Anilinopyrimidine Fungicides Research Articles

Mitochondrial Inner Membrane ABC Transporter Bcmdl1 Is Involved in Conidial Germination, Virulence, and Resistance to Anilinopyrimidine Fungicides in Botrytis cinerea.

Botrytis cinerea causes gray mold on thousands of plants, leading to huge losses in production. Anilinopyrimidine (AP) fungicides have been applied to control B. cinerea since the 1990s. Although resistance to AP fungicides was detected soon after their application, the mechanism of AP resistance remains to be elucidated. In this study, a sexual cross between resistant and sensitive isolates was performed, and the genomes of parental isolates and progenies were sequenced to identify resistance-related single nucleotide polymorphisms (SNPs). After screening and verification, mutation E407K in the Bcmdl1 gene was identified and confirmed to confer resistance to AP fungicides in B. cinerea. Bcmdl1 was predicted to encode a mitochondrial protein that belonged to a half-type ATP-binding cassette (ABC) transporter. Although Bcmdl1 was a transporter, it did not mediate resistance to multiple fungicides but mediated resistance specifically to AP fungicides. On the other hand, reductions in conidial germination and virulence were observed in Bcmdl1 knockout transformants compared to the parental isolate and complemented transformants, illustrating the biological functions of Bcmdl1. Subcellular localization analysis indicated that Bcmdl1 was localized in mitochondria. Interestingly, the production of ATP was reduced after cyprodinil treatment in Bcmdl1 knockout transformants, suggesting that Bcmdl1 was involved in ATP synthesis. Since Mdl1 could interact with ATP synthase in yeast, we hypothesize that Bcmdl1 forms a complex with ATP synthase, which AP fungicides might target, thereby interfering with the metabolism of energy. IMPORTANCE Gray mold, caused by B. cinerea, causes huge losses in the production of many fruits and vegetables. AP fungicides have been largely adopted to control this disease since the 1990s, and the development of resistance to AP fungicides initiates new problems for disease control. Due to the unknown mode of action, information on the mechanism of AP resistance is also limited. Recently, mutations in mitochondrial genes were reported to be related to AP resistance. However, the mitochondrial process of these genes remains to be elucidated. In this study, we identified several AP resistance-related mutations by quantitative trait locus sequencing (QTL-seq) and confirmed that mutation E407K in Bcmdl1 conferred AP resistance. We further characterized the expression patterns, biological functions, subcellular localization, and mitochondrial processes of the Bcmdl1 gene. This study deepens our understanding of the mechanism of resistance to and mode of action of AP fungicides.

Site-directed transformants with E407K substitution in Bcmdl1 possesses different fitness from field anilinopyrimidine resistant isolates with E407K mutation in Botrytis cinerea

Botrytis cinerea is the causal agent of devastating disease gray mold on numerous crops worldwide. To control gray mold, anilinopyrimidine (AP) fungicides have been widely applied since the 1990s. However, the development of resistance in B. cinerea brought a new challenge to this disease control. Due to the unknown mode of action, the mechanism of AP resistance is still ambiguous. In our previous study, mutation E407K in Bcmdl1 was identified to be associated with AP resistance. Since this mutation is the major mechanism of AP resistance in our cases, it is essential to investigate the fitness of E407K strains before designing anti-resistance management strategies. Besides using field-resistant isolates with the E407K mutation, strains with E407K substitution obtained by site-directed mutagenesis were also used to estimate the specific effect of this mutation or substitution on fitness. The fitness of E407K strains were evaluated by determining mycelial growth, sporulation, conidial germination, virulence, acid production, osmotic and oxidative sensitivity, and sclerotial production and viability. Field resistant isolates with E407K mutation produced fewer sclerotia on intermediate medium (IM) but more conidia on PDA when compared with sensitive isolates, whereas site-directed transformants with E407K substitution did not show any fitness costs. The competitive ability of E407K strains was also evaluated on apple fruit using conidial mixtures at three initial ratios of resistant and sensitive isolates at 1:9, 1:1, and 9:1, respectively. Similar with fitness, impaired competitive ability was observed in field resistant isolates but not site-directed transformants at all initial ratios tested. These results indicated that field strains associated with AP resistance suffer a fitness penalty not linked directly to the E407K substitution in Bcmdl1.

