Carrier-free self-assembled nanoparticles based on prochloraz and fenhexamid for reducing toxicity to aquatic organism.

Nanoformulations of pesticides are an effective way to increase utilization efficiency and alleviate the adverse impacts on the environments caused by conventional pesticide formulations. However, the complex preparation process, high cost, and potential environmental risk of nanocarriers severely restricted practical applications of carrier-based pesticide nanoformulations in agriculture. Herein, carrier-free self-assembled nanoparticles (FHA-PRO NPs) based on fenhexamid (FHA) and prochloraz (PRO) were developed by a facile co-assembly strategy to improve utilization efficiency and reduce toxicity to aquatic organism of pesticides. The results showed that noncovalent interactions between negatively charged FHA and positively charged PRO led to core-shell structured nanoparticles arranged in an orderly manner dispersing in aqueous solution with a diameter of 256 nm. The prepared FHA-PRO NPs showed a typical pH-responsive release profile and exhibited excellent physicochemical properties including low surface tension and high max retention. The photostability of FHA-PRO NPs was improved 2.4 times compared with free PRO. The FHA-PRO NPs displayed superior fungicidal activity against Sclerotinia sclerotiorum and Botrytis cinerea and longer duration against Sclerotinia sclerotiorum on potted rapeseed plants. Additionally, the FHA-PRO NPs reduced the acute toxicity of PRO to zebrafish significantly. Therefore, this work provided a promising strategy to develop nanoformulations of pesticides with stimuli-responsive controlled release characteristics for precise pesticide delivery.

Stimulation of estrogen receptor-alpha by hydroxyanilide fungicide, fenhexamid promotes lipid accumulation in 3 T3-L1 adipocyte.

Fenhexamid are fungicides that act against plant pathogens by inhibiting sterol biosynthesis. Nonetheless, it can trigger endocrine disruption and promote breast cancer cell growth. In a recent study, we investigated the mechanism underlying the lipid accumulation induced by fenhexamid hydroxyanilide fungicides in 3 T3-L1 adipocytes. To examine the estrogen receptor alpha (ERα)-agonistic effect, ER transactivation assay using the ERα-HeLa-9903 cell line was applied, and fenhexamid-induced ERα agonist effect was confirmed. Further confirmation that ERα-dependent lipid accumulation occurred was provided by treating 3 T3-L1 adipocytes with Methyl-piperidino-pyrazole hydrate (MPP), an ERα-selective antagonist. Fenhexamid mimicked the actions of ERα agonists and impacted lipid metabolism, and its mechanism involves upregulation of the expression of transcription factors that facilitate adipogenesis and lipogenesis. Additionally, it stimulated the expression of peroxisome proliferator-activated receptor (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1 (SREBP1) and significantly elevated the expression of fatty acid-binding protein 4 (FABP4). In contrast, in combination with an ERα-selective antagonist, fenhexamid suppressed the expression of adipogenic/lipogenic transcription factors. These results suggest that fenhexamid affects the endocrine system and leads to lipid accumulation by interfering with processes influenced by ERα activation.

Chilean Botrytis cinerea Isolates with Reduced Sensitivity to Fludioxonil Exhibit Low to Null Fitness Penalties.

