Monitoring Corynespora cassiicola Resistance to Boscalid, Trifloxystrobin, and Carbendazim in China.

Cucumber leaf spot (CLS), caused by Corynespora cassiicola, is a serious disease of greenhouse cucumbers. With frequent use of existing fungicides, C. cassiicola has developed resistance to some of them, with serious implications for the control of CLS in the field. With a lack of new fungicides, it is necessary to use existing fungicides for effective control. Therefore, this study monitored the resistance of C. cassiicola to three commonly used and effective fungicides, boscalid, trifloxystrobin, and carbendazim, from 2017 to 2021. The frequency of resistance to boscalid showed an increasing trend, and the highest frequency was 85.85% in 2020. The frequency of resistance to trifloxystrobin was greater than 85%, and resistance to carbendazim was maintained at 100%. Among these fungicides, strains with multiple resistance to boscalid, trifloxystrobin, and carbendazim were found, accounting for 32.00, 25.25, 33.33, 43.06, and 37.24%, respectively. Of the strains that were resistant to boscalid, 87% had CcSdh mutations, including seven genotypes: B-H278L/Y, B-I280V, C-N75S, C-S73P, D-D95E, and D-G109V. Also, six mutation patterns of the Ccß-tubulin gene were detected: E198A, F167Y, E198A&M163I, E198A&F167Y, M163I&F167Y, and E198A&F200C. Detection of mutations of the CcCytb gene in resistant strains showed that 98.8% were found to have only the G143A mutation. A total of 27 mutation combinations were found and divided into 14 groups for analysis. The resistance levels differed according to genotype. The development of genotypes showed a complex trend, increasing from 4 in 2017 to 13 in 2021 and varying by region. Multiple fungicide resistance is gradually increasing. Therefore, it is necessary to understand the types of mutations and the trend of resistance to guide the use of fungicides to achieve disease control.

Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

Co-exposure to boscalid and amoxicillin inhibited the degradation of boscalid and aggravated the threat to the earthworm.

The extensive application of pesticides and antibiotics in agricultural production makes it possible for them to coexist in farmland, and the interaction of the two pollutants can lead to changes in environmental behavior and toxicity, creating uncertainty risks to soil and soil organisms. In this study, we explored the environmental behavior and the effects of earthworms under co-exposure to amoxicillin and boscalid and further explored the accumulation and toxic effects on earthworms. The results showed that amoxicillin increased the adsorption of boscalid in soil and inhibited its degradation. In addition, we noticed that the co-exposure of amoxicillin and boscalid caused intestinal barrier damage, which increased the bioaccumulation of earthworms for boscalid and led to more severe oxidative stress and metabolic disorders in earthworms. In summary, our findings indicate that amoxicillin can increase the ecological risk of boscalid in the environment and imply that the encounter between antibiotics and pesticides in the environment can amplify the toxic effects of pesticides, which provides new insights into the ecological risks of antibiotics.

Resistance of the Ginseng Gray Mold Pathogen, Botrytis cinerea, to Boscalid and Pyraclostrobin Fungicides in China.

Gray mold caused by Botrytis cinerea severely threatens the yield of ginseng (Panax ginseng). Various categories of fungicides have been utilized to control gray mold on this crop. In this study, the resistance of 102 isolates of B. cinerea from 11 commercial ginseng-growing regions in China to fungicides was examined. A total of 32.4% were resistant to boscalid, with EC50 values that ranged from 12.26 to 235.87 µg/ml, and 94.1% were resistant to pyraclostrobin, with EC50 values that ranged from 5.88 to 487.72 µg/ml. Except for sdhA and sdhD, the amino acid substitutions of P225F, P225L, N230I, H272Y, and H272R in the sdhB subunit from 24 (4 sensitive [S] and 20 resistant [R]), 5 (1 S and 4 R), 1 (S), 1 (R), and 8 (4 S and 4 R) strains, respectively, and the concurrent amino acid substitutions of G85A + I93V + M158V + V168I in the sdhC subunit from 5 (4 S and 1 R) strains were identified. A G143A substitution in cytochrome b was identified in 96 isolates that were resistant to pyraclostrobin and three that were sensitive to it. The Bcbi-143/144 intron was identified in the other three isolates sensitive to pyraclostrobin, but it was absent in the isolates that harbored the G143A mutation. The results showed that the populations of B. cinerea on ginseng have developed strong resistance to pyraclostrobin. Therefore, it is not recommended to continue using this fungicide to control gray mold on P. ginseng. Boscalid is still effective against most isolates. However, to prevent fungicide resistance, it is recommended to use a mixture of boscalid with other categories of fungicides.

Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

Ternary Mixture of Azoxystrobin, Boscalid and Pyraclostrobin Disrupts the Gut Microbiota and Metabolic Balance of Honeybees (Apis cerana cerana).

There is a growing risk of pollinators being exposed to multiple fungicides due to the widespread use of fungicides for plant protection. A safety assessment of honeybees exposed to multiple commonly used fungicides is urgently required. Therefore, the acute oral toxicity of the ternary mixed fungicide of ABP (azoxystrobin: boscalid: pyraclostrobin = 1:1:1, m/m/m) was tested on honeybees (Apis cerana cerana), and its sublethal effect on foragers' guts was evaluated. The results showed that the acute oral median lethal concentration (LD50) of ABP for foragers was 12.6 µg a.i./bee. ABP caused disorder of the morphological structure of midgut tissue and affected the intestinal metabolism; the composition and structure of the intestinal microbial community was perturbed, which altered its function. Moreover, the transcripts of genes involved in detoxification and immunity were strongly upregulated with ABP treatment. The study implies that exposure to a fungicide mixture of ABP can cause a series of negative effects on the health of foragers. This work provides a comprehensive understanding of the comprehensive effects of common fungicides on non-target pollinators in the context of ecological risk assessment and the future use of fungicides in agriculture.

Selected Fungicides as Potential EDC Estrogenic Micropollutants in the Environment.

An increasing level of pesticide exposition is being observed as a result of the consumption of large amounts of fruits, vegetables and grain products, which are key components of the vegetarian diet. Fungicides have been classified as endocrine-disrupting compounds, but their mechanisms of action have not yet been clarified. The effect of boscalid (B), cyprodinil (C) and iprodione (I) combined with Tamoxifen (T) and 17ß-estradiol (E2) on cell viability, cell proliferation, reporter gene expression, ROS content, the cell membrane's function, cell morphology and antioxidant enzymes gene expression in MCF-7 and T47D-KBluc cell lines were investigated. The cell lines were chosen due to their response to 17ß -estradiol. The selected fungicides are commonly used in Poland to protect crops against fungi. Our results revealed that the studied fungicides caused significant increases in cell viability and proliferation, and estrogenic activity was present in all studied compounds depending on their concentrations. Oxidative stress activated uncontrolled cancer cell proliferation by inducing ROS production and by inhibiting antioxidant defense. Our findings verify that the studied fungicides could possibly exhibit endocrine-disrupting properties and exposure should be avoided.

Risk and molecular mechanisms for boscalid resistance in Penicillium digitatum.