Mepanipyrim induces visual developmental toxicity and vision-guided behavioral alteration in zebrafish larvae

Mepanipyrim, an anilinopyrimidine fungicide, has been extensively used to prevent fungal diseases in fruit culture. Currently, research on mepanipyrim-induced toxicity in organisms is still very scarce, especially visual developmental toxicity. Here, zebrafish larvae were employed to investigate mepanipyrim-induced visual developmental toxicity. Intense light and monochromatic light stimuli-evoked escape experiments were used to investigate vision-guided behaviors. Meanwhile, transcriptomic sequencing and real-time quantitative PCR assays were applied to assess the potential mechanisms of mepanipyrim-induced visual developmental toxicity and vision-guided behavioral alteration. Our results showed that mepanipyrim exposure could induce retinal impairment and vision-guided behavioral alteration in larval zebrafish. In addition, the grk1b gene of the phototransduction signaling pathway was found to be a potential aryl hydrocarbon receptor (AhR)-regulated gene. Mepanipyrim-induced visual developmental toxicity was potentially related to the AhR signaling pathway. Furthermore, mepanipyrim-induced behavioral alteration was guided by the visual function, and the effects of mepanipyrim on long and middle wavelength light-sensitive opsins may be the main cause of vision-guided behavioral alteration. Our results provide insights into understanding the relationship between visual development and vision-guided behaviors induced by mepanipyrim exposure.

Impact of SDH Mutations in Alternaria solani on Recently Developed SDHI Fungicides Adepidyn and Solatenol.

Early blight, caused by Alternaria solani, is observed annually in all midwestern potato production areas. The use of foliar fungicides remains a primary management strategy. However, A. solani has developed reduced sensitivity or resistance to many single-site fungicides such as quinone outside inhibitor (QoI, FRAC group 11), succinate dehydrogenase inhibitor (SDHI, FRAC group 7), demethylation inhibitor (DMI, FRAC group 3), and anilinopyrimidine (AP, FRAC group 9) fungicides. Boscalid, fluopyram, solatenol, and adepidyn are EPA-registered SDHI fungicides used commercially on a variety of crops, including potato. Five SDH mutations have been characterized previously in A. solani that affect the efficacy of boscalid while only one of these mutations has been demonstrated to negatively affect fluopyram efficacy. Conidial germination assays were used to determine if a shift in sensitivity has occurred in these SDHI fungicides. A. solani isolates collected prior to the commercial application of SDHI fungicides (baseline) were compared with recently collected isolates (nonbaseline). Greenhouse evaluations were conducted also to evaluate the efficacy of boscalid, fluopyram, solatenol, and adepidyn on A. solani isolates possessing individual SDH mutations. Additionally, field trials were conducted to determine the effects of application of these SDHI fungicides on the frequency of SDH mutations. Fluopyram, solatenol, and adepidyn had high intrinsic activity against A. solani when compared with boscalid, based on invitro assays. The application of adepidyn and solatenol resulted in greater early blight control than the application of boscalid and fluopyram in greenhouse experiments. Molecular characterization of A. solani isolates collected from the field trials determined that the frequency of the H134R-mutation can increase in response to more recently developed SDHI fungicides. In contrast, the H278R/Y- and H133R-mutations decreased to the point of being nearly absent in these field experiments.

Hygienic assessment of working conditions under use of anilinopyrimidine fungicide