The main phytosanitary problem for table grape production in Chile is gray mold caused by the fungus Botrytis cinerea. To manage this issue, the primary method utilized is chemical control. Fludioxonil, a phenylpyrrole, is highly effective in controlling B. cinerea and other plant pathogens. Consistently, there have been no field reports of reduced efficacy of fludioxonil; however, subpopulations with reduced sensitivity to fludioxonil are on the rise globally, as per increasing reports. Our study involved a large-scale evaluation of B. cinerea's sensitivity to fludioxonil in the Central Valley of Chile's primary table grape production area during the growing seasons from 2015 to 2018. Out of 2,207 isolates, only 1.04% of the isolates (n = 23) exceeded the sensitivity threshold value of 1 µg/ml. Remarkably, 95.7% are concentrated in a geographic region (Valparaíso Region). Isolates with reduced sensitivity to fludioxonil showed growth comparable with sensitive isolates and even more robust growth under nutritional deficit, temperature, or osmotic stress, suggesting greater environmental adaptation. When table grape detached berries were stored at 0°C, isolates less sensitive to fludioxonil caused larger lesions than sensitive isolates (2.82 mm compared with 1.48 mm). However, the lesions generated by both types of isolates were equivalent at room temperature. This study found no cross-resistance between fludioxonil and fenhexamid, an essential fungicide integrated with fludioxonil in Chilean B. cinerea control programs. All the Chilean isolates with reduced sensitivity to fludioxonil were controlled by the fludioxonil/cyprodinil mixture, a commonly employed form of fludioxonil. The cyprodinil sensitivity in the isolates with reduced sensitivity to fludioxonil explains their low field frequency despite their null fitness penalties. However, the emergence of fludioxonil-resistant isolates inside the Chilean B. cinerea population demands a comprehensive analysis of their genetic bases, accompanied by monitoring tools that allow the permanence of field fludioxonil efficacy.

Postinfection Application of Fenhexamid at Lower Doses in Conjunction with Captan Slowed Fungicide Resistance Selection in Botrytis cinerea on Detached Grape Berries.

Fungicide resistance is a limiting factor in sustainable crop production. General resistance management strategies such as rotation and mixtures of fungicides with different modes of action have been proven to be effective in many studies, but guidance on fungicide dose or application timing for resistance management remains unclear or debatable. In this study, Botrytis cinerea and the high-risk fungicide fenhexamid were used to determine the effects of fungicide dose, mixing partner, and application timing on resistance selection across varied frequencies of resistance via detached fruit assays. The results were largely consistent with the recent modeling studies that favored the use of the lowest effective fungicide dose for improved resistance management. In addition, even 10% resistant B. cinerea in the population led to about a 40% reduction of fenhexamid efficacy. Overall, our findings show that application of doses less than the fungicide label dose, mixture with the low-risk fungicide captan, and application postinfection seem to be the most effective management strategies in our controlled experimental settings. This somewhat contradicts the previous assumption that preventative sprays help resistance management.

Improved procedure for square-wave voltammetric sensing of fenhexamid residues on blueberries peel surface at the anodically pretreated boron-doped diamond electrode.

BACKGROUND: Fungicide fenhexamid (FH) has a high residual concentration on fruits and vegetables, thus, it is of high importance to monitor the level of FH residues on foodstuff samples. So far, the assay of FH residues in selected foodstuff samples has been conducted by electroanalytical methods on sp2 carbon-based electrodes that are well-known to be susceptible to severe fouling of the electrodes surfaces during electrochemical measurements. As an alternative, sp3 carbon-based electrode such as boron-doped diamond (BDD) can be used in the analysis of FH residues retained on the peel surface of foodstuff (blueberries) sample. RESULTS: In situ anodic pretreatment of the BDDE surface was found to be the most successful strategy to remediate the passivated BDDE surface by FH oxidation (by)products, and the best validation parameters, i.e., the widest linear range (3.0-100.0 µmol L-1), the highest sensitivity (0.0265 µA L µmol-1) and the lowest limit of detection (0.821 µmol L-1), were achieved on the anodically pretreated BDDE (APT-BDDE) in a Britton-Robinson buffer, pH 2.0, using square-wave voltammetry (SWV). The assay of FH residues retained on blueberries peel surface was performed on the APT-BDDE using SWV, and the obtained concentration of FH residues of 6.152 µmol L-1 (1.859 mg kg-1) was found to be below the maximum residue value fixed for blueberries by the European Union regulations (20 mg kg-1). SIGNIFICANCE AND NOVELTY: In this work, a protocol based on a very easy and fast foodstuff sample preparation procedure combined with the straightforward pretreatment approach of the BDDE surface was elaborated for the first time for the monitoring of the level of FH residues retained on the peel surface of blueberries samples. The presented reliable, cost-effective, and easy-to-use protocol could find its application as a rapid screening method for the control of food safety.

