Sterol Biosynthesis-inhibiting Fungicides Research Articles

Genetic algorithm multiple linear regression and machine learning-driven QSTR modeling for the acute toxicity of sterol biosynthesis inhibitor fungicides

Sterol Biosynthesis Inhibitors (SBIs) are a major class of fungicides used globally. Their widespread application in agriculture raises concerns about potential harm and toxicity to non-target organisms, including humans. To address these concerns, a quantitative structure-toxicity relationship (QSTR) modeling approach has been developed to assess the acute toxicity of 45 different SBIs. The genetic algorithm (GA) was used to identify key molecular descriptors influencing toxicity. These descriptors were then used to build robust QSTR models using multiple linear regression (MLR), support vector regression (SVR), and artificial neural network (ANN) algorithms. The Cross-validation, Y-randomization test, applicability domain methods, and external validation were carried out to evaluate the accuracy and validity of the generated models. The MLR model exhibited satisfactory predictive performance, with an R2 of 0.72. The SVR and ANN models obtained R2 values of 0.7 and 0.8, respectively. ANN model demonstrated superior performance compared to other models, achieving R2cv and R2test values of 0.74 and 0.7, respectively. The models passed both internal and external validation, indicating their robustness. These models offer a valuable tool for risk assessment, enabling the evaluation of potential hazards associated with future applications of SBIs.

Synergistic effects of three sterol biosynthesis inhibiting fungicides on the toxicity of a pyrethroid and neonicotinoid insecticide to bumble bees

Understanding how different pesticides influence bee health is inhibited by a limited knowledge about the interactions between different compounds to which bees are simultaneously exposed. Although research has demonstrated synergistic effects of some sterol biosynthesis inhibiting (SBI) fungicides on the toxicity of certain insecticides to bees, a high degree of variability exists in the relatively few SBI fungicide-insecticide interactions tested. Furthermore, most research has been conducted on honey bees, Apis mellifera, limiting our understanding of pesticide synergisms in native wild bees. We tested the effects of the SBI fungicides difenoconazole, myclobutanil, and fenhexamid on acute contact toxicity of the insecticides thiamethoxam (neonicotinoid) and bifenthrin (pyrethroid) to the common eastern bumble bee, Bombus impatiens. Based on range-finding trials, we selected a single dosage of each pesticide, with insecticides approximating LD20 values and fungicides approximating their maximum sublethal levels. We found that the triazole SBI fungicide difenoconazole interacted synergistically with bifenthrin, with a maximum synergy ratio of 1.48, while the triazole myclobutanil interacted synergistically with both bifenthrin and thiamethoxam, with maximum synergy ratios of 11.0 and 2.38, respectively. However, the hydroxyanilide SBI fungicide fenhexamid did not exhibit a synergistic effect on either insecticide. Our results suggest that certain SBI fungicides, and potentially those from particular SBI classes, may be particularly harmful to bumble bee health when co-exposure to insecticides occurs, despite their low toxicity when experienced in isolation.

Evolution of cross-resistance to medical triazoles in Aspergillus fumigatus through selection pressure of environmental fungicides.

Resistance to medical triazoles in Aspergillus fumigatus is an emerging problem for patients at risk of aspergillus diseases. There are currently two presumed routes for medical triazole-resistance selection: (i) through selection pressure of medical triazoles when treating patients and (ii) through selection pressure from non-medical sterol-biosynthesis-inhibiting (SI) triazole fungicides which are used in the environment. Previous studies have suggested that SI fungicides can induce cross-resistance to medical triazoles. Therefore, to assess the potential of selection of resistance to medical triazoles in the environment, we assessed cross-resistance to three medical triazoles in lineages of A. fumigatus from previous work where we applied an experimental evolution approach with one of five different SI fungicides to select for resistance. In our evolved lines we found widespread cross-resistance indicating that resistance to medical triazoles rapidly arises through selection pressure of SI fungicides. All evolved lineages showed similar evolutionary dynamics to SI fungicides and medical triazoles, which suggests that the mutations inducing resistance to both SI fungicides and medical triazoles are likely to be the same. Whole-genome sequencing revealed that a variety of mutations were putatively involved in the resistance mechanism, some of which are in known target genes.