Introduction. An assortment of fungicides in agriculture is constantly being updated due to creating new highly effective but moderately toxic pesticide formulations that minimize the risks for those working with them. The study aimed to research working conditions and assess the risk for workers during the application of pyrimethanil-based plant protection products in agriculture using various technologies. Materials and methods. Working conditions when using preparations based on pyrimethanil separately or in a mixture (pyrimethanil+fluopyram) in field conditions: during ground boom spraying of field crops; air blast spraying of horticultural crops; knapsack spraying of sheltered ground tomato (greenhouse), field and horticultural crops in individual subsidiary plots (PSP). Air samples of the working area were gathered in the breathing zone of those working with pesticides (operators and users) using PU-4E aspiration devices coupled with filters. Before and after work, swab samples were taken from common areas of the operator’s and user’s skin, and pyrimethanil and fluopyram were identified in air and swab samples. Total risk for the complex (inhalation and dermal) exposure (SFsum) was calculated by summing the safety factors of pesticide inhalation (SFinh) and dermal (SFd) income of pesticides (MU 1.2.3017-12). The absorbed dose risk (SFab) was determined by the ratio of the absorbed exposure dose of pesticides and the permissible daily exposure level for the operator (PDELO, mg/kg) and the acceptable daily intake (ADI, mg/kg bw). Results. The established safety factors when assessing the complex effect of pyrimethanil by exposure (SFexp - 0.08-0.11) and by absorbed dose (SFad - 0.002-0.007) allow considering the health risks for workers and consumers as permissible, subject to strict adherence to regulations and safety measures. Conclusion. The obtained results are the basis for recommending pyrimethanil-based formulations for use in agricultural production in Russia.

Pyrimethanil Sensitivity and Resistance Mechanisms in Penicillium digitatum.

Pyrimethanil is an anilinopyrimidine (AP) fungicide that is highly effective in controlling green mold caused by Penicillium digitatum but has not yet been registered in China to control postharvest diseases of citrus. In this study, baseline sensitivity of P. digitatum to pyrimethanil was established based on the effective concentrations for 50% inhibition (EC50) values of 127 isolates collected from five major citrus-growing regions of China. The distribution of these EC50 values was unimodal but with a long right tail. The mean ± SD EC50 value was 0.137 ± 0.046 μg/ml, and the minimum and maximum were 0.073 and 0.436 μg/ml, respectively. Pyrimethanil in potato dextrose agar (PDA) at 0.20 μg/ml decreased methionine production in the mycelia by 21.6% and reduced the activity of cell wall-degrading enzymes cellulase and pectinase by 9.1 and 32.8%, respectively. Twelve pyrimethanil-resistant mutants were obtained by consecutive subculturing of 12 arbitrarily selected sensitive isolates on pyrimethanil-amended PDA for four generations, and the resistance factors ranged from 69 to 3,421. There was no cross-resistance between pyrimethanil and prochloraz (r = 0.377, P = 0.123). Compared with their parental isolates, pyrimethanil-resistant mutants had reduced pathogenicity to citrus fruit but higher tolerance to hydrogen peroxide. No differences were detected in tolerance to NaCl, CaCl2, Congo red, or sodium dodecyl sulfate. The exogenous addition of methionine into PDA partially alleviated pyrimethanil toxicity to the sensitive isolates but had no significant effect on toxicity to the resistant mutants. Sequencing of cystathionine γ-synthase encoding genes CGS1 and CGS2, the potential target genes for pyrimethanil, showed that there was no nucleotide mutation in the coding region of CGS of the pyrimethanil-resistant mutants. However, the relative expression of CGS1 and CGS2 of the pyrimethanil-resistant mutants was reduced by 42.5 and 57.4%, respectively. These results have important implications for applications of pyrimethanil to control P. digitatum and for understanding the modes of action and resistance mechanisms of pyrimethanil.

Residues of anilinopyrimidine fungicides and suspected metabolites in wine samples

The coexistence of the anilinopyrimidine fungicides pyrimethanil (PYR) and cyprodinil (CYP), and suspected metabolites in wine samples was investigated by liquid chromatography (LC) with tandem mass spectrometry (MS/MS), based on triple quadrupole (QqQ) and quadrupole time-of-flight (QTOF) MS instruments. For the first time, quantitative data obtained after solid-phase extraction (SPE) of wine samples have demonstrated the systematic presence of 4-hydroxyanilino derivatives of PYR and CYP in wines containing residues of parent fungicides, at concentrations from 0.2 to 58 ng mL−1. Higher concentration ratios (hydroxylated derivative/active fungicide) were measured in red than in white wines, particularly in case of PYR. On average, the concentrations of PYR-4OH were twice those measured for PYR in red wines. A targeted search of hydroxyl derivatives in wine extracts by LC-QTOF-MS showed the existence of additional hydroxylation positions in the pyrimidine ring and/or in the alkyl substituents bond to this cycle in the structure of both anti-botrytis fungicides. Moreover, free and glycosylated forms of the hydroxylated metabolites for both fungicides coexist in wine samples. In case of CYP, it is proved that hydroxylated and glycosylated metabolites are already present in grapes before vinification.