The antifungal potential of the chelating agent EDTA against postharvest plant pathogen Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungus that causes gray mold, a major postharvest disease of fruits and vegetables. Chemical fungicides remain the main solution to control Botrytis disease, but concerns have raised about their safety to environment and human health, and there is an increasing need for development of more effective and less toxic treatments. In this study the divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) exhibited marked antifungal activity against B. cinerea, including inhibition of spore germination, mycelial growth, infection cushion formation, stimulation of cell death, and impairment of fungal virulence. These adverse effects of EDTA could be reversed by the addition of calcium ion, implying that metal ion chelation is involved in the fungicidal mechanism. Bean leaf and tomato fruit protection assay indicated that EDTA treatment led to a significant reduction of infection by B. cinerea. Furthermore, the antifungal activity of EDTA was significantly enhanced when used in combination with fenhexamid. These findings suggest that EDTA could be a promising tool to control B. cinerea, and application of EDTA may reduce the use of conventional chemical fungicides.

A fungicide, fenhexamid, is involved in the migration and angiogenesis in breast cancer cells expressing estrogen receptors.

Fenhexamid (Fen) is used to eradicate gray mold of fruits and vegetables leading to greater detection of its residual concentration in wine than other fungicides. Here, we further investigated the malign influence of Fen on the migration and angiogenesis via regulation of the estrogen receptor (ER) and phosphoinositide 3-kinase (PI3K) pathways in breast cancer models. ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells were exposed to 17ß-estradiol (E2, 10-9 M), Fen (10-5 M and 10-7 M), ICI 182,780 (ICI; an ER antagonist, 10-8 M) or/and Pictilisib (Pic; a PI3K inhibitor, 10-7 M), and subsequently subjected to migration assay, live cell motility monitoring, trans-chamber assay, immunofluorescence, angiogenesis assay, tumor spheroid formation, and Western blot analysis. In MCF-7 cells, E2 and Fen induced cell migration by regulating the cell migration-related proteins. Although expressions of N-cadherin and Vimentin remained unchanged E2 and Fen induced the decrease of E-cadherin and Occludin in the immunofluorescence assay and Western blot analysis. In addition, Fen increased vessel formation in HUVEC cells. Furthermore, Fen treatment induced the formation of larger and denser tumor spheroids in MCF-7 cells. Western blot further confirmed the increased expressions of vascular endothelial growth factor (VEGF) and sex-determining region Y-box 2 (SOX2) after exposure to Fen. We conclude that Fen plays an important role as an endocrine-disrupting chemical in breast cancer migration and metastasis through the regulation of ER and PI3K signaling pathways.

Lack of confirmatory data following Article 12 MRL reviews for 2,4-d, fenhexamid and iprovalicarb.

The European Commission mandated EFSA to issue a statement concerning confirmatory data that were not submitted by the set deadline by the applicant following Article 12 reviews under Regulation (EC) No 396/2005 for the following substances/commodity combination: 2,4-d on buckwheat and other pseudo-cereals, fenhexamid on kiwis, iprovalicarb on lettuces, escaroles/broad-leaved endives and roman rocket/rucola. EFSA prepared a statement containing a final conclusion on the completeness of the data necessary to support the existing tentative maximum residue levels (MRLs) and indications to risk managers whether or not the tentative MRLs currently established by Regulation (EC) No 396/2005 could be maintained. The statement was circulated to Member States for consultation via a written procedure before finalisation.

Agricultural mulching and fungicides-impacts on fungal biomass, mycotoxin occurrence, and soil organic matter decomposition.

