Mode of action of the phenylpyrrole fungicide fenpiclonil in Fusarium sulphureum

In the last few decades, plant disease control has become heavily dependent on fungicides. Most modem fungicides were discovered by random synthesis and empirical optimization of lead structures. In general, these fungicides have specific modes of action and meet modem enviromnental criteria. A disadvantage of modem fungicides is the potency of plant pathogens to acquire fungicide resistance. This phenomenon urges the agrochemical industry to search for chemicals with new modes of action. Natural products often have unique modes of action and can be used as lead structures in chemical synthesis programmes. One natural lead structure is the antifungal antibiotic pyrrolnitrin, produced by several Pseudomonas species. Its use in an optimization programme of CIBA-GEIGY A.G., Basel, Switzerland, led to the development of the highly active phenylpyrrole fungicides fenpiclonil (CGA 142705) and fludioxonil (CGA 173506). The elucidation of the mode of action of fenpiclonil is the topic of this thesis.Fenpiclonil is toxic to representatives of Ascomycetes, Basidiomycetes and Deuteromycetes. Its effect on several physiological processes was studied using the fungus Fusarium sulphureum as a sensitive target organism. The EC 50 of fenpiclonil to radial growth on PDA and mycelial growth in Czapek Dox liquid medium is 0.5 and 4 μM, respectively.Fenpiclonil accumulates to a high level in mycelium of F. sulphureum and in artificial liposomes. The accumulation appears to be the result of a physico-chemical partitioning of the fungicide over lipids in mycelium and the medium. Accumulation is reversible as the fungicide is readily released from mycelium by washing with water. The fungus does not metabolize fenpiclonil upon incubation for 24 hours.At its EC 50 (4 μM), feripiclonil does not immediately affect oxygen consumption, nuclear division, and DNA-, RNA-, protein-, chitin-, ergosterol- and (phospho)lipid biosynthesis. However, accumulation of amino acids and sugars is instantaneously inhibited at concentrations ranging from 4.2- 42 μM), The reduction in accumulation is accompanied by an increased accumulation of the membrane potential probe tetraphenylphosphonium(TPP +) bromide (TPP +) and a marginal change of the proton gradient probe propionic acid. This suggests that the biochemical mechanism of action of fenpiclonil may be related to membrane dependent transport processes. The increased accumulation of TPP +is probably the result of changes of potentials over membranes of various cell compartments rather than from plasma membrane hyperpolarization. The fungicide neither influences membrane fluidity in artificial liposomes nor amino acid accumulation in bacterial vesicles. Thus, accumulation of the fungicide does not aspecifically effect functioning of various types of membranes.At its EC 15 (0.4 μM), fenpiclonil selectively inhibits accumulation and incorporation of various monosaccharides into macromolecules of F. sulphureum . This effect was not observed with a fenpiclonil-resistant laboratory isolate of the fungus. Various less active structural analogues of fenpiclonil also inhibit accumulation and incorporation ot monosaccharides into macromolecules, but to a lesser extent. Strongest inhibition (58%) was observed for the incorporation of [U- 14C]glucose in hyphal wall glycan fractions. Biosynthesis of these glycans is catalysed by glycan synthases in the plasma membrane. However, fenpiclonil does not directly interfere with these enzymes, since their activity is not inhibited in a cell-free assay. Furthermore, the precursor of glycans, uridinediphosphoglucose, does not accumulate upon fenpiclonil treatment. All effects described were observed within 15 min of incubation with the fungicide, and indicate that the mechanism of action of fenpiclonil may be related to glucose metabolism.Fenpiclonil (0.4 μM) inhibits the production of [ 14C]carbon dioxide in mycelium incubated in a medium with glucose as the carbon source. However, no differential effect on [ 14 C]carbon dioxide production with glucose labelled at different carbon positions was observed. The fungicide does not inhibit [ 14C]carbon dioxide production using acetate as the carbon source. These results indicate that metabolisation of glucose is neither affected by inhibition of pyruvate dehydrogenase activity nor of enzymes in the TCA-cycle. Therefore, the site of action of fenpiclonil is most likely located in early steps of glycolysis.Fenpiclonil inhibits the accumulation of 2-deoxy[U- 14C]glucose in starved mycelium loaded with 2-deoxyglucose. Fungicide treatment results within one min of incubation in an increased content of 2- deoxy[U- 14C]glucose and a decreased content of 2-deoxy [U- 14C]glucose-6-phosphate. The fungicide does not effect cell-free phosphorylation of glucose and the mycelial ATP concentration. These results indicate that the mode of action of fenpiclonil is due to inhibition of transport-associated phosphorylation of glucose. Inhibition of glucose phosphorylation will cause a cascade of metabolic events leading to the toxic action of the fungicide. A major event may be the accumulation of polyols which was not observed in an osmotically-sensitive and fenpiclonil resistant laboratory isolate of the fungus