Development and validation of an immunochromatography test strip for rapid detection of pyrimethanil residues

ABSTRACT Pyrimethanil is one of anilinopyrimidine fungicides widely used in crop protection and frequently found in a variety of crops, including fruits, vegetables, and cereals. This paper describes the development of the first reported immunochromatography test strip (ICTS) based on colloidal gold-labeled monoclonal antibody for the detection of pyrimethanil. Under optimal conditions, the visual cut-off value of ICTS for pyrimethanil was 40 ng/mL. Using a portable strip reader, the 50% inhibition concentration and calculated LOD of pyrimethanil in the PBS samples were 4.8 and 0.9 ng/mL, respectively. When used to analyse fortified samples of fruit and vegetable, acceptable recovery rates of 83.3–105.7% with the coefficient of variation values below 11.7% were obtained. Analysis of blindly fortified samples by the proposed ICTS gave similar results to those of the ic-ELISA and HPLC-MS/MS methods. These results indicate the proposed ICTS is a rapid, simple analytical method for quantitative on-site monitoring of pyrimethanil in foodstuffs.

Development of a direct competitive enzyme-linked immunosorbent assay for determination of the fungicide mepanipyrim and its metabolite.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for determination of anilinopyrimidine fungicide mepanipyrim in vegetables. Two derivatives of mepanipyrim and mepanipyrim propanol type metabolite which carried carboxy acid were synthesized and conjugated with keyhole limpet hemocyanin. BALB/c mice were immunized to prepare anti-mepanipyrim monoclonal antibodies (MoAbs) by obtained conjugates. The dc-ELISAs based on the prepared MoAbs, MPP107 and MPP204, showed working ranges between 0.12 and 1.8 ng/mL with mepanipyrim for MPP107, 0.12 and 2.4 ng/mL with mepanipyrim for MPP204, and 0.2 ng/mL and 5.7 ng/mL with the mepanipyrim propanol type for MPP204. The dc-ELISAs showed the sufficient sensitivity to determine the mepanipyrim residues for the MRLs of 1-15 mg/kg among the majority of vegetables and fruits in Japan. Recovery and/or correlation results from HPLC suggested that the dc-ELISAs would be applicable to the residue analysis of mepanipyrim and its propanol type in vegetables.

In Vitro, in Vivo and in Field Sensitivity of Venturia inaequalis to Anilinopyrimidine Fungicides with Different Types of Scab Management and Degree of Control

The sensitivity of Venturia inaequalis to anilino-pyrimidine (AP) fungicides (pyrimethanil and cyprodinil) was studied on populations sampled from apple trees with different fungicide and APs histories: 1) apple trees in uncultivated areas never exposed to fungicides (wild types), 2) commercial orchards with many years of good scab control with APs (well-controlled); 3) populations in experimental Centre where an initial good activity decreased after several years of intensive use (poorly controlled). The in vitro sensitivity tests were performed on monoconidial isolates, while those in vivo with curative or preventive applications on populations inoculated on apple seedlings. In the field, the infection on apple trees was assessed as severity of attack on leaves. The results evidenced that the sensitivity in vitro to pyrimethanil and cyprodinil showed a progressive lower reduction from wild types to well controlled, ending with poorly controlled populations. In vivo tests the same moderate decrease of sensitivity and activity (with a wide range) was more evident with curative, respect preventive applications, as noticed also with an important DMI (difenoconazole). In field trials, the populations intensively treated with APs showed, after some years, a reduction of control, that was confirmed in the following years. These activity reductions were sensibly less strong respect those evidenced by strobilurins, both in field and in vitro tests. The particular characteristic of resistance to AP fungicides is discussed.

Anilinopyrimidine Resistance in Botrytis cinerea Is Linked to Mitochondrial Function.

