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Fungicide Enhancement of Nitrogen Fixation and Colonization of Phaseolus vulgaris by Rhizobium phaseoli.

The number and weight of pods and the weight and nitrogen content of the tops of beans (Phaseolus vulgaris) derived from seeds inoculated with a thiram-resistant strain of Rhizobium phaseoli were increased if the seeds were treated with thiram before sowing in soil. A greater percentage of the nodules on 21-day-old plants were derived from the resistant strain, more nodules were formed, and these nodules were more effective in the presence of the fungicide than in its absence. These differences in nodule numbers were no longer present in 56-day-old plants, and only a small percentage of the nodules contained the resistant strain. The abundance of the fungicide-tolerant R. phaseoli increased rapidly soon after planting the seed and subsequently fell markedly, but the rate of decline was less if the seeds had been treated with the chemical. Protozoa also proliferated if thiram had not been applied to the seed, but their numbers were deleteriously influenced by thiram. Bdellovibrio, bacteriophages, and lytic micro-organisms acting on R. phaseoli were rare under these conditions. Ciliates and flagellated protozoa were initially suppressed by planting thiram-coated bean seeds in nonsterile soil, but the former were inhibited longer than the latter and the ciliate numbers never fully recovered if the seeds were treated with the fungicide. The resistant strain grew well in sterile soil also inoculated with a protozoa-free mixture of soil microorganisms whether thiram was added or not, but after an initial rise in numbers, its abundance fell if the mixture contained protozoa; the rate of this fall was delayed by the fungicide. The numbers of R. phaseoli were consistently less in sterile soil inoculated with the rhizobium plus a mixture of soil microorganisms containing ciliates and other protozoa than if the inoculum contained other protozoa but no ciliates. These results suggest that a suppression of protozoa, and possibly especially the ciliates, accounts for the enhanced growth of beans and the greater initial frequency of nodules formed by the thiram-resistant R. phaseoli in the presence of this fungicide. Thiram applied to uninoculated seed enhanced bean growth if thiram-resistant R. phaseoli were present in soil.

Journal Article↗

Aneuploidy in sperm and exposure to fungicides and lifestyle factors. ASCLEPIOS. A European Concerted Action on Occupational Hazards to Male Reproductive Capability.

Fungicides include chemicals that are known aneugens. The purpose of the present study was to investigate whether occupational exposure to these and other agricultural pesticides induces aneuploidy in human sperm. The contribution of lifestyle factors (smoking and alcohol consumption) to the frequency of aneuploid sperm was evaluated as well. The effects of age and sperm concentration were analyzed as confounders. Spermatozoa from 30 healthy farmers were studied before and after exposure to fungicides, using fluorescence in situ hybridization (FISH). Ten thousand spermatozoa were scored per semen sample to determine the disomy and diploidy frequencies for chromosomes 1 and 7. Exposure to fungicides was not associated with sperm aneuploidy. Smoking was significantly associated with sperm carrying an extra chromosome 1 and with diploid sperm as well as with the aggregate frequency of aneuploid sperm. Alcohol consumption, sperm concentration, and age showed inconsistent results before and after the season of exposure to fungicides. For low-level exposures, such as occupational exposures, the sensitivity of the sperm-FISH method may not be sufficient. The present study supports earlier ones showing that smoking can increase aneuploidy in human sperm.

Adult↗

Rapid determination of fungicides in fruit juices by micellar electrokinetic chromatography: use of organic modifiers to enhance selectivity and on-column high-salt stacking to improve sensitivity.

A rapid, reliable method for the multiresidue analysis of eight commonly used fungicides by micellar electrokinetic chromatography (MEKC) was developed. Excellent separation of the eight fungicides (carbendazim, metalaxyl, captan, procymidone, folpet, captafol, vinclozolin and iprodione) is achieved within about 10 min by using optimized electrophoretic conditions that include the addition of a mixture of organic modifiers to the running buffer for improved resolution. The sensitivity of the method is enhanced by using an enrichment step that involves on-column high-salt stacking. Limits of detection in the microgram-per-liter region and relative standard deviations from 2.1 to 5.9% are thus obtained for the fungicides without detracting from peak resolution. These results reveal that the high-salt stacking method provides highly improved sensitivity and enables highly flexible adjustment of the selectivity of the separation method. Also, the method surpasses other stacking alternatives used in MEKC and affords routine analyses of fruit juice containing fungicides at trace levels following a straightforward sample treatment. The robustness of the high-salt stacking method as demonstrated in this work makes MEKC methods involving stacking procedures an attractive choice for routine analyses.