Plastic and straw coverage (PC and SC) are often combined with fungicide application but their influence on fungicide entry into soil and the resulting consequences for soil quality are still unknown. The objective of this study was to investigate the impact of PC and SC, combined with fungicide application, on soil residual concentrations of fungicides (fenhexamid, cyprodinil, and fludioxonil), soil fungal biomass, mycotoxin occurrence, and soil organic matter (SOM) decomposition, depending on soil depth (0-10, 10-30, 30-60 cm) and time (1 month prior to fungicide application and respectively 1 week, 5 weeks, and 4 months afterwards). Soil analyses comprised fungicides, fusarium mycotoxins (deoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, and zearalenone), ergosterol, soil microbial carbon and nitrogen, soil organic carbon, dissolved organic carbon, and pH. Fludioxonil and cyprodinil concentrations were higher under SC than under PC 1 week and 5 weeks after fungicide application (up to three times in the topsoil) but no differences were observed anymore after 4 months. Fenhexamid was not detected, presumably because of its fast dissipation in soil. The higher fludioxonil and cyprodinil concentrations under SC strongly reduced the fungal biomass and shifted microbial community towards larger bacterial fraction in the topsoil and enhanced the abundance and concentration of deoxynivalenol and 15-acetyldeoxynivalenol 5 weeks after fungicide application. Independent from the different fungicide concentrations, the decomposition of SOM was temporarily reduced after fungicide application under both coverage types. However, although PC and SC caused different concentrations of fungicide residues in soil, their impact on the investigated soil parameters was minor and transient (< 4 months) and hence not critical for soil quality.

Fenhexamid induces cancer growth and survival via estrogen receptor-dependent and PI3K-dependent pathways in breast cancer models.

Fenhexamid (Fen), a fungicide used to treat gray mold of fruits and vegetables, is reported to function as an endocrine disrupting chemical via the estrogen receptors (ER), despite low-toxicity of the pesticide. In this study, we elucidated that the disrupting effects of Fen are exerted via the ER and phosphatidylinositol 3-kinase (PI3K) pathways in breast cancer models. The WST assay, live cell monitoring, cell cycle analysis, colony formation assay, apoptotic analysis by JC-1 dyeing, and Western blot analysis were applied in ER positive MCF-7 and ER negative MDA-MB-231 breast cancer cells, after exposure to 17ß-estradiol (E2), Fen, ICI 182,780 (ICI; an ER antagonist) and/or Pictilisib (Pic; a PI3K inhibitor). Exposure to E2 and Fen induced the cell growth and survival ability of MCF-7 cells by increasing the S-phase cells and regulating the cell cycle-related proteins (Cyclin D1 and E1, p21 and p27). In addition, E2 and Fen treatment resulted in elevated levels of the survival-related proteins (Survivin and PCNA), and inhibited apoptosis by increasing the mitochondrial membrane potential and regulating the apoptosis-related proteins (BAX, BCL-2, and Caspase-9). These changes were reversed to the same level as the control group when exposed to their respective inhibitors, thereby indicating that the changes are exerted via the ER and PI3K pathways. In particular, co-treatment with these inhibitors induced greater inhibition than single treatment. Conversely, no alterations were observed in the ER-negative MDA-MB-231 breast cancer cells. Taken together, these results indicate that Fen promotes the growth of breast cancer cells via the ER and/or PI3K pathways, similar to the E2 mechanism. Although a relatively safe pesticide, Fen possibly exerts its influence as an endocrine disrupting chemical in ER-positive breast cancer cells via the ER and PI3K pathways.

Rapid monitoring of fungicide fenhexamid residues in selected berries and wine grapes by square-wave voltammetry at carbon-based electrodes.

A completely new electroanalytical method for the determination of fenhexamid (FNX) residues in fruit samples has been developed. This method is based on anodic oxidation of fungicide in Britton-Robinson buffer (pH 4) containing 10% (v/v) methanol using square-wave voltammetry when five different carbon-based electrodes were tested. An electrochemical behaviour of FNX was studied on a glassy carbon electrode using cyclic voltammetry, while glassy carbon paste electrode was selected for analytical purposes. Linear range for FNX from 3.96 to 49.50 µmol L-1 characterized by coefficient of determination of 0.9964, sensitivity of 0.176 µA L µmol-1, and detection limit of 1.32 µmol L-1 were calculated. Results acquired from analyses of blueberries and wine grapes were compared to those obtained by a reference chromatographic method, and a satisfactory agreement has been reached. Finally, it seems that the present voltammetric approach could find its application in food quality control as screening assay.

