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Effect of fungicides on growth and on DNA, RNA and protein anabolism of a Rhizobium sp.

Eleven fungicides were examined for their effect on the growth and anabolism of Rhizobium sp. in pure culture. Of these, only 4 were found to inhibit growth of rhizobia at a concentration as low as 10 micrograms/ml. Growth inhibition by these fungicides appeared to be primarily due to inhibition of respiration, although thiophanate and anilazine also affected DNA and RNA synthesis, respectively. Inhibition is, however, transitory and the bacterium has the ability to overcome this initial inhibition without mutation.

Antifungal Agents↗

Decreased fertility, increased dominant lethals, skeletal malformations induced in the mouse by Ziram fungicide.

The toxic effect of the Ziram fungicide (Zn-dimethyldithiocarbamate) on fertility, its lethal and teratogenic potential were tested on two mouse strains, C3H and AK. The fungicide was administered by gavage to male mice in daily doses of 0.2 mg% and 0.1 mg% along three weeks, then the mice were mated with normal females. Ziram induced changes in the testes and meiotic chromosomes. From among the mated females, 80 per cent of the C3H mice and 20 per cent of the AK remained unfertilized. The dominant lethals have a higher incidence in the AK strain. The skeletal malformation induced were kyphosis, scoliosis, sternum ossification failure; retardation in skeletal development is more obvious in the AK strain.

Abnormalities, Drug-Induced↗

Influence of phospholipids from Listeria monocytogenes and Aspergillus fumigatus on the bactericidal and fungicidal activity of mouse peritoneal macrophages.

Investigations were carried out on an influence of phospholipids from A. fumigatus and L. monocytogenes on the bactericidal and fungicidal activity of mouse peritoneal macrophages. L. monocytogenes bacilli and C. neoformans fungi served as the experimental microorganisms. Phospholipids from A. fumigatus and L. monocytogenes were shown to significantly increase macrophage activity under in vivo and in vitro conditions. This was accompanied by an intensified bactericidal and fungicidal activity of extracts from the lysosomal fraction of phagocyte examined. Phospholipid-stimulated macrophages were observed to liberate bactericidal factors into the culture fluid. Lymphocytes had no influence upon this process.

Animals↗

Increased frequencies of sister chromatid exchanges and micronuclei in "in vitro" lymphocyte cultures treated with the fungicides thiram and ziram.

The two fungicides analysed in this paper, Thiram (TH) and Ziram (ZI) are among the most common residues found in fruit and vegetables sold in Italy. These compounds were tested in "in vitro" lymphocyte cultures, from 16 individuals, at different concentrations (Thiram 5 x 10(-8) M; 5 x 10(-7) M; 5 x 10(-6) M and Ziram 1 x 10(-7) M; 1 x 10(-6) M; 1 x 10(-5) M) in order to ascertain if they are able to modify sister chromatid exchange and/or micronuclei frequencies. Both fungicides produced a significant increase in SCE and Micronuclei frequencies. The two highest doses of TH and ZI were found to be cytotoxic for most of the individuals and were not included in the statistical analysis. With regard to the Proliferation Rate Index (i.e. the number of first, second, third or more mitoses), no difference was found between controls and treated cultures.

Adult↗

In vivo studies on possible adverse effects on reproduction of the fungicide methyl thiophanate.

The fungicide methyl thiophanate (MT), widely used to control some of the most common fungal diseases in crops, is metabolized in animals into benzimidazole compounds, including the well-known reproductive toxicant carbendazim. However, standard toxicological tests did not indicate that MT may cause testicular toxicity and/or embryotoxicity, which are typical effects of many benzimidazoles. In the present study some aspects of the MT potential for reproductive toxicity have been assayed by means of two non-conventional models. Following the oral administration of 700 and 1000 mg kg(-1) body wt. for five consecutive days, short-term testicular toxicity was examined in the B6C3F1 mouse through specific parameters (sperm head count, specific enzyme activities, histopathology on days 3-35 post-dosing). In spite of the high doses administered, none of the testicular parameters examined, including histopathology, showed significant alterations as compared to controls at any time post-dosing. Pregnant CD rat dams were administered orally the limit dose of 650 mg kg(-1) body wt. day(-1) during preimplantation (gestational day or GD 2-5) or peri-implantation (GD 6-9) phases; embryos and adnexa were evaluated morphologically on GD 12 as a window for the early observation of embryotoxicity. Evident maternal toxicity was present in both treated groups, whereas only marginal reductions of the growth of embryos and adnexa were observed. A full understanding of MT toxicology will need more quantitative data on metabolism, including plasma kinetics and dosimetry of carbendazim at the relevant targets. Nevertheless, the absence of any clear-cut effect on a number of specific endpoints may provide reassurance that no further testing of MT is needed with regard to testicular toxicity or embryotoxicity.

Administration, Oral↗

[Synthesis of the isocamphene series. 33. The synthesis of fungicidal norbornane derivatives].