Crop protection anilinopyrimidine (AP) fungicides were introduced more than 20 years ago for the control of a range of diseases caused by ascomycete plant pathogens, and in particular for the control of gray mold caused by Botrytis cinerea. Although early mode of action studies suggested an inhibition of methionine biosynthesis, the molecular target of this class of fungicides was never fully clarified. Despite AP-specific resistance having been described in B. cinerea field isolates and in multiple other targeted species, the underlying resistance mechanisms were unknown. It was therefore expected that the genetic characterization of resistance mechanisms would permit the identification of the molecular target of these fungicides. In order to explore the widest range of possible resistance mechanisms, AP-resistant B. cinerea UV laboratory mutants were generated and the mutations conferring resistance were determined by combining whole-genome sequencing and reverse genetics. Genetic mapping from a cross between a resistant field isolate and a sensitive reference isolate was used in parallel and led to the identification of an additional molecular determinant not found from the characterized UV mutant collection. Together, these two approaches enabled the characterization of an unrivaled diversity of resistance mechanisms. In total, we report the elucidation of resistance-conferring mutations within nine individual genes, two of which are responsible for almost all instances of AP resistance in the field. All identified resistance-conferring genes encode proteins that are involved in mitochondrial processes, suggesting that APs primarily target the mitochondria. The functions of these genes and their possible interactions are discussed in the context of the potential mode of action for this important class of fungicides.

Parasitic Fitness of Fungicide-Resistant and -Sensitive Isolates of Alternaria solani.

Resistance to chemistries of the succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicides has developed rapidly in populations of Alternaria solani, the cause of early blight of potato. Reduced sensitivity to the anilinopyrimidine (AP) fungicide pyrimethanil has also been identified recently, determining that resistance to three chemical classes of fungicides is present within the A. solani population. Although no mutations have been characterized to confer resistance to APs, in A. solani five point mutations on three AsSdh genes have been determined to convey resistance to SDHIs, and the substitution of phenylalanine with leucine at position 129 (F129L) in the cytb gene confers resistance to QoIs. The objective of this study was to investigate the parasitic fitness of A. solani isolates with resistance to one or more of these chemical classes. A total of 120 A. solani isolates collected from various geographical locations around the United States were chosen for in vitro assessment, and 60 of these isolates were further evaluated in vivo. Fitness parameters measured were (i) spore germination in vitro, (ii) mycelial expansion in vitro, and (iii) aggressiveness in vivo. No significant differences in spore germination or mycelial expansion (P = 0.44 and 0.51, respectively) were observed among wild-type and fungicide-resistant isolates in vitro. Only A. solani isolates possessing the D123E mutation were shown to be significantly more aggressive in vivo (P < 0.0001) compared with wild-type isolates. These results indicate that fungicide-resistant A. solani isolates have no significant fitness penalties compared with sensitive isolates under the parameters evaluated regardless of the presence or absence of reduced sensitivity to multiple chemical classes. Results of these studies suggest that A. solani isolates with multiple fungicide resistances may compete successfully with wild-type isolates under field conditions.

Sensitivity of &lt;i&gt;Botrytis cinerea&lt;/i&gt; to fungicides used in New Zealand wine grape spray programmes

Fungicide resistance development in Botrytis cinerea threatens the efficacy of anilinopyrimidine (AP) fungicides, which have been used for botrytis bunch rot control in New Zealand vineyards for about 20 years. A 2016 survey of 33 vineyards in Gisborne, Hawke's Bay and Marlborough showed that the majority of 670 isolates tested in agar-based assays were sensitive to the AP cyprodinil (EC50 &lt; 1 mg/litre), although 18% showed low-resistance (EC50 1-10 mg/litre) and 10% showed medium-resistance (EC50 &gt;10 mg/litre). There was high cross-resistance to the APs cyprodinil and pyrimethanil. The survey also established baseline sensitivity to the succinate dehydrogenase inhibitor (SDHI) fungicide boscalid and &gt;90% of isolates were sensitive (EC50 &lt; 5 mg/litre). There was a low degree of cross-resistance between the SDHIs boscalid and fluopyram. There was also high sensitivity to fludioxonil (0.009 to 0.018 mg/litre) and to fenhexamid (0.49 to 0.76 mg/litre).

A class-selective immunoassay for simultaneous analysis of anilinopyrimidine fungicides using a rationally designed hapten.