1-Propanol↗

Recognizing acute health effects of substitute fungicides: are first-aid reports effective?

Recently, many British Columbia sawmills stopped using traditional chlorophenate anti-sapstain fungicides and substituted 2-(thiocyanomethylthio) benzothiazole (TCMTB) and copper-8-quinolinolate (Copper 8). We conducted a cross-sectional study with two aims: to ascertain which acute health effects, if any, were associated with the use of the substitute fungicides; and to determine the effectiveness of first-aid records as a means of detecting acute health outcomes. Workers in five coastal sawmills were asked to complete a self-administered questionnaire about symptoms considered potentially related and unrelated to fungicide exposure, and about injuries commonly reported in sawmills. In addition, we collected first-aid records from the mills, and asked senior workers to estimate the duration of exposure to fungicides for each job. Symptoms found to be consistently elevated in TCMTB mills included dry skin around the eyes, blood-stained mucus from the nose, nose bleed, peeling skin, burning or itching skin, and skin redness or rash. No symptoms were consistently elevated in the Copper 8 mills. Symptoms related to TCMTB exposure were recorded only 12 times in first-aid logs during the study period (versus 335 questionnaire self-reports). This low symptom-recording frequency may be a function of established patterns of first-aid use in which illness symptoms are reported less frequently than injuries.

Acute Disease↗

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens.

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schröt, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.

Amino Acid Substitution↗

A new method for assessing foliar uptake of fungicides using Congo Red as a tracer.

In order to develop a new method for measuring foliar uptake of fungicides, Congo Red was selected as a tracer, and optimum procedures were established for washing, extracting and analyzing it and fungicides from leaf surfaces. Congo Red, a water-soluble dye, was not absorbed into cucumber or rice leaf, even in the presence of various surfactants, and was completely washable from leaf surfaces by aqueous acetonitrile solutions. Congo Red and fungicides in washings were quantified to calculate the amount of foliar uptake of the latter, by comparing the ratio to Congo Red. The optimum concentration of Congo Red in a formulation should be established in order to minimize its influence on fungicide uptake. Although Congo Red has proved to be useful with a conventional droplet application method, it will give more realistic and practical results with the spraying method used in the present study.

Acetonitriles↗

Synthesis and fungicidal activity of a series of novel aryloxylepidines.

A series of novel (hetero) aryloxylepidine derivatives was devised as hybrid structures of the phenoxyquinoline and phenethoxyquin(az)oline fungicides. Synthesis of these targets required the development of several new routes to derivatised 4-hydroxymethylquinolines, and subsequent coupling with phenols or haloarenes. The aryloxylepidines generally showed moderate broad-spectrum fungicidal activity across several diseases of cereals. Substitution of the quinoline ring with chlorine at the 7- and/or 5-positions gave molecules with high levels of protectant activity against Erysiphe graminis f sp tritici (powdery mildew of wheat), but this did not improve the level of fungicidal activity against other diseases. In vitro activity against mitochondrial electron transport complex I (MET) derived from Ustilago maydis showed that 8-fluorolepidine analogues were moderately active at this target site, while the more fungicidally active 7- and 5,7-substituted compounds were inactive. This indicates that MET is not the primary target of these highly active powdery mildewicides.

Ascomycota↗

Mechanisms of resistance to fungicides in field strains of Botrytis cinerea.

Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.

Botrytis↗

Synthesis and fungicidal activity of ethaboxam against Oomycetes.

This study describes the chemical synthesis and intrinsic fungicidal activity of ethaboxam [(RS)-N-(alpha-cyano-2-thenyl)-4-ethyl-2-(ethylamino)-1,3-thiazole-5-carboxamide], a new Oomycetes fungicide. In in vitro tests, ethaboxam showed inhibitory activity against isolates of Phytophthora and some Pythium spp, with MIC values ranging from 0.1 to 0.5 mg litre(-1) for nine isolates of Phytophthora infestans (Montagne) de Bary and from 1.0 to 5.0 mg litre(-1) for eight isolates of Phytophthora capsici Leonian. In tests to determine time and concentration for complete inactivation of each pathogen (five isolates of P infestans and five isolates of P capsici), ethaboxam inactivated all isolates of P infestans within 48h at 10 mg litre(-1) and those of P capsici within 96 h at 10 mg litre(-1). Ethaboxam effectively suppressed development of tomato late blight caused by P infestans and pepper Phytophthora blight caused by P capsici in the studies conducted to determine its preventive, curative, persistent and systemic activity. These results show that ethaboxam has desirable fungicidal characteristics as an Oomycetes fungicide.