The succinate dehydrogenase inhibitor (SDHI) fungicide boscalid is an excellent broad-spectrum fungicide but has not been registered in China to control Penicillium digitatum, the causal agent of green mold of citrus. The present study evaluated the risk and molecular mechanisms for boscalid resistance in P. digitatum. Resistance induction with four arbitrarily selected sensitive isolates of P. digitatum by ultraviolet (UV) irradiation on conidia plated on boscalid-amended potato dextrose agar (PDA) and consecutive growing on boscalid-amended PDA produced five highly resistant isolates with EC50 values greater than 1000 µg/mL and two resistant isolates with EC50 lower than 200 µg/mL. Boscalid resistance of the five mutants with EC50 values above 1000 µg/mL was stable after successive transfers on PDA for 16 generations. However, for the other two mutants with EC50 lower than 200 µg/mL, the EC50 values decreased significantly after successive transfers. There was significant cross-resistance between boscalid and carboxin (r = 0.925, P < 0.001), but no significant cross-resistance was detected between boscalid and fludioxonil (r = 0.533，P = 0.095) or between boscalid and prochloraz (r = -0.543，P = 0.088). The seven resistant mutants varied greatly in the mycelia growth, sporulation, pathogenicity, and sensitivities to exogenous stresses including NaCl, salicylhydroxamic acid (SHAM), and H2O2. Alignment of the deduced amino acid sequence showed that there was no point mutation in the target enzyme succinate dehydrogenase (Sdh) subunits SdhA, SdhC, or SdhD in each of the seven resistant mutants, and the mutation of a conserved histidine residue to tyrosine (H243Y) in the subunit SdhB (i.e., iron­sulfur protein) occurred in only three highly resistant isolates. Molecular docking indicated that mutation H243Y could not prevent the binding of boscalid into the quinone-binding site of SDH in the presence of the heme moiety. However, for SDH without the heme moiety, boscalid could bind into a deeper site with a much higher affinity, and the mutation H243Y spatially blocked the docking of boscalid into the deeper site. This may be the molecular mechanism for boscalid resistance caused by SdhB-H243Y mutation.

Fungicide Sensitivity of Colletotrichum Species Causing Bitter Rot of Apple in the Mid-Atlantic U.S.A.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed ß-tubulin E198A mutations, and two (<1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic U.S.A. (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and 13 fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the eastern U.S.A. suggested that EC25 values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC50 values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.

Dissipation Residue Behaviors and Dietary Risk Assessment of Boscalid and Pyraclostrobin in Watermelon by HPLC-MS/MS.

Fungicides containing active ingredients of boscalid and pyraclostrobin have been widely applied in watermelon disease control. To provide data for avoiding health hazards caused by fungicides, we investigated its terminal residues and evaluated the dietary risk. In this work, watermelon samples were collected from field sites in six provinces and analyzed with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The average recoveries of boscalid and pyraclostrobin in the watermelon matrix were 97-108% and 93-103%, respectively, with the relative standard deviations (RSDs) ≤ 9.1%. The limits of quantifications (LOQs) were 0.01 and 0.005 mg/kg for boscalid and pyraclostrobin. Twenty-one days after applying the test pesticide with 270 g a.i./ha, the terminal residues of boscalid and pyraclostrobin were all below 0.05 mg/kg and below the maximum residue limits (MRLs) recommended by European Food Safety Authority (EFSA). According to the national estimated daily intake (NEDI), the risk quotients (RQs) of boscalid and pyraclostrobin were 48.4% and 62.6%, respectively. That indicated the pesticide evaluated in watermelon exhibited a low dietary risk to consumers. All data provide a reference for the MRL establishment of boscalid in watermelon for China.

A Method for the Examination of SDHI Fungicide Resistance Mechanisms in Phytopathogenic Fungi Using a Heterologous Expression System in Sclerotinia sclerotiorum.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

Development of Boscalid Resistance in Botrytis cinerea and an Efficient Strategy for Resistance Management.

Among succinate dehydrogenase inhibitors, only boscalid has been registered in China for controlling gray mold. In Shandong Province of China, it has been more than a decade since the first use of boscalid to control gray mold. In the current study, we monitored the resistance development process of Botrytis cinerea to boscalid, identified the mutation types that occurred in boscalid-resistant isolates, and proposed an original application technique to delay resistance development. A total of 720 B. cinerea isolates collected from tomato and cucumber in Shandong Province from 2014 to 2019 were determined to be sensitive to boscalid. The results showed that the sensitivity of the B. cinerea isolates to boscalid declined gradually over time, with a mean half maximal effective concentration of 0.3 ± 0.02 mg/liter in 2014 and 6.39 ± 1.66 mg/liter in 2019. The proportion of resistant isolates quickly increased from 0.81% in 2014 to 28.97% in 2019. Mutations of P225F, N230I, H272Y, and H272R in the SdhB subunit were responsible for boscalid resistance. Four concurrent mutations (G85A, I93V, M158V, and V168I) in the SdhC subunit were first discovered in Shandong Province, but they did not affect the level of boscalid resistance. Interestingly, this study found that the fruit dipping application, a precise topical application technique, could delay the development of boscalid resistance. Therefore, this application technique provides a new method for resistance management of B. cinerea.