The development of multianalyte immunoassays constitutes a main research issue in the field of bioanalytical techniques. In the present study, class-specific antibodies against the three members of the anilinopyrimidine family of fungicides (pyrimethanil, cyprodinil and mepanipyrim) were raised by using a bioconjugate of a rationally designed hapten [5-(6-methyl-2-(phenylamino)pyrimidin-4-yl)pentanoic acid]. Highly sensitive immunoassays were developed for the generic determination of these compounds, using the competitive enzyme-linked immunosorbent assay (ELISA). Particularly, a direct antibody-coated competitive ELISA afforded identical sensitivity for the three anilinopyrimidines, with IC50 values of 0.26, 0.27 and 0.25 μg L-1 for pyrimethanil, cyprodinil and mepanipyrim, respectively. This immunoassay was fully characterized and applied to the multianalyte determination of anilinopyrimidine fungicides in white and red wines, with a limit of quantification of 1 μg L-1, average recoveries from 93.1 to 114.4%, and relative standard deviations lower than 20%. Commercial wine samples were analyzed and those containing detectable anilinopyrimide residues were verified by a reference chromatographic technique.

Spatial and Temporal Sensitivity of Alternaria Species Associated With Potato Foliar Diseases to Demethylation Inhibiting and Anilino-Pyrimidine Fungicides.

Early blight and brown spot, caused by Alternaria solani and Alternaria alternata, respectively, are important foliar diseases of potato, affecting both tuber yield and quality. Most of the commercial cultivars lack resistance; therefore, the application of foliar fungicides remains a primary disease management strategy. Baseline sensitivities of A. solani to difenoconazole and metconazole (demethylation inhibitors) using mycelial growth assay exhibited similar intrinsic activity against the pathogen with mean EC50 (the effective concentration at which the fungal growth is inhibited by 50%) values of 0.09 μg/ml. However, the sensitivity of individual baseline A. solani isolates to each fungicide varied substantially, resulting in very low and nonsignificant correlation coefficients among fungicides. Mean EC50 values for baseline A. alternata isolates in response to difenoconazole and metconazole were 0.14 and 0.26 μg/ml, respectively. The sensitivity of the majority of A. solani and A. alternata isolates collected from 2010 to 2014 from various potato production states was consistent with baseline isolates, therefore, these potato pathogens remain sensitive to the two demethylation inhibitor chemistries used to manage it. Baseline sensitivity assays of pyrimethanil (anilino-pyrimidine) also indicated great intrinsic activity against both foliar pathogens with mean EC50 values of 0.44 and 0.35 μg/ml for A. solani and A. alternata, respectively. Although A. alternata remains largely sensitive to pyrimethanil, 6 out of 245 A. solani isolates collected from 2010 to 2014 exhibited reduced-sensitivity to the fungicide in in vitro assays. Reduced-sensitive isolates were not controlled at most pyrimethanil doses except at 100 μg/ml in greenhouse in vivo efficacy tests. These chemistries remain valuable options for fungicide rotation programs in areas of high disease pressure.

Mepanipyrim (Pesticides).

The Food Safety Commission of Japan (FSCJ) conducted a risk assessment of mepanipyrim (CAS No.110235-47-7), an anilinopyrimidine fungicide, based on results from various studies. Major adverse effects of mepanipyrim observed were hepatocellular hypertrophy, hepatocellular degeneration, and increased kidney weight in rats. Neither reproductive toxicity, teratogenicity nor genotoxicity was observed. Mepanipyrim (parent compound only) was identified as a chemical for the residue definition for dietary risk assessment in agricultural products. FSCJ adopted the no-observed-adverse-effect level (NOAEL) of 7.34 mg/kg bw/day, obtained in a two-year combined chronic/carcinogenicity study in rats, appropriate for specification of an acceptable daily intake (ADI). An ADI was thus specified as 0.073 mg/kg bw/day, applying a safety factor of 100 to the NOAEL. The lowest NOAEL for adverse effects that would be likely to be elicited by a single oral administration of mepanipyrim was 400 mg/kg bw obtained in an acute neurotoxicity study in rats. Consequently, FSCJ specified an acute reference dose (ARfD) of 4 mg/kg bw, applying a safety factor of 100 to the NOAEL.