Characterization of the Populations of Botrytis cinerea Infecting Plastic Tunnel-Grown Strawberry and Tomato in the Hubei Province of China.

A total of 707 isolates of Botrytis were collected from plastic tunnel-grown strawberry and tomato in the Hubei province of China. They were identified based on the specific molecular markers. Diversity of the B. cinerea (Bc) isolates was evaluated by typing the transposable elements (Boty, Flipper) and the mating types (MAT1-1, MAT1-2), as well as by determining virulence on tobacco (Nicotiana benthamiana) and fenhexamid sensitivity in agar medium. The results showed that 706 isolates (99.9%) were Bc and 1 isolate (0.1%) was B. pseudocinerea. The Bc isolates (n = 706) were classified into four transposable element types, Vacuma (3.1%), Boty (9.6%), Flipper (18.4%), and Transposa (68.8%). The strawberry and tomato subpopulations of Bc had significantly different (P < 0.05) compositions of the four transposable element types. The overall ratio of MAT1-1 to MAT1-2 deviated from 1:1 (n = 706; P = 0.0002), and MAT1-2 (56.9%) predominated over MAT1-1 (43.1%). In 7 of 12 geographic subpopulations, the ratio of MAT1-1 to MAT1-2 matched 1:1; however, in the remaining five geographic subpopulations, the ratio of MAT1-1 to MAT1-2 did not match 1:1. Results of the biological characterizations showed that most Bc isolates were highly sensitive or sensitive to fenhexamid, and the majority of Bc isolates were highly virulent or virulent on tobacco. Moreover, the relationship between genetic diversity and biological characteristics was analyzed. The results achieved during this study are helpful for understanding of the populations of B. cinerea.

Evaluation of Callus Cultures to Elucidate the Metabolism of Tebuconazole, Flurtamone, Fenhexamid, and Metalaxyl-M in Brassica napus L., Glycine max (L.) Merr., Zea mays L., and Triticum aestivum L.

Plant cell cultures can be used to identify the metabolic degradation of pesticides in crops. Therefore, Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Triticum aestivum L. were used to elucidate the metabolic degradation of the following pesticides: tebuconazole, flurtamone, fenhexamid, and metalaxyl-M. Callus cultures were treated with 10 µM of the named pesticides by passive diffusion out of the nutrition agar while young plants were hydroponically exposed to it. After 14 days, the comparison of in planta and in vitro experiments showed that the metabolic degradation is well described by in vitro callus cultures. The intracellular uptake of all pesticides and a broad spectrum of exemplarily hydroxylated and conjugated metabolites were detectable. Overall, the comparability of the nature of residues out of both experiments with the regulatory guideline metabolism studies could be demonstrated. Therefore, we recommend it as a potential screening tool to elucidate the metabolism of pesticides in crops.

Interaction of Metamitron and Fenhexamid with Ca2+ -Montmorillonite Clay Surfaces: A Density Functional Theory Molecular Dynamics Study.

Metamitron (Meta), an herbicide, and fenhexamid (Fen), a fungicide, are authorized by the European Union to be used in agriculture. This article reports theoretical calculations about Meta and Fen in interaction with a clay surface: a Ca-montmorillonite (Mont). Conformational searches have been performed thanks to Car-Parrinello molecular dynamics simulations from which geometries have been extracted. Interaction and adsorption energies have been calculated for isomers of Meta or Fen in interaction with Mont to understand the relative stability of various kinds of complexation. Substantial adsorption energies are comparable for Meta and Fen: around -40 kcal/mol. For Fen-Mont, the CO monodentate family is surprisingly the lowest in energy. Moreover, the 10 lowest-energy isomers involve complexation on Fen carbonyl oxygens. The Meta-Mont lowest-energy family, N-N, does not involve π delocalization breaking within Meta. At the same time, the stronger the interaction energy is, the larger the structural modifications within Mont are, particularly concerning the interacting cation distance to the surface. The non-negligible charge transfer and the magnitude of the adsorption energy speak in favor of the chemisorption of the pesticide on the surface. © 2019 Wiley Periodicals, Inc.