Fungicides, Industrial↗

Plant sterol 14 alpha-demethylase affinity for azole fungicides.

Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14 alpha-demthylase (CYP51) than the plant orthologue. Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out. S. bicolor CYP51 was purified to homogenity as determined by SDS--PAGE and specific heme content. Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations. Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC(50) values. The implications for fungicide mode of action and application are discussed.

Cytochrome P-450 Enzyme Inhibitors↗

Effects of temperature and fungicides on Chrysosporium pannorum (Link) Hughes.

C. pannorum was isolated in low frequencies and only in the colder months from untreated soil and leaves and from those treated with Captan, Dicloran and Thiram. The fungus was isolated throughout the year as the major species from Verdasan-treated substrates and it occurred with increased frequencies immediately after application of the fungicide. The abundance of C. pannorum on Verdasan-treated substrates was attributed to the ability of the fungus to utilise the fungicide rather than to the absence of faster growing competitor species. C. pannorum was shown to be a slow growing and moderately cellulolytic fungus with maximum rates of growth, germination and cellulose clearing at between 15 degrees and 20 degrees C. Increasing concentrations of the fungicides retarded or prevented growth and activity of the fungus. C. pannorum could grow, germinate and clear cellulose in higher concentrations of Verdasan than could other species studied. C. pannorum was more tolerant of HgCl2 than of Verdasan. The fungus could detoxify up to 3 micrograms/ml active ingredient of Verdasan (= 120 micrograms/ml of the formulated fungicide) in liquid culture.

Aniline Compounds↗

Effects of fungicides on Aspergillus fumigatus.

Aspergillus fumigatus was the most frequently isolated thermophilous fungus from green leaf surfaces. The application of fungicides significantly reduced the frequency of its occurrence there. A. fumigatus was relatively tolerant to fungicides. On Captan-, Thiram-, and Verdasan-treated leaves, A. fumigatus constituted 66%--80% of the total number of isolates obtained at 45 degrees C from each treatment while Dicloran did not depress the percentages. At 45 degrees C, A. fumigatus was found to be strongly cellulolytic with a slow rate of radial extension on YpSs agar and rapid rate of mycelial growth in Czapek Dox liquid medium. Increasing concentrations of all four fungicides reduced or prevented growth, sporulation, starch depletion and cellulose clearing of A. fumigatus. The fungus could tolerate higher concentrations of HgCl2 than of Verdasan. 2.5 microgram/ml of the four fungicides altered the rates of mycelial growth but not the maximum amount of mycelial dry weight attained.

Aspergillus fumigatus↗

Fungicide inhibition of aflatoxins, diacetoxyscirpenol and zearalenone production.

The effect of fungicides on the production of aflatoxin by Aspergillus flavus IMI 89717, diacetoxyscirpenol and zearalenone by Fusarium graminearum was studied. In a yeast extract-sucrose medium, dicloran, iprodione and vinclozolin fungicides significantly inhibited mycelial growth of A. flavus at 250 ppm and significantly decreased aflatoxin production at 100, 250 and 500 ppm, respectively. In potato-dextrose broth, these fungicides diminished the mycelial growth of F. graminearum and production of diacetoxyscirpenol and zearalenone at 100 ppm. Sensitivity of toxigenic mycelia to fungicides increased approximately five-fold in a yeast extract-starch medium with an appreciable reduction in sugar uptake and alpha-amylase activity.

Aflatoxins↗

Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants.

Progeny from transgenic broccoli (cv. Green Comet) expressing a Trichoderma harzianum endochitinase gene were used to assess the interaction between endochitinase and the fungicide Bayleton in the control of Alternaria brassicicola. In vitro assays have shown synergistic effects of endochitinase and fungicides on fungal pathogens. Our study examined the in planta effects of endochitinase and Bayleton, individually and in combination. Two month old transgenic and non-transgenic plants were sprayed with ED50 levels of Bayleton and/or inoculated with an A. brassicicola spore suspension. Disease levels in non-sprayed transgenic plants were not statistically different from sprayed transgenic plants nor from sprayed non-transgenic controls. Thus endochitinase-transgenic plants alone provided a significant reduction of disease severity, comparable to the protection by fungicide on non-transgenic plants. Comparison of the expected additive and observed effects revealed no synergism between endochitinase and Bayleton (at ED50 level), and usually less than an additive effect. Some transgenic lines sprayed with fungicide at doses higher than ED50 showed resistance similar to the non-sprayed transgenic lines, again suggesting no synergistic effect. Lack of synergism may be due to incomplete digestion of the cell wall by endochitinase, so that the effect of Bayleton at the cell membrane is not enhanced.