Monoclonal antibody-based immunoassays for cyprodinil residue analysis in QuEChERS-based fruit extracts

Cyprodinil is among the most common agrochemical residues found in highly perishable fruits, such as strawberries. In the present study, high-affinity monoclonal antibodies to this anilinopyrimidine fungicide were raised for the first time with the aim to produce valuable immunochemical analytical assays. Cyprodinil bioconjugates and the generated novel monoclonal antibodies were employed for sensitive competitive immunoassay development in two different formats. The limits of detection of the optimized assays were 20 and 30ngL−1 for the indirect and direct assay, respectively. Influence over assay parameters of different physicochemical factors was studied. Strawberry samples were extracted following the recommended QuEChERS procedure for pesticide residues in food, and analyzed by the optimized immunoassays. Recoveries and coefficients of variation from fortified samples were within standard values. In addition, the obtained immunochemical results with naturally contaminated samples were statistically comparable, according to Deming regression analysis, to those of a reference chromatographic method.

Crystal structure of cyprodinil.

In the title compound, C14H15N3 (systematic name: 4-cyclo­propyl-6-methyl-N-phenyl­pyrimidin-2-amine), which is the anilino­pyrimidine fungicide cyprodinil, the dihedral angles between the planes of the central pyrimidine ring and the terminal phenyl ring and the mean plane of the cyclo­propane ring system are 14.52 (11) and 88.79 (10)°, respectively. In the crystal, weak π–π inter­actions [3.8551 (11) Å] connect the dimers into chains along the b-axis direction.

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using direct and indirect formats, achieving IC50 values around 0.15 μg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and cider as model food processed matrices. The obtained recoveries were from 90% to 120%, and the limit of quantification was in the 1–5 μg/L range. Finally, a monitoring study (n = 150) was performed to estimate the occurrence and the concentration of cyprodinil in commercial wine and cider products from different origins. We found that 28% of the analysed wine samples contained cyprodinil residues at levels higher than 5 μg/L.

Location-Specific Fungicide Resistance Profiles and Evidence for Stepwise Accumulation of Resistance in Botrytis cinerea

The fungicide resistance profiles to seven chemical classes of fungicides were investigated in 198 Botrytis cinerea isolates from five blackberry fields and 214 B. cinerea isolates from 10 strawberry fields of North and South Carolina. Populations of B. cinerea tended to have a single dominant, location-specific resistance profile that consisted of resistance to multiple fungicides in fields sprayed weekly with site-specific fungicides. The most prevalent profile in blackberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, and boscalid. The most prevalent resistance profile found in conventional strawberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. A statistical model revealed that multifungicide resistance patterns did not evolve randomly in populations from both crops. Instead, strains resistant to thiophanate-methyl were more likely to acquire resistance to pyraclostrobin, the resulting dual-resistant population was more likely to acquire resistance to boscalid, the resulting triple-resistant population was more likely to acquire resistance to cyprodinil, and the resulting quadruple-resistant population was more likely to acquire resistance to fenhexamid (strawberry population only) compared with random chance. Resistance to iprodione and fludioxonil evolved from a pool of strains with different fungicide resistance profiles. Resistance to thiophanate-methyl, pyraclostrobin, boscalid, and fenhexamid in blackberry isolates was, without exception, based on target gene mutations, including E198A and E198V in β-tubulin, G143A in cytochrome b, H272Y and H272R in SdhB, and F412I in Erg27, respectively. A new genotype associated with fenhexamid resistance was found in one strain (i.e., Y408H and deletion of P298). Fungicide-resistant strains were present but rare in an unsprayed blackberry field, where some unique phenotypes, including low and medium resistance to fludioxonil, had emerged in the absence of fungicide pressure. The isolates resistant to fludioxonil had effective dose that inhibited 50% of mycelial growth values of 0.16 μg/ml (low resistance) and 0.32 and 0.38 μg/ml (medium resistance) and were also resistant to the anilinopyrimidine fungicide cyprodinil, indicating that this and similar phenotypes will eventually be selected by continued applications of the fludioxonil + cyprodinil premixture Switch. This study shows that multifungicide-resistant phenotypes are common in conventionally maintained strawberry and blackberry fields and that resistance to multiple fungicides evolved from stepwise accumulation of single resistances.