Acaricide, Fungicide and Drug Interactions in Honey Bees (Apis mellifera)

BackgroundChemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects.Methodology/Principal FindingsLaboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested.Conclusions/SignificanceInteractions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an important role. Evidence of non-transivity, year-to-year variation and induction of detoxication enzymes indicates that pesticide interactions in bees may be as complex as drug interactions in mammals.

The Allele-Specific Probe and Primer Amplification Assay, a New Real-Time PCR Method for Fine Quantification of Single-Nucleotide Polymorphisms in Pooled DNA

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

The sterol biosynthesis inhibitor molecule fenhexamid impacts the vegetative compatibility of Glomus clarum

The vegetative compatibility of the arbuscular mycorrhizal fungus (AMF) Glomus clarum MUCL 46238 was evaluated after continuous exposure to fenhexamid, a sterol biosynthesis inhibitor (SBI). Three lineages of this AMF were cultured in vitro for five generations in association with Ri T-DNA transformed carrot roots in the presence of 0, 5 or 10mgl(-1) of fenhexamid. Whatever the AMF generation, fenhexamid at 5 and 10mgl(-1) had no significant impact on the number of spores produced. However, vegetative compatibility tests (VCT) conducted with spores from the three lineages, in the presence of 10mgl(-1) of fenhexamid, impacted the anastomosis process. At this concentration, the morphology of the germ tubes was modified. In addition, nitrotetrazolium-trypan blue staining revealed that 10mgl(-l) of fenhexamid significantly reduced the probability of fusion between the germ tubes regardless of the culture conditions (i.e. absence or presence of fenhexamid) preceding the VCT. Our results demonstrated that spore production was not affected by fenhexamid, while anastomosis between germ tubes was decreased. This suggested that high concentrations, accumulation or repeated application of this SBI fungicide may impact the community structure of AMF in soil.

Characterization of a CuZn superoxide dismutase gene in the arbuscular mycorrhizal fungus Glomus intraradices

To gain further insights into the mechanisms of redox homeostasis in arbuscular mycorrhizal fungi, we characterized a Glomus intraradices gene (GintSOD1) showing high similarity to previously described genes encoding CuZn superoxide dismutases (SODs). The GintSOD1 gene consists of an open reading frame of 471 bp, predicted to encode a protein of 157 amino acids with an estimated molecular mass of 16.3 kDa. Functional complementation assays in a CuZnSOD-defective yeast mutant showed that GintSOD1 protects the yeast cells from oxygen toxicity and that it, therefore, encodes a protein that scavenges reactive oxygen species (ROS). GintSOD1 transcripts differentially accumulate during the fungal life cycle, reaching the highest expression levels in the intraradical mycelium. GintSOD1 expression is induced by the well known ROS-inducing agents paraquat and copper, and also by fenpropimorph, a sterol biosynthesis inhibitor (SBI) fungicide. These results suggest that GintSOD1 is involved in the detoxification of ROS generated from metabolic processes and by external agents. In particular, our data indicate that the antifungal effects of fenpropimorph might not be only due to the interference with sterol metabolism but also to the perturbation of other biological processes and that ROS production and scavenging systems are involved in the response to SBI fungicides.

Arbuscular mycorrhiza partially protect chicory roots against oxidative stress induced by two fungicides, fenpropimorph and fenhexamid.

