiveness of 20-25% approximately; however, in the highest concentration applied, they caused phytotoxic effects. As an alternative strategy of plant defence, an experiment was performed to silence the GFP-fluorescence of transformant 208m2 as a model for silencing fungal genes responsible for plant infection via the transgenic host. Two types of ?T0? transgenic tomato plants were produced from ?Hellfrucht? cultivar, one carrying the gfp-gene and the other carrying inverted repeat fragment of the gfp-gene (gpg). GFP-expressing plants could be identified by their green fluorescence under UV-A light. gpg-transformants were verified by PCR analysis of their genomic DNA and the formation of siRNAs by Northern blotting. In order to test the silencing effects in the plants Agrobacteria harbouring the gfp-gene or gpg-construct in a binary vector were infiltrated into tomato leaves. Leaves of agro-infiltrated (gfp) wild type plants were found to fluoresce, but no green fluorescence appeared when gpg-tomato leaves were infiltrated, due to the production of siRNAs resulting in gene silencing. The green fluorescence of gfp-tomato leaves disappeared gradually starting from the major vein expanding to small veins after infiltration of gpg-Agrobacteria due to the same phenomenon. However, no systemic silencing over the whole plant was observed; this may be due to the comparatively low expression of the gfp-gene in the?T0? plants used. However, the GFP in the P. infestans transformant 208m2 was not found to be silenced when leaves of gpg-plants were infected; this might be due to unsuccessful passing of siRNAs through haustorium?s membrane system, or due to the comparatively low amount of siRNAs in the gpg-tomato tissues.

K1 Phytophthora infestans, Erwinia amylovora
PP Hohenheim
PB Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
UL http://opus.uni-hohenheim.de/volltexte/2008/222