Resistance to Boscalid in Botrytis cinerea From Greenhouse-Grown Tomato.

Gray mold, caused by the fungus Botrytis cinerea Pers ex Fr., is one of the most destructive spoilage diseases, severely affecting tomato production in Henan Province, China. Spraying fungicides from the flowering to the harvest stage is a necessary measure to reduce losses associated with B. cinerea infection. However, B. cinerea has developed resistance to fungicides in many countries. Boscalid is a succinate dehydrogenase inhibitor (SDHI) fungicide and was registered for the control of gray mold. In this study, a total of 269 B. cinerea isolates were collected from tomato in commercial greenhouses in different locations of Henan Province in 2014 and 2015. The sensitivity and resistance of B. cinerea field isolates were determined based on mycelial growth. The effective concentration 50 ranged from 0.11 to 15.92 µg/ml and 0.16 to 8.54 µg/ml, in 2014 and 2015, respectively. The frequency of low resistance to boscalid was 12.6 and 7.6%, and moderate resistance was 2.7 and 1.3% in 2014 and 2015, respectively. No highly resistant isolates were found in Henan Province, China. Mycelial growth, mycelial dry weight, spore production, and pathogenicity were not significantly different between resistant and sensitive phenotypes of the B. cinerea isolates. The results of cross-resistance testing showed no correlation between boscalid and carbendazim, procymidone, pyrimethanil, fluazinam, or fluopyram. In this study, the succinate dehydrogenase genes B (sdhB), C (sdhC), and D (sdhD) were analyzed and compared in sensitive and low-resistance and moderately resistant B. cinerea isolates to boscalid. Results showed that point mutations occurred simultaneously at sdhC amino acid positions 85 (G85A), 93 (I93V), 158 (M158V), and 168 (V168I) in 4 out of 10 sensitive isolates and 23 of 26 low-resistance and 5 of 5 moderately resistant B. cinerea isolates to boscalid. No point mutations were found in the sdhB and sdhD genes of all isolates. Furthermore, no point mutations were found in sdhB, sdhC, and sdhD genes in 3 of 26 low-resistance B. cinerea isolates to boscalid. Therefore, we speculate that the simultaneous point mutations in the sdhC gene may not be related to the resistance of B. cinerea to boscalid. These results suggested that there might be a substitution mechanism for the resistance of B. cinerea to the SDHI fungicide boscalid.

SDHI Fungicide Toxicity and Associated Adverse Outcome Pathways: What Can Zebrafish Tell Us?

Succinate dehydrogenase inhibitor (SDHI) fungicides are increasingly used in agriculture to combat molds and fungi, two major threats to both food supply and public health. However, the essential requirement for the succinate dehydrogenase (SDH) complex-the molecular target of SDHIs-in energy metabolism for almost all extant eukaryotes and the lack of species specificity of these fungicides raise concerns about their toxicity toward off-target organisms and, more generally, toward the environment. Herein we review the current knowledge on the toxicity toward zebrafish (Brachydanio rerio) of nine commonly used SDHI fungicides: bixafen, boscalid, fluxapyroxad, flutolanil, isoflucypram, isopyrazam, penthiopyrad, sedaxane, and thifluzamide. The results indicate that these SDHIs cause multiple adverse effects in embryos, larvae/juveniles, and/or adults, sometimes at developmentally relevant concentrations. Adverse effects include developmental toxicity, cardiovascular abnormalities, liver and kidney damage, oxidative stress, energy deficits, changes in metabolism, microcephaly, axon growth defects, apoptosis, and transcriptome changes, suggesting that glycometabolism deficit, oxidative stress, and apoptosis are critical in the toxicity of most of these SDHIs. However, other adverse outcome pathways, possibly involving unsuspected molecular targets, are also suggested. Lastly, we note that because of their recent arrival on the market, the number of studies addressing the toxicity of these compounds is still scant, emphasizing the need to further investigate the toxicity of all SDHIs currently used and to identify their adverse effects and associated modes of action, both alone and in combination with other pesticides.