The present work examined the oxidative stress induced by different concentrations (0.02 and 0.2 mg l-1) of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) in non-target chicory root colonised or not by Glomus intraradices in a monoxenic system. The fungicides were found to cause oxidative damage by increasing lipid peroxidation measured by malondialdehyde production in non-colonised roots. Detoxification of the H(2)O(2) product was measured at 0.2 mg l-1 of fenpropimorph by an increase in peroxidase activities suggesting an antioxidant capacity in these roots. Moreover, this study pointed out the ability of arbuscular mycorrhiza to alleviate partially the oxidative stress in chicory roots, probably by lowering reactive oxygen species concentrations, resulting from increases in antioxidant defences. Our results suggest that the enhanced fungicide tolerance in the AM symbiosis could be related to less cell membrane damage.

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2 mg l −1) and fenhexamid (0.02; 0.2; 2; 20 mg l −1), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Δ 5-sterols were replaced by unusual compounds such as 9β,19-cyclopropylsterols (24-methylpollinastanol), Δ 8,14-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Δ 8-sterols (Δ 8 sitosterol) and Δ 7-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected. Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.

Effect of the new pyrazole carboxamide fungicide penthiopyrad on late leaf spot and stem rot of peanut

Management of early leaf spot (Cercospora arachidicola Hori.), late leaf spot [Cercosporidium personatum (Berk. & MA Curtis) Deighton] and stem rot (Sclerotium rolfsii Sacc.) of peanut (Arachis hypogaea L.) in the southeastern USA is heavily dependent upon sterol biosynthesis inhibitor (SBI) and quinone outside inhibitor (QoI) fungicides. Effective new fungicides with different modes of action could improve overall disease control and extend the utility of the current fungicides. Penthiopryad is a pyrazole carboxamide fungicide being evaluated for use on peanut. Field experiments were conducted from 2004 to 2007 to determine the effect of a range of rates (0-0.36 kg AI ha(-1)) of penthiopyrad on leaf spot and stem rot and the relative efficacy of penthiopyrad and current fungicide standards chlorothalonil, tebuconazole and azoxystrobin. Leaf spot control in plots treated with penthiopyrad at 0.20 kg AI ha(-1) or higher was similar to or better than that for the chlorothalonil standard. The incidence of stem rot for all penthiopyrad treatments was usually less than that for the tebuconazole or azoxystrobin standard treatments. Pod yields for all penthiopyrad treatments were similar to or higher than those for the respective standards. Penthiopyrad has excellent potential for management of late leaf spot and stem rot of peanut, and may complement current SBI and QoI fungicides.

Effects of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) on the development of an arbuscular mycorrhizal fungus

The effects of different concentrations (0.2, 2, 20, 200 mg l −1) of two sterol biosynthesis inhibitor (SBI) fungicides, i.e. fenpropimorph and fenhexamid, were evaluated on the spore germination, germ tube elongation, sporulation, and root colonization of Glomus intraradices grown monoxenically in association with transformed carrot roots. The percentage of germinated spores incubated on the SBI fungicides and the length of the germ tubes decreased with increasing concentrations of both fungicides. However, for spore germination this impact was fungistatic rather than fungicidal. Extraradical mycelium architecture and spore production in contact with the SBI fungicides were also strongly impacted at high concentration (20 mg l −1). Conversely, the colonization of roots developing in the fungicide-free compartment, but interconnected with the extraradical mycelium developing on the SBI fungicides, appeared unaffected. Our results demonstrated that the monoxenic culture system could be used as a standardized, reproducible technique to compare the impacts of different molecules on arbuscular mycorrhizal fungi, and for the initial screening of new candidate molecules before registration.

Influence of Fungicide Residues on the Primary Fermentation of Young Lager Beer

The effect of four sterol biosynthesis-inhibiting fungicides added to the pitching wort on the evolution of several organoleptic parameters during the primary fermentation of young lager beer was assessed. Pyrimidine (nuarimol and fenarimol) and triazole (myclobutanil and propiconazole) fungicides were individually supplied to the pitching wort to obtain a concentration of 1 mg/L. A marked influence in the fermentation rate was observed for the samples with propiconazole residues. From the fourth day onward, the fermentation prematurely ceased (stuck fermentation), and therefore, statistical significant differences were found in fermented extract, alcohol content, fermentable carbohydrates, pH, color, and total polyphenol and flavonoid contents of beer. Myclobutanil residues are only influenced in the total polyphenol and flavonoid contents, while differences in the analyzed parameters were not noticeable for the samples containing nuarimol and fenarimol residues in comparison with the blank sample.