Starting from the corresponding amines the syntheses of various norbornyl ureas with potential fungicidal activity are described. These new compounds possess a tricyclo[5.2.1.0(2,6)]decane, or an isocamphenilane, a camphenilane, an exo- and endo- dimethylnorbornane or a bicyclo[2.2.2]octane carbon skeleton. With the exception of the first class all other derivatives bear a geminal dimethyl group--important because of its shielding effect--at C-3 of the bicyclus. Some ureas, especially 6 and the acetamide 5, are active against phytopathogenic fungi.

Ascomycota↗

Cytotoxicity and mutagenicity of the fungicides captan and folpet in cultured mammalian cells CHO/HGPRT system).

The cytotoxicity and mutagenicity of the fungicides captan and folpet were determined in the CHO/HGPRT system which utilizes Chinese hamster ovary cells and resistance to 6-thioguanine to estimate mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus. Treatment of cultures with each compound for 5 hr is serum-free medium resulted in reproducible, significant, concentration-dependent increase in the frequency of 6-thioguanine-resistant mutants.

Animals↗

Disturbances in rat smooth-muscle induced by a substance with fungicide action.

The fungicide substance QAS [N,N,N',N'-tetramethyl-N,N'-di(8,15-dichloropentadeca-5,10- dien)ethylenediamine methylsulphate] has a biphasic effect on the spontaneous electrical and mechanical activity of smooth-muscle samples of rat corpus and guinea-pig taenia coli. During the first phase of QAS application membrane depolarization and increased spontaneous spike frequency were recorded. The tone of the preparations (resting tone) increased transiently. Calcium ion-entry blockers did not affect the contractile effect of QAS, but K(+)-channel blockers and some modulators of the second messenger system abolished or decreased it. During the second phase depolarization increased progressively, spike frequency decreased and the increase in resting tone recorded during the first phase was eliminated. The results reveal that the long-lasting depolarizing effect of QAS (in concentrations used for plant protection) probably inactivates the entry of Ca2+ into the smooth-muscle cells and disturbs Ca2+ homeostasis.

Animals↗

[Inhibition of chitin synthetase of Mucor rouxii in vitro by fungicides and other compounds].

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.

Antifungal Agents↗

Anomalous cell wall synthesis in Mucor mucedo (L.) Fres. induced by some fungicides and other compounds related to the problem of dimorphism.

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.

Antifungal Agents↗

Characterization of the sorption of an anthranilate fungicide in soil using thermal analytical and mineralogical techniques.

The sorption of pesticides to soil particles has implications for their distribution and fate in the soil environment. A batch equilibrium technique was used to investigate sorption of the fungicide AEC623892 to intact and hydrogen-peroxide-treated whole soils and several particle-size fractions isolated from them. Sorption of AEC623892 to the soil as a whole was low. K(f oc) values measured in the whole soils were 169.2 and 41.9 ml g(-1) for Soil A and Soil B respectively. The highest values of K(f oc) were measured in soil particle-size fractions <53 microm (266.5 ml g(-1) in the 2-20 microm fraction of Soil A; 471.9 ml g(-1) in the 20-53 microm fraction of Soil B). Sorption was most irreversible in the 2-20 microm fractions. Overall, treatment of soil particle-size fractions with hydrogen peroxide resulted in lower values of K(f oc) (112.3 ml g(-1) in Soil A whole soil and 30.9 ml g(-1) in Soil B whole soil). In both soils, the maximum sorption among hydrogen-peroxide-treated samples was observed in the <2 microm fraction (166.6 and 311.0 ml g(-1) for Soil A and Soil B, respectively). Investigation of the mineralogical composition of the soils suggested that the clay mineralogy (dominated by kaolinite and illite) is less likely to account for the differences in sorption observed than differences in the characteristics of the soil organic matter. Thermal analysis of the different soil fractions indicated that hydrogen peroxide treatment preferentially removed aliphatic fractions of organic matter, but had less effect on lignin-like, aromatic fractions.

Adsorption↗

In vitro selection of an effective fungicide against Armillaria mellea and control of white root rot of grapevine in the field.

Armillaria mellea (Vahl ex Fr) Kummer is an aggressive pathogen which causes white root rot in a wide range of hosts. Most chemicals tested so far against Armillaria, both in vitro and in the field, have not been effective in reducing fungal growth and/or preventing plant decline and mortality. In the present work the effects of four DMI (sterol demethylation inhibitor) fungicides, cyproconazole, hexaconazole, propiconazole and tetraconazole, and another six downwardly mobile systemic chemicals, azoxystrobin, cubiet (copper bis(ethoxy-dihydroxy-diethylamino)sulfate), fosetyl-Al, potassium phosphite, sodium tetrathiocarbonate (STTC) and 2-(thiocyanomethylthio)benzothiazole (TCMTB), on the mycelial growth of A. mellea were compared and evaluated; the product yielding the best results in in vitro experiments was selected to determine its efficacy in preventing decline and mortality of grapevines in the field. Best results on in vitro fungal growth reduction were obtained with the four azoles tested, in particular with cyproconazole and hexaconazole, achieving 67-72% mycelial growth inhibition at the lowest dose. Results obtained in the field showed that a dose of 50 mg AI litre(-1) of cyproconazole once or twice a year was efficient in controlling the disease even in vines seriously affected by the pathogen. However, further research is required to study minimum effective doses, residual effects and the convenience of the application of annual dressings in damaged vineyards, so as to gradually reduce the pathogen inoculum potential in soil and control the disease while reducing chemical residues in the plant and preventing development of fungal resistance.