The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines.

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

Exogenous 24-epibrassinolide activates detoxification enzymes to promote degradation of boscalid in cherry tomatoes.

BACKGROUND: Boscalid is often used to extend the storage time of postharvest cherry tomato. Pesticide residue has become an issue of food safety. This study sought to investigate the spatial distribution of boscalid residue in cherry tomato fruits and to determine the effect of 24-epibrassinolide (EBR) in promoting boscalid degradation. RESULTS: Boscalid could quickly penetrate into cherry tomatoes, but mainly remained in the peel. The migration of boscalid from the peel into the core was a time-consuming and complex process during storage. After 72 h, boscalid residues in the pulp and the core began to accumulate gradually. The exogenous application of EBR activated peroxidase, glutathione reductase and glutathione S-transferase, and effectively promoted the degradation of boscalid by a maximum decrease of 44.8% in peel, 54.0% in pulp and 71.2% in core. CONCLUSION: As one of the common pesticides, boscalid had a strong ability to enter the cherry tomato and thus become a potential risk for public consumption. Therefore, rational use of pesticides is recommended. The results of this study indicate that the possible risk of boscalid residue could be alleviated by EBR pretreatment through activating detoxification enzymes. © 2020 Society of Chemical Industry.

Activity of the Novel Succinate Dehydrogenase Inhibitor Fungicide Pydiflumetofen Against SDHI-Sensitive and SDHI-Resistant Isolates of Botrytis cinerea and Efficacy Against Gray Mold.

Succinate dehydrogenase inhibitor (SDHI) fungicides are currently the most frequently used fungicides for controlling gray mold. However, isolates of Botrytis cinerea resistant to SDHI fungicides have emerged in the field. Pydiflumetofen is a new SDHI fungicide that can control a variety of fungal diseases, but its efficacy against gray mold and whether the activity of pydiflumetofen is affected by the current SDHI-resistant isolates is currently unknown. The sensitivity of 291 single-spore B. cinerea isolates collected from 2017 to 2019 to pydiflumetofen was determined by spore germination inhibition assays. The mean EC50 value (fungicide concentration resulting in a 50% inhibition compared with that of the control) of pydiflumetofen was 0.06 ± 0.01, 0.07 ± 0.02, and 0.05 ± 0.02 mg/liter in 2017, 2018, and 2019, respectively. There was no significant difference in the sensitivity of B. cinerea to pydiflumetofen among the 3 years. Furthermore, pydiflumetofen at 300 mg/liter effectively controlled gray mold on cucumber leaves (80.9%), and its efficacy was superior to that of boscalid at 400 mg/liter (42.7%). The isolates carrying P225F, N230I, H272Y, and H272R mutations in the SdhB subunit were associated with the less sensitivity of B. cinerea to SDHI fungicides. After establishing the baseline sensitivity of B. cinerea to pydiflumetofen (EC50 of 0.03 ± 0.003 mg/liter), we found that the P225F and H272Y mutant isolates showed low to moderate levels of resistance to pydiflumetofen, and the H272R and N230I mutant isolates showed low levels of resistance. The reduced sensitivity to pydiflumetofen resulted from the positive correlation of pydiflumetofen with the other four SDHI fungicides (i.e., boscalid, fluopyram, isopyrazam, and benzovindiflupyr). These results suggest that pydiflumetofen provides effective control for the management of gray mold but must be used with caution.

Occurrence and Extent of Boscalid Resistance in Populations of Alternaria alternata from California Pistachio Orchards.