The hydroxyanilide fenhexamid, a new sterol biosynthesis inhibitor fungicide efficient against the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea).

Fenhexamid, a recently developed botryticide, is shown here to inhibit sterol biosynthesis. When the fungus Botryotinia fuckeliana was grown in the presence of fenhexamid, the ergosterol content was reduced, and three 3-keto compounds, 4 alpha-methylfecosterone, fecosterone and episterone, accumulated, suggesting an inhibition of the 3-keto reductase involved in C-4 demethylation. Thus, fenhexamid belongs to a new, promising class of sterol biosynthesis inhibitors not previously used in agriculture or in medicine.

The effect of soil moisture content on the sorption of five sterol biosynthesis inhibiting fungicides as a function of their physicochemical properties

We investigated the sorption of five widely used sterol biosynthesis inhibitor fungicides (SBIs: flusilazole, propiconazole, epoxiconazole, fenpropimorph and prochloraz) on a loam soil to assess availability of the SBI residues that are usually left in soil after crop treatments. We focused particularly on the soil moisture content effect, which is poorly documented and is difficult to investigate under realistic conditions. SBI sorption was determined (using diuron as a reference) at two soil moisture contents (26.1% and 46.6% w/w) over a period of 3 weeks using a direct soil solution sampling method. After 24 h of contact, <1% of each applied fungicide was recovered in the soil solution. Despite their low availability in the liquid phase, long-term sorption was observed for all the compounds, reducing concentrations in the soil solution and doubling the value of the partition coefficient. Significant effects of soil moisture on long-term sorption were observed, depending on the properties of the chemicals and the sorption mechanisms. Wershaw's humus model (humic substances have a membrane-like structure) was adapted to fit our observations. Low soil moisture content is assumed to modify the structure of humic substances and to generate hydrophobic surfaces, which favour sorption of hydrophobic fungicides (flusilazole, propiconazole and epoxiconazole). This effect is likely to decrease with the increase in the hydrophobic character of non-ionic pesticides. It becomes adverse for the more hydrophilic compounds (diuron), which are more sorbed at high soil moisture content due to their higher affinity for hydrophilic regions of humus and to diffusion. Soil moisture effects are more complex when compounds are likely to be protonated in soil. Weakly basic compounds (prochloraz) may partition rapidly into the liquid-like interior of humus at low soil moisture content but increased diffusion at high soil moisture content may cause additional sorption by ion exchange at colloid surfaces. Strongly basic compounds (fenpropimorph) may essentially adsorb due to ionic interactions with colloids, and their sorption is enhanced at high soil moisture content due to diffusion. Consequences for environmental fate and biological activity of pesticides are briefly discussed. © 2000 Society of Chemical Industry

Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus,Nectria haematococca:ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ14-reductase and Δ8Δ7-isomerase, whereas tridemorph was a better inhibitor of Δ8Δ7-isomerase than of the Δ14-reductase. Large accumulations of Δ8,14- or Δ8-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca. © 1998 Society of Chemical Industry

Effects of flusilazole, a sterol biosynthesis inhibitor fungicide, on the free and bound aroma fraction of Muscat of Alexandria wines

&lt;p style="text-align: justify;"&gt;Vines from Muscat of Alexandria cultivar were subjected to flusilazole treatments, a sterol biosynthesis inhibitor fungicide (SBI), over 2 years. Effects of these treatments on the corresponding wines were assessed by analyzing the free and bound aroma fractions using selective retention on Amberlite XAD-2 and gas chromatography-mass spectrometry. Quantitative changes in the composition of the free and bound fractions of the treated wines were observed. Levels of varietal, prefermentative and fermentative compounds were moditied and a regulatory mechanism for sterol synthesis in yeast involving the monoterpenes could be hypothesized.&lt;/p&gt;