Transcriptome and Resistance-Related Genes Analysis of Botrytis cinerea B05.10 Strain to Different Selective Pressures of Cyprodinil and Fenhexamid.

The pathogen Botrytis cinerea is a very dangerous pathogen that infects many economically important crops such as grape, strawberry, tomato, and eggplant. Cyprodinil, a pyrimidine amine fungicide, and fenhexamid, an amide fungicide, are new reagents for controlling gray mold with special efficacy. It is necessary to understand the change trends in the toxicological and physiological characteristics of B. cinerea with successive selective pressures of cyprodinil and fenhexamid to elongate the serving life of these fungicides for effective disease control. The toxicities of cyprodinil and fenhexamid at successive concentrations of EC25, EC50 and EC75 on B. cinerea strain BO5.10 were assayed along with mycelial growth-inhibition capacity. The results showed that the EC50 value of the cyprodinil-treated F27 strain increased approximately 18-fold, whereas of which in the fenhexamid-treated F27 strain increased only 3-fold compared with that of the F0 strain. The conductivities and glycerinum contents of the strains resistant to cyprodinil and fenhexamid were obviously enhanced; in contrast, the oxalic acid contents were decreased compared with those in the F0 strain. The transcriptomes of the F27 control (T01), cyprodinil-treated (T02) and fenhexamid- treated (T03) strains were analyzed, and the expression levels of functional genes in the T02 and T03 strains were significantly increased compared with those in the T01 strain; these results were further validated using qRT-PCR. The results indicated that the relative expression of two genes encoding mixed-functional oxidases (MFOs) BC1G_16062 and BC1G_16084, two genes encoding transmembrane proteins BC1G_12366 and BC1G_13768, two genes encoding Zinc finger proteins BC1G_13764 and BC1G_10483,one gene encoding citrate synthase enzyme BC1G_09151, one gene encoding gluconolactonase BC1G_15612 in the T02 and T03 strains and one gene encoding lysophospholipids enzyme BC1G_04893 in the T3 strain increased substantially compared with that in the T1 strain (P < 0.01). Functional prediction analysis of upregulated gene expression and structural verification was also performed, and the results showed that BC1G_10483 was a ZnF_C2HC transcriptional regulator interacting with the Sp1 element of these genes to respond to the pressures from cyprodinil and fenhexamid. Our results could contribute to a better understanding of the resistance mechanism of B. cinerea against cyprodinil and fenhexamid.

Elucidation of intermolecular interaction of bovine serum albumin with Fenhexamid: A biophysical prospect.

Fenhexamid, as a hydroxyanilide, is widely applied to control Botrytis cinerea for protecting crops and fruits. But it could adversely affect human and animals health due to accumulation of residues in food production. Here, the affinity characteristics of fenhexamid on bovine serum albumin (BSA) was studied via a series of spectroscopic methods such as steady-state fluorescence spectroscopy, ultraviolet spectroscopy (UV), synchronous fluorescence spectroscopy (SFS), 3D fluorescence spectroscopy, and fourier transform infrared spectroscopy (FT-IR). The experimental results illustrated that the fluorescence quenching mechanism of BSA induced by fenhexamid was a static quenching. The binding constant (Kb) of fenhexamid with BSA was 2.399 × 104 M-1 at 298 K and the combination ratio was about 1:1. The competitive experiment demonstrated that fenhexamid was binding on the BSA at site II (subdomain IIIA), which was confirmed by the molecular docking studies. The negative values of thermodynamic parameter (ΔH0, ΔS0 and ΔG0) revealed that the reaction of fenhexamid with BSA could proceed spontaneously, the van der Waals force and hydrogen bonding interaction conducted the main effect, and the binding process was enthalpy-driven. What's more, the 8-Anilino-1-naphthalenesulfonate (ANS) and sucrose binding studies were also performed and further verified the binding force between BSA and fenhexamid.

