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Lipid peroxidation has been observed in the interaction of fungi with several groups of unrelated fungicides. However, the primary events that initiate these reactions appear to be quite different from each other and are thus far poorly understood. The stimulation of lipid peroxidation is not a general effect of fungicide action and is specific to dicarboximide fungicides (DCOFs) and aromatic hydrocarbon fungicides (AHFs). Other antifungal inhibitors such as aldimorph, tridemorph, dichlo-fluanid, and metalaxyl had no effects on lipid peroxidations at concentrations sufficient to inhibit growth. The induction of lipid peroxidation by DCOFs and AHFs is most likely not caused via a direct conversion of these fungicides to free organic radicals. A prerequisite for the peroxidation of polyunsaturated fatty acids in living cells is either the presence of an active enzyme, or a more general oxidative stress situation caused by free radicals or by reduced oxygen specimens.

Udgivelsesdato: oktober

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.

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.

Aromatic hydrocarbon fungicides represent an older and chemically heterogeneous group of fungicides consisting of an aromatic ring system. They have been in use for a long time, but have lost their importance. Some of them are still valuable compounds in tropical countries and in use for controlling storage diseases. Many of them could be replaced by newer compounds with a broader antifungal spectrum, better physical properties, and higher activity. Keywords: aromatic hydrocarbon fungicides; hexachlorobenzene; chlorinated nitrobenzenes; chloroneb; dicloran; o-phenylphenol; diphenyl; biphenyl

Les effets d'une incubation du systeme racinaire a differentes temperatures (5 a 35 °C) pendant 4 mois, sur la croissance aerienne de plusieurs especes ligneuses forestieres europeennes, ont ete analyses. D'importantes differences interspecifiques ont ete mises en evidence dans cette reponse. Les optima thermiques de croissance en biomasse totale allaient de 15 °C (Picea abies, Larix decidua, Pseudotsuga menziesii, Betula verrucosa) a 25 °C (Quercus robur, Carpinus betulus), voire 30 °C (Pinus nigra). Un refroidissement des racines de Juglans regia a 2 °C a resulte dans une diminution rapide et durable de l'assimilation nette de CO 2 , mais seulement d'une baisse limitee de conductance stomatique et de transpiration. La respiration etait stimulee apres quelques jours. Les consequences ecologiques de ces differences des optima thermiques sont discutees en regard de la distribution des especes et de leurs reactions a des accroissements de temperature dus a l'effet de serre.

A strong induction of lipid peroxidation by sterol C-14 demethylation inhibitor (DMI) fungicides could be demonstrated in Mucor mucedo and Ustilago maydis. Lipid peroxidation and growth inhibition were rather well correlated. This is possible especially in Mucor because growth is not affected by inhibition of sterol C-14 demethylation. DMI fungicides seem to exhibit a secondary mechanism of action in sensitive fungi which leads to fungicidal effects through destruction of membranes. Growth inhibition as well as lipid peroxidation in Mucor and Ustilago can be alleviated in most DMI (with the exception of prochloraz) by addition of the radical scavenger α-tocopherol. The fungicide dichlofluanid does not induce a lipid peroxidation at growth inhibiting concentrations, such as that which occurs under the influence of trimorphamide and the unrelated fungicide drazoxolon.

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.

With the help of active types of oxygen, extracellular enzymes and mechanical processes,Botrytis cinerea is capable of infecting plant tissue. Whereas no clear correlation was found between the activities of protease, pectolytic or other cell wall degrading enzymes and virulence, a positive correlation between pathogenicity and the intensity of active types of oxygen released was apparent in some isolates. It is assumed that these toxins result from the activity of glucose or xylose oxidases. Antioxidants inhibited the infection. It is concluded from these results, that active types of oxygen play a decisive role in the infection process.

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.

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.

Carboxin (= vitavax = DCMO) is rapidly oxidized by hydrogen peroxide or H2O2 generating systems as riboflavin, flavin enzymes with or without substrate, to the more water soluble nontoxic sulfoxid (DCMOS). Illumination enhances the inactivation. Peroxidase is without effect. Isolated mitochondria from sensitive as well as from insensitive fungal species have the ability to oxidize DCMO, which leads to a leakage of the compounds mainly in form of DCMOS from the mitochondria. Among several fungal species investigated only Ustilago maydis in vivo oxidizes DCMO to a higher degree. The further oxidation of DCMOS to the corresponding dioxid (DCMOD = plantvax) occurs only under certain conditions to a limited extent. Proteases are unable to attack the - CONH-bounding. It is assumed that in vivo mainly flavin enzymes are responsible for the rapid oxidative inactivation of DCMO (vitavax).