Alternaria↗

The fungicidal and phytotoxic properties of benomyl and PPM in supplemented agar media supporting transgenic arabidopsis plants for a Space Shuttle flight experiment.

Fungal contamination is a significant problem in the use of sucrose-enriched agar-based media for plant culture, especially in closed habitats such as the Space Shuttle. While a variety of fungicides are commercially available, not all are equal in their effectiveness in inhibiting fungal contamination. In addition, fungicide effectiveness must be weighed against its phytotoxicity and in this case, its influence on transgene expression. In a series of experiments designed to optimize media composition for a recent shuttle mission, the fungicide benomyl and the biocide "Plant Preservative Mixture" (PPM) were evaluated for effectiveness in controlling three common fungal contaminants, as well as their impact on the growth and development of arabidopsis seedlings. Benomyl proved to be an effective inhibitor of all three contaminants in concentrations as low as 2 ppm (parts per million) within the agar medium, and no evidence of phytotoxicity was observed until concentrations exceeded 20 ppm. The biocide mix PPM was effective as a fungicide only at concentrations that had deleterious effects on arabidopsis seedlings. As a result of these findings, a concentration of 3 ppm benomyl was used in the media for experiment PGIM-01 which flew on shuttle Columbia mission STS-93 in July 1999.

Agar↗

Effect of the fungicide Prochloraz-Mn on the cell wall structure of Verticillium fungicola.

The chemical structure of the cell wall of two isolates of Verticillium fungicolacollected from diseased fruit bodies of the commercial mushroom Agaricus bisporus treated with the fungicide Prochloraz-Mn was analyzed. The isolates were obtained during different periods of time and grown in the absence and presence of the LD(50) values of the fungicide for V. fungicola. In addition, another V. fungicola isolate collected previous to the routine utilization of Prochloraz-Mn but grown under the same conditions was also analyzed. The overall chemical composition of the cell wall from the three isolates showed detectable differences in their basic components, with a significant decrease in the protein content in fungicide-treated cells. This inhibitory effect was partially compensated by an increase in neutral and/or aminated carbohydrates and was accompanied by appreciable modifications of polysaccharide structure, as deduced after methylation analysis and gas-liquid chromatography-mass spectrometry (GLC-MS). Moreover, differences in hyphal morphology caused by the fungicide were observed by transmission electron microscopy (TEM).

Cell Wall↗

Induction of numerical chromosomal aberrations during DNA synthesis using the fungicides nimrod and rubigan-4 in root tips of Vicia faba L.

The 2 fungicides nimrod and rubigan-4 were tested for genotoxicity using Vicia faba root tips as the biological test system. Treating lateral roots with different concentrations of each fungicide for different periods showed that both fungicides were able to produce numerical but not structural chromosomal aberrations. The percentage of total aberrations in root tips exposed to nimrod reached 54.39% at 250 ppm for 4 h, and 64.69% in root tips exposed to rubigan-4 at 250 ppm for 6 h. The types of numerical chromosomal aberrations produced by both fungicides included: binucleate cells, c-metaphases, sticky chromosomes, polyploid cells, and laggards. Recovery experiments for 24, 48, and 96 h showed no significant differences between the percentage of total aberrations in treated and control groups.

Chromosome Aberrations↗

1,8-Naphthalic anhydride antidote enhances the toxic effects of captan and thiram fungicides on Azospirillum brasilense cells.

The effects of ten fungicides, six herbicides and four insecticides on the nitrogen-fixing bacterium Azospirillum brasilense were examined. The fungicides captan and thiram were the most toxic among the compounds tested. Cell growth and nitrogenase activity of the bacterium were markedly inhibited by low concentrations of the two fungicides. Antidote 1,8-naphthalic anhydride increased by a factor of 2 the cellular level of glutathione. The addition of the antidote in the presence of captan or thiram caused a similar increase in the glutathione content, but at the same time enhanced the toxicity of the two fungicides.

Azospirillum brasilense↗