The role of fenhexamid on the proliferation of ovarian cancer BG-1 cells.

To study the influence of fenhexamid in pesticide residue to the human ovarian cancer BG-1 cell proliferation. Detecting the effectiveness of 17ß-estradiol, fenhexamid and Fulvestrant to BG-1 cell proliferation by MTT, and detecting the expression levels of cyclin D1 and cyclin E by Western blot. Fenhexamid can promote BG-1 cell proliferation for its estrogen-like effect. On the other hand, it can help to improve the expression levels of cyclin D1 and cyclin E in BG-1 cells which is regulated by ER-dependent pathway. And 17ß-estradiol is also regulated by the same way. The existence of fenhexamid can promote ovarian cancer cell proliferation, so for patients with ovarian cancer, fenhexamid in pesticide residue may make medical conditions worse.

Modification of the existing maximum residue levels for fenhexamid in various crops.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Bayer CropScience SAS submitted a request to the competent national authority in Italy to modify the existing maximum residue levels (MRL) for the active substance fenhexamid in plums, blueberries, cranberries, currants, gooseberries and beans with pods. The data submitted in support of the request were found to be sufficient to derive MRL proposals for all crops under consideration. Adequate analytical methods for enforcement are available to control the residues of fenhexamid in plant matrices. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of fenhexamid according to the reported agricultural practices is unlikely to present a risk to consumer health.

A new transformant selection system for the gray mold fungus Botrytis cinerea based on the expression of fenhexamid-insensitive ERG27 variants.

The gray mold fungus Botrytis cinerea features a wide host range and causes severe economic losses, making it an important object for molecular research. Thus far, genetic modification of the fungus mainly is relied on two selection systems (nourseothricin and hygromycin), while other selection systems hold significant disadvantages. To broaden the spectrum of available molecular tools, a new selection system based on the cheap and widely used fungicide fenhexamid (hydroxyanilide group) was established. Fenhexamid specifically targets the 3-ketoreductase ERG27 from the ergosterol biosynthesis pathway. We generated a set of expression vectors suitable for deletion or expression of genes of interest (GOIs) in B. cinerea based on fenhexamid-insensitive ERG27 variants. Expression of BcERG27F412I and Fusarium fujikuroi ERG27 in the sensitive B. cinerea strain B05.10 causes resistance towards fenhexamid (fenR) and allows for the selection of transformants and their genetic purification. A modified split-marker approach facilitates the site-specific integration and expression of GOIs at the bcerg27 locus. No undesired secondary phenotypes regarding virulence, stress responses, the formation of reproductive structures or conidial germination were observed in strains expressing fenhexamid-insensitive ERG27 variants. Thus, the fenR system represents a third reliable selection system for genetic modifications of fenhexamid-sensitive B. cinerea strains.

Preharvest Interval Periods and their relation to fruit growth stages and pesticide formulations.

The aim of this study was to evaluate the effect of pesticide formulations and fruit growth stages on the Pre-harvest Interval Period (PHI). Results showed that pesticide formulations did not affect the initial deposit and dissipation rate. However, the fruit growth stage at the application time showed a significant effect on the above-mentioned parameters. Fruit diameter increases in one millimeter pesticide dissipation rates were reduced in -0.033mgkg-1day-1 (R2=0.87; p<0.001) for grapes and -0.014mgkg-1day-1 (R2=0.85; p<0.001) for apples. The relation between solar radiation, air humidity and temperature, and pesticide dissipation rates were dependent on fruit type. PHI could change according to the application time, because of the initial amount of pesticide deposit in the fruits and change in the dissipation rates. Because Maximum Residue Level are becoming more restrictive, it is more important to consider the fruit growth stage effects on pesticide when performing dissipation studies to define PHI.