Chloroneb (1,4-dichloro-2,5-dimethoxybenzene) is a fungicide with moderate systemic properties and selective activity toward certain fungi (e.g., Phytophthora, Pythium, Botrytis, Mucor ). Although the compound is metabolized by some fungi to the toxic oxidation products DCMP (2,5-dichloro-4-methoxyphenol) and DCHQ (2,5-dichlorohydroquinone) the fungitoxic principle is chloroneb per se. It causes a lysis of the inner mitochondrial membrane of sensitive fungi, attacks the nuclear membrane and also other membranes at higher doses. Oxygen consumption by isolated mitochondria slowly decreases, but the respiratory control quotient remains nearly constant. No specific uncoupling activity or inhibition of the respiratory chain could be detected. Chloroneb is bound to the mitochondrial membrane in sensitive organisms, and a good correlation exists between binding capacity, damage to mitochondrial structure and function, and growth inhibitory effects. DCMP has a nearly identical effect, but some additional uncoupling activity whereas DCHQ is different in its mechanism of action. A comparison of a chloroneb-sensitive strain of Mucor mucedo and a resistant strain with respect to the amino acid composition of their mitochondrial membranes demonstrated a significantly diminished content of tyrosine in the resistant strain, which may be associated with the decreased binding capacity for chloroneb. Although the reason for mitochondrial destruction is not yet elucidated, counteracting effects of piperonylbutoxide and α-tocopherol acetate suggest that a radical mechanism may be involved.

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.

83 fungal species were tested for their correlation between carboxin sensitivity and glycolytic ability regarding the taxonomic position of the test fungi. Besides basidiomycetes the ascomycetous orders Helotiales, Sordariales, Chaetomiales, and Dothiorales showed carboxin sensitivity which was coupled with lacking fermentation and acid production while carboxin resistant taxa as a rule behaved vice versa. Furthermore, hexose monophosphate participation was high and phosphofructokinase activity was low in carboxin sensitive fungi. It is concluded that response to carboxin and glucose metabolism are coupled characters but without strict causal connection. In yeast-like fungi carboxin sensitivity may indicate their taxonomic position within the basidiomycetes.

1. Hinsichtlich der Tyrosinase-Bildung lassen sich bei holzzerstorenden Pilzen drei Typen unterscheiden: A. Das Enzym ist standig im Mycel vorhanden, B. das Enzym tritt nur in jugendlichem Mycel, oft nur in der Zuwachszone auf, C. das Enzym wird bevorzugt von alterndem Mycel gebildet und kann in jungem Mycel durch Stoffwechselgifte induziert werden. 2. Tyrosin und phenolische Substrate losen bei Fomes marginatus keine Tyrosinase-Bildung aus. 3. Eine Enzym-Induktion last sich durch Applikation von Stoffwechselgiften erreichen, die die oxydative Phosphorylierung entkoppeln oder in anderer Weise die Bildung von ATP storen. 4. Eine Tyrosinase-Induktion erfolgt daher haufig, aber nicht notwendigerweise bei einem Uberwiegen abbauender Prozesse, sofern die Syntheseleistungen des Mycels nicht vollig gestort sind. 5. Zur Erklarung dieses Phanomens wird eine metabolische Repression der Enzymbildung im jugendlichen Mycel angenommen, die durch die Gifteinwirkung aufgehoben wird.

The favoured inhibition of basidiomycetes by oxathiin can be confirmed with yeast like organisms. A possible common base for the sensitivity against oxathiins is probably the dominance of the hexosemonophosphate shunt (HMP) in sensitive organisms, although the primary site of attack is the respiration system. Sensitive and insensitive species don't differ in the overall accumulation of 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilid DCMO), which is, as total molecule, responsible for the toxic effect. A degradation results in a detoxication.

The selective effect of oxathiin compounds (DCMO) on basidiomycetes does not depend on stronger accumulation of the fungicide by sensitive fungi. Uptake of DCMO is closely related to the lipid content of a fungus. It is shown by electron microscopy that within 120 min DCMO damages the mitochondrial system and the vacuolar membrane, while plasmalemma, nuclear membrane and ER remain unchanged. In isolated mitochondria of sensitive fungi DCMO causes a similar inhibition of respiration as in whole cells. Succino-dehydrogenase (SDH) is inhibited to the same extent, while NADH-dehydrogenase is only weakly inhibited and malate-dehydrogenase not at all. Inhibition of SDH is reduced with increasing temperature. Soluble SDH is insensitive to DCMO. The point of attack of DCMO is considered to be SDH in situ or a closely following reaction.

Bacillus subtilis forms a xylanase complex which does not depend on induction by xylane or xylose in the medium. The enzyme production is influenced by the kind of the carbon source, probably due to catabolic repression. The regulation of enzyme formation is complex in nature, because the C/N/P-Relation in the medium, cell mass, and growth phase influence the amount of enzyme in the culture fluid. Maximal enzyme production was achieved at high but balanced amounts of C/N/P, although growth was strongly reprimed under these conditions. A formation of cellulase, aryl-β-xylosidase and aryl-β-glucosidase could not be observed.

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.

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.

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.

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.

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.