Lack of Correlation Between Fitness and Resistance to Sterol Biosynthesis-Inhibiting Fungicides inPyrenophora teres

Fitness costs associated with resistance to sterol biosynthesis-inhibiting fungicides (SBIs) were investigated by calculating correlations between SBI-resistance phenotypes and fitness phenotypes in Pyrenophora teres. Correlations between resistance to the SBIs triadimenol and propiconazole and two components of fitness were estimated with P. teres isolates randomly sampled from populations in North Dalcota and Bavaria, Germany. The fitness components, latent period (the time from inoculation to the first appearance of a conidium) and sporulation (total sporulation per lesion), were determined quantitatively with detached barley leaf sections inoculated with P. teres conidia in the greenhouse [...]

Genetic Correlations in Resistance to Sterol Biosynthesis-Inhibiting Fungicides inPyrenophora teres

We investigated cross-resistance in Pyrenophora teres to five sterol biosynthesis-inhibiting fungicides (SBIs) by calculating genetic correlations in resistances. Two approaches were used in separate experiments to estimate the correlations: In one method, progeny sampled from crosses of P. teres were used to determine among-family genetic correlations; in the other method, replicated isolates campled from different P. teres populations were used to calculate among-isolate genetic correlations [...]

Reduced sensitivity of Botrytis cinerea to two sterol biosynthesis‐inhibiting fungicides: fenetrazole and fenethanil

Isolates of Botrytis cinerea having reduced sensitivity to the sterol biosynthesis‐inhibiting (SBI) fungicides fenetrazole and fenethanil were obtained from one out of four sites from which isolates were tested. Reduced sensitivity was associated with poor disease control by fenetrazole, which had been applied with dichlofluanid. Conidial germination and hyphal growth of B. cinerea from the four sites were tested in vitro on media amended with the fungicides. Following fenetrazole or fenethanil treatment, at the site where control had failed, populations of B. cinerea were detected with higher EC50 and EC90 values than at the three other sites. Germination of conidia of B. cinerea was markedly inhibited by 1.0 μg/ml fenetrazole and 0.5–1.0 μg/ml fenethanil. The frequency of isolates insensitive to 1.0 μg/ml fenetrazole or to 0.5 μg/ml fenethanil was 3.4 and 1.8 times higher, respectively, at the site where control had failed, compared with another site where SBI fungicides had never been applied to control grey mould. Grey mould caused by selected isolates of B. cinerea which expressed the phenotype of low sensitivity to SBI fungicides in leaves of tomato, pepper and Senecio cineraria was not controlled by either fenetrazole or fenethanil (1 .5–3.0 μg/ml). However, up to 100% disease reduction was obtained when leaves infected by sensitive isolates were treated with the fungicides.

Effects of Sterol Biosynthesis Inhibitor Fungicides on Growth and Sterol Composition of Ustilago maydis, Botrytis cinerea and Pyrenophora teres

AbstractThe effects of the sterol biosynthesis inhibitor (SBI) fungicides fenarimol, fenpropimorph, imazalil, prochloraz, propiconazole and triadimenol on growth and sterol composition of Ustilago maydis, Botrytis cinerea and Pyrenophora teres, grown from spores or sporidia in liquid culture, were determined. Growth of U. maydis was only slightly inhibited by SBI fungicides at concentrations which caused considerable changes in both sterol content and composition. Conversely, in B. cinerea and P. teres, growth was strongly inhibited under conditions where ergosterol was still the predominant sterol, suggesting that, in these two fungi, growth may be more sensitive to SBI fungicides than overall sterol production. Demethylase inhibitor fungicides behaved as a homogeneous group in their effects on growth and on sterol profiles of the three fungi studied.