Natural products with antifungal properties as alternative to copper in agriculture

Abstract

Copper salts are widely used in agriculture due to their activity against a broad range of plant pathogens. However, copper accumulates in soils, leading to ecological issues. Therefore, there is a strong demand for substitutes, especially in organic farming, where no efficient alternatives to copper are currently available. An in-house library containing more than 3000 extracts from fungal and plant origin was screened in vitro against three major agricultural pathogens, specifically grapevine downy mildew (Plasmopara viticola), potato and tomato late blight (Phytophthora infestans), and apple scab (Venturia inaequalis). From this screening, several plant extracts with significant activity were selected for further investigation in this thesis. They include the ethyl acetate extracts of Juncus effusus (Juncaceae) medulla, Magnolia officinalis (Magnoliaceae) bark, Verbesina lanata Asteraceae) leaves, Iryanthera megistocarpa (Myristicaceae) leaves, and the petroleum ether extracts of Styrax tonkinensis and Styrax paralleloneurum/benzoin (Styracaceae) resins. The active compounds were identified by an approach referred to as HPLC-activity profiling. Subsequently, the identified compounds were isolated by several chromatographic methods and characterised by diverse spectroscopic techniques, such as HR-ESI-MS, 1D and 2D microprobe NMR, ECD, optical rotation, and X-ray crystallography. The ethyl acetate extract of J. effusus medulla showed strong in vitro inhibitory activity against the three aforementioned pathogens. The active compound was identified as dehydroeffusol. On grapevine and apple seedlings, efficacies up to 100 % were reached with the extract and the purified compound. S. tonkinensis resin (Siam benzoin) and Sumatra benzoin (resin from Styrax paralleloneurum or Styrax benzoin) exhibited promising in vitro inhibitory activity against the three pathogens. On grapevine seedlings, Siam benzoin and Sumatra benzoin dissolved in ethanol were reaching an efficacy of 100% at a concentration of 1 mg/mL. The active compounds were coniferyl benzoate in Siam benzoin and p-coumaryl cinnamate in Sumatra benzoin. The activities of the two resins were also significant against V. inaequalis and Marssonina coronaria on apple seedlings, and against P. infestans on tomato seedlings. In field trials on grapevine, Siam benzoin reduced the infestation by downy mildew (P. viticola) and powdery mildew (Uncinula necator, anamorph Oidium tuckeri). These results led to the submission of an European Patent Application in view of a potential commercial development. The ethyl acetate extract of M. officinalis bark showed high in vitro activity against the three pathogens. The active compounds were identified as the two neolignans, magnolol and honokiol. The efficacies on grapevine and apple seedlings were similar to copper, whereas on tomato seedlings the efficacy was lower. The activity against apple scab could not be confirmed on field trials. In contrast, the efficacy of M. officinalis extract formulated as a wettable powder was equivalent to the reference organic fungicide. The ethyl acetate extract of V. lanata leaves exhibited promising in vitro activity against P. viticola. At a concentration of 1 mg/mL, the extract reduced the leaf surface infestation by 82% compared to the non-treated control. Sixteen eudesmane sesquiterpenes with a cinnamoyloxy group were isolated. Among them, eight were new congeners. Nine of these compounds were tested in vitro against P. viticola and five showed MIC100 values <10 μg/mL. The ethyl acetate extract of I. megistocarpa leaves exhibited good activity in vitro against the three pathogens. The leaf surface infestation on seedlings at an extract concentration of 1 mg/mL was reduced by 87% on grapevine and by 68% on tomato. Tests on apple seedlings are ongoing. From the extract, two dihydrochalcones and eight flavonolignans including several stereoisomers were isolated. Against each pathogen, the two most active compounds showed MIC100 values ≤2.3 μg/mL. Seven of the flavonolignans possess three different planar structures with several configurations along a side chain. Due to the possible free rotation, their stereochemistry could not be established by NMR analysis. Suitable crystals were only obtained for one compound allowing the determination of its relative configuration by X-ray diffraction analysis. Further crystallisation attempts should be performed with the other compounds, possibly after the preparation of suitable derivatives. The results of this thesis confirm that plant-derived products represent promising candidates for the development of new organic pesticides which could enable to significantly reduce the use of copper. In further steps, toxicity studies will have to be performed with the plant extracts and pure compounds to ensure product safety. Further field trials with optimised extract formulations should be also carried out.