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In connection with investigations on the mechanism of action of the systemic fungicide carboxin a new hypothesis was developed on the mechanism of the cyanide-insensitive pathway of respiration, which is widely distributed in fungi und higher plants, and on the nature of the alterative terminal oxidase. Based on own experimental results which are in concordance with the properties of the alternative pathway described in the literature, it is assumed that the nonheme-iron-sulfurprotein (FeSPp) of the succinodehydrogenase, which is located on the main route of the cytochrome mediated respiration, is the alternative terminal oxidase itself. This property seems to be based on the ability for autoxidation in some organisms where the Fe-atoms are oxygenized and reach the maximal coordination number of 6. By this mechanism it is explainable that carboxin which also attacks the normal electron flow at the FeSPp of the succinodehydrogenase is able to inhibit simultaneously under certain circumstances the cyanide-insensitive respiration organisms sensitive to carboxin.

Comparative investigations on the inhibition of chitin synthetase of Mucor rouxii revealed that in contrast to results obtained in vivo, only few fungicides and other compounds inhibit the enzyme in vitro. Besides the well-known effect of polyoxin D, an inhibition was demonstrated for terrazol, tridemorph, hinosan, and formulated preparations of dimilin (PH 60--40, 60--38). The latter preparations, however, showed growth inhibition also with fungal species which do not synthesize chitin and the pure compounds are ineffective. Inhibition by phospholipase C, unsaturated fatty acids, and the reversibility of the inhibition caused by terrazol after addition of procain-hydrochloride demonstrates that phospholipids are essential for the activity of the enzyme, whereas sterols seem to be ineffective. Action of trypsin, PCNB, pentachlorophenol, and some similar compounds results in significantly increased activity, which in the case of trypsin could be due to the hydrolysis of a protein inhibitor. Hinosan inhibits the enzyme indirectly in a still unexplored manner.

The influence of the fungicidic compound beta-thujaplicin (beta-isopropyl-tropolone) on the energy transformation processes of oxidative phosphorylation was investigated in isolated rat liver mitochondria with succinate (plus rotenone) as substrate. To elucidate the observed strong inhibition of active respiration by beta-thujaplicin three possibilities were assayed: the inhibition of 1) transport processes across the inner mitochondrial membrane for inorganic phosphate, adenine nucleotides, or succinate, 2) electron flux along the respiratory chain, and 3) mitochondrial ATPase. In this respect a remarkable inhibition of both Pi transport and the translocation of adenine nucleotides could not be observed. However, the effective suppression of the DNP-induced ATPase by beta-thujaplicin explains the pronounced inhibition of active respiration. An impairment of succinate transport and the measured partial inhibition of the terminal respiratory chain at the level of cytochrome oxidase contribute to the less marked inhibition of the uncoupled respiration. The ability of beta-thujaplicin to extract mitochondrial Mg++ and the prevention of the effects of beta-thujaplicin by an excess of Mg++ in the medium suggest a common mode of action of beta-thujaplicin as a lipophilic chelator of Mg++ and other divalent cations.

It is assumed that the fungistatic effect of terrazol in Mucor mucedo is induced by a liberation of phospholipases within within the mitochondria and perhaps at other membranes. In isolated mitochondria a rapid formation of lysolecithin can be demonstrated at low concentrations of terrazol. This would explain the lytic symptoms in mitochondria visible by electron microscopy. Lipid peroxidation could not be demonstrated. The only antidots at present known for the growth inhibiting effect of terrazol and on its ultrastructural effects are impure saccharose, which seems to contain an unknown factor, and procain hydrochloride or to a lesser extent lidocain, which are well-known inhibitors of phospholipases. The pathological thickening of the cell wall induced by terazol seems to be an unspecific side effect reflecting a diminished phosphorylating activity of the mitochondria.

The effect of PCNB in various concentrations on the ultrastructure of Mucor mucedo and phytophthora cactorum was analyzed after an incubation period of 2 hours. The most striking effect in both fungi was a diffuse lysis of the internal structure of the mitochondria which differs markedly from the lysis induced by etridiazol (terrazol). Moreover an enlargement of the perinuclear space and an increased formation of vacuoles was observed. In Mucor mucedo, but not in Phytophthora cactorum a pathological thickening of the cell wall was observed. Although after 2 hours incubation with PCNB Phytophthora gave similar ultrastructural reactions in the mitochondria as Mucor, in growth experiments on agar dishes this species was 5-10 times less sensitive to PCNB compared to Mucor.

Analogs of the natural substance (E)-hex-2-en-1-al which occurs naturally in green plants and is known to have microbiocidal properties were synthesized and studied with respect to their antifungal properties. Saturated and unsaturated aldehydes, alcohols, carbon acids and enolacetate were tested against various fungi by applying these compounds either over the gaseous phase or in suspension. The following ranking was observed on the basis of fungicidal activity: enolacetate of 2-ethylhex-2-enoic acid greater than 2-ethylhex-2-en-1-al greater than 2-ethylhex-2-en-1-al greater than 2-ethylhex-2-en-1-ol. Saturated analogs showed the same order of activity as the unsaturated ones, however, they were less efficient. The effects on the ultrastructural changes were analyzed in Mucor mucedo exposed to concentrations of the analogs that inhibited growth. Main effects were observed on mitochondrial and nuclear membranes. The kind of destruction caused by enolacetate of 2-ethylhex-2-en-1-al differed from the other substances tested. Aspects of the mode of action are discussed.

Higher concentrations of terrazol inhibit the growth of a large number of fungi, but oomycetes are most sensitive. At medium concentrations (ED50 = 10 ppm) in Mucor mucedor several alterations of ultrastructure are recognizable even after short incubation periods. Significant observations are vacuolization of the mitochondrial cristae, invagination of the cytoplasmic membrane, and thickening of the cell wall. Later on, numerous vacuoles in the cytoplasm are visible. Primary effects on the cell membrane (destructive effect), on O2-consumption, on fermentation, or on nucleic acid synthesis could not be demonstrated. Lipid synthesis under the influence of terrazol showed characteristic changes. The synthesis of triglycerides and sterolesters was inhibited while the synthesis of free fatty acids and phospholipids was stimulated. Addition of lipids, vitamines, amino acids, or nucleic acids did not counteract the effect of terrazol. Further investigations are necessary to elucidate the primary mechanism of action.

Terrazol, a systemic fungicide showing high specifity to oomycetes, inhibits the apical growth of hyphae and promotes at lower concentrations the thickening of the cell wall in Mucor mucedo. As revealed by ultrastructural analysis, particularly the fine structure of some membrane systems is influenced. In the first place the inner membrane of the mitochondria is attacked leading to a complete lysis of mitochondria. However, the sensitivities within a given population are different. The plasmalemma enlarges, forms several invaginations, partly redraws from the cell wall, but remains intact. Only after an extensive treatment with relatively high concentrations of terrazol the nuclear envelope shows vesicles between the double membranes. The mechanism of action of terrazol is discussed.

An anomalous cell wall thickening in Mucor mucedo is induced already within 60-120 min by some fungicides (etridiazol, chloroneb, pentachloronitrobenzene, dicloran, drazoxolon, biphenyl) as well as with a N2-atmosphere or high concentrations of glucose, but not with 2,4-dinitrophenol, chlorinated phenols, dichlofluanid and antimycin A. This effect seems to be identical to the change from the mycelial (M-) to the yeast (Y-) form in dimorphic fungi, which can be achieved by culture conditions as well as by addition of chemicals. The cause seems to be a specific, complex change in the metabolic state. A scheme of regulation is presented which explains most of the experimental results described till now.