Agaricales↗

A non-Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis.

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.

Acrylates↗

Evaluation of Erysiphe graminis f sp tritici field isolates for resistance to strobilurin fungicides with different SNP detection systems.

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.

Drug Resistance, Fungal↗

Synthesis and structure-activity study of fungicidal anilinopyrimidines leading to mepanipyrim (KIF-3535) as an anti-Botrytis agent.

A series of 2-anilinopyrimidines was prepared and their fungicidal activities against Botrytis cinerea Pers were examined. The activity fell sharply with any substitution on the anilinobenzene ring. Substituents at the 5-position of the pyrimidine ring greatly reduced the activity. Substituents such as chloro, methoxy, methylamino, methyl or 1-propynyl were well tolerated at the 4- and 6-positions of the pyrimidine ring. Among these substituents, the combination of methyl and 1-propynyl groups was the most favourable. 2-Anilino-4-methyl-6-(1-propynyl)pyrimidine (KIF-3535), which showed excellent activity and no significant phytotoxicity, was finally selected for development and has been given the common name mepanipyrim.

Botrytis↗

Measured and computed volatilisation of the fungicide fenpropimorph from a sugar beet crop.

Depending on their vapour pressure, volatilisation can be one of the main pathways of emission of pesticides into the environment. The volatilisation of fenpropimorph was studied in a field experiment in which the fungicide was sprayed onto a sugar beet crop. Volatilisation rates were calculated by measuring the concentration gradient in air, using the Aerodynamic and Bowen Ratio methods. A simplified computation model was used to simulate pesticide volatilisation, together with the concurrent processes of penetration into the plant leaves and phototransformation. Input data for the model had already been obtained by carrying out a wind-tunnel study with fenpropimorph, whereby field conditions were imitated. The computations yielded a reasonable description of the level and rate of decline of fenpropimorph volatilisation in the first 4 h after spraying. The continued volatilisation 2 and 3 days after spraying could be described by assuming that a fraction of the deposit was poorly exposed with comparatively low rates of the decline processes. In the first 3 days, penetration of fenpropimorph into the plant leaves was computed to be the main route for the pesticide (52% of the dosage), with substantial contributions from volatilisation (12%) and phototransformation (11%). The computation model can be developed further as a tool for extrapolating results on volatilisation from small-scale experiments to field conditions, but this requires more information on the effect of environmental conditions on the model parameters.

Air Pollutants↗

Teratogenic effects of the fungicide dinocap in the mouse.

The teratogenic potential of the fungicide dinocap was evaluated in CD-1 mice. Pregnant mice were dosed by intubation with dinocap in corn oil on gestation days 7-16. Doses used were 0, 5, 10, 20, 40, 80, and 120 mg/kg/day, based on day 6 weight. Dams were killed on day 18, at which time fetuses were counted, weighed, and preserved for necropsy or skeletal examination. The highest dose killed 80% of the dams dosed, while 29% of the dams in the 80 mg/kg group died during dosing. There was no dose-related maternal mortality at lower doses. Net maternal weight gain was affected only at 80 mg/kg/day. There were no live fetuses at 120 mg/kg/day. The number of live fetuses per litter was decreased and resorptions increased at 80 mg/kg. Dose-related decreases in gravid uterus weight and fetal weight were significant at all doses of dinocap. Cleft palate was found in fetuses at 5(1/234; 0.4%), 20(46/195; 23.6%), 40(140/185; 75.5%), and 80(63/85; 74.1%) mg/kg/day. There was a dose-related increase in supernumerary ribs and a low frequency of exencephaly and umbilical hernia at high doses. This study shows that dinocap is teratogenic in the CD-1 mouse at doses well below those causing maternal toxicity.

Animals↗

In vitro transforming effect of the fungicides metalaxyl and zineb.

The cytotoxic effects and the transforming properties of two fungicides, metalaxyl and zineb, whose mutagenic or carcinogenic activity has not been clarified yet, were analyzed in the in vitro BALB/c 3T3 cell transformation test both in the presence and in the absence of an exogenous metabolizing system. Zineb was completely detoxified when the exogenous metabolizing system was added to the target cells to increase their inherent metabolic capacity. Metalaxyl induced cell transformation at any assayed dosage, i.e., 500, 250, and 50 micrograms/ml, in the presence of bioactivation, and at the highest dosage (500 micrograms/ml) in the absence of bioactivation. The transforming effect was detectable only in the level-II transformation cultures and it was likely linked to the induction of additional cell proliferation which allowed obtaining the transformation amplification in these experimental conditions.

3T3 Cells↗