Fungicide, nanoparticles, and their combined effect on ectomycorrhizal fungi as non-target organisms

Abstract

Sorption can alter the availability of compounds and could explain different findings on pesticide action in laboratory and field conditions. In this thesis, the influence of sorption on the toxicity on non-target organisms was studied. Tailor-made nanoparticles (NPs) and propiconazole were used as model compounds. Propiconazole is a widely used fungicide that inhibits the growth of pathogenic fungi and non-target organisms like ectomycorrhizal fungi. As test organisms, three ectomycorrhizal fungi were utilized that originate from distinct fungal phyla. The effect of sorption on toxicity was studied in liquid and solid experimental systems. In submerged cultures, three major findings were obtained. First, all three tested fungi responded less sensitively to propiconazole in liquid than in solid cultivation. Second, in the presence of fungi, the amount of freely available propiconazole declined rapidly. In addition, different degradation metabolites were found in varying quantities in the filtrate of each species, which indicates a species-specific fungicide degradation. Third, a combined supply of propiconazole and high NP amounts resulted in slightly increased dry weight levels of the fungi and in greatly increased propiconazole levels in the mycelium. It is assumed that NPs sorbed on the fungal surface and contributed to the dry weight although growth inhibition itself was low. Further, the presence of NPs might trigger fungal uptake of propiconazole or its sorption on the fungal cell. The fungal cell wall might possess a higher sorption affinity for propiconazole than NPs. Due to strive for equilibrium, propiconazole might be continuously released from NPs and cause an elevated attachment on the mycelium. Another possible explanation is that NPs could elicit ROS formation, membrane damage and increased propiconazole permeability. Fungal cultivation on agar medium and continuous growth monitoring allowed the study of differentiated sorption conditions under sterile conditions. Here, NPs significantly reduced the toxicity of propiconazole in all tested fungi. The observed growth parameters imply reduced fungicide availability in the presence of NPs due to sorption. This was corroborated by HPLC analyses. Propiconazole toxicity occurred in a dose-dependent manner. The higher the applied NP dosage, the lower was the freely available fungicide fraction, and the higher was the fungal growth. Unlike in Ascomycetes, gene expression following an azole treatment has rarely been studied in Basidiomycetes. Existing data on erg6 and erg11 gene expression is severely limited and contradicting (Stammler et al. 2009, Lee et al. 2010). In this respect, the present thesis is the first study to expose a Basidiomycete fungus to propiconazole. This thesis was able to verify that upon exposure to propiconazole, transcription levels of both erg6 and erg11 were significantly increased in L. bicolor. The levels of upregulation correlated very closely. Therefore, the usage of erg6 and erg11 as marker genes can be recommended. Moreover, NPs mitigated the detrimental effect of propiconazole on the fungal ability to form ectomycorrhizas with poplar.