Alternaria late blight (ALB) caused by Alternaria spp. is an annual disease problem in California pistachio and requires repeated applications of fungicides to prevent significant losses of pistachio foliage and nut quality. From 2003 onward, the succinate dehydrogenase inhibiting fungicide boscalid has played a key role in ALB management. The development of boscalid resistance in A. alternata populations was monitored from 2005 to 2012 in pistachio producing areas in California. A total of 1,765 single-spore isolates, collected from commercial and experimental pistachio orchards with or without a history of boscalid exposure, were tested in a radial growth assay in agar media amended with the discriminatory dose of 10 µg/ml of boscalid. The frequency of boscalid-resistant isolates in 2005 was 12% but increased significantly and remained stable toward the end of the survey period. Most of the resistant isolates exhibited a high level of resistance (R) to boscalid with percent of mycelial growth inhibition (PGI) values between 0 and 50%, whereas significantly fewer isolates had an intermediate level (IR) of resistance (50 < PGI < 75%). The frequency of sensitive (S) isolates (75 < PGI < 100%) was generally the highest in orchards with no history of boscalid usage, whereas mean incidences of boscalid-resistant populations of Alternaria were 81, 92.4, 80.2, and 98%, in 2006, 2007, 2011, and 2012, respectively, in orchards that received a high number (at least three per season) of boscalid spray applications. In comparison, none to relatively low frequencies (0 to 12%) of resistance were observed in populations with no or limited exposure to Pristine, suggesting an air-movement of resistant spores through wind from treated to nontreated areas. In 2012, boscalid-resistant isolates were found practically in every sampled location in all counties, with the orchards in Fresno, Madera, Tulare, and King Counties being the locations with the highest frequencies of resistance (100%). Monitoring of A. alternata AaSDHB, AaSDHC, and AaSDHD mutations in 286 boscalid-resistant phenotypes identified 11 mutations, leading to amino acid substitutions in AaSDHB (seven mutations: H277Y/R/L, P230A/R, N235D/T), AaSDHC (one mutation: H134R), and AaSDHD (three mutations: D123E, H133R/P), with AaSDHB mutations being the most prevalent (80%) ones throughout the survey period. The majority of isolates carrying these mutations exhibited the R phenotype toward boscalid. The increased prevalence of boscalid resistance in populations of A. alternata is a likely contributing factor to the inability of pistachio farmers to successfully control ALB with Pristine. Other factors implicated in the rapid and widespread occurrence of A. alternata boscalid-resistant populations in California pistachios are further discussed.

Dissipation pattern and residual levels of boscalid in cucumber and soil using liquid chromatography-tandem mass spectrometry.

To stipulate the rationale of spraying doses and to determine the safe interval period of boscalid suspension concentrate (SC), the degradation dynamics and residual levels were investigated in cucumber and soil using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Field trials were conducted according to Chinese Guideline on pesticide residue trials. Following application, the degradation kinetics was best ascribed to first-order kinetic models with half-life of 2.67-9.90 d in cucumber. Spraying boscalid SC at 1.5-fold the recommended dosage yield terminal residues, which are clearly lower than the maximum residue limit (MRL) established by China (MRL =5 mg.kg-1) in cucumber. At variance, the dissipation dynamics in soil did not fit to first-order kinetics and the half-life was more than 17 days, the finding which denotes that the degradation behavior of boscalid in soil proceeds slowly. It has therefore been shown that boscalid is safe for use on cucumbers under the recommended dosage.

Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin.

As a managed agricultural pollinator, the western honeybee Apis mellifera frequently encounters agrochemicals as contaminants of nectar and pollen. One such contaminant, the fungicide boscalid, is applied at bloom in orchards for fungal floral pathogen control. As an inhibitor of complex II in the mitochondrial electron transport chain of fungi, boscalid can potentially interfere with high energy-demanding activities of bees, including flight. We designed an indoor flight treadmill to evaluate impacts of ingesting boscalid and/or quercetin, a ubiquitous phytochemical in bee food that also affects mitochondrial respiration. Boscalid reduced the wingbeat frequencies of foragers during flight but did not alter the duration of flight. At the colony level, boscalid ingestion may thereby affect overall health by reducing forager efficiency. The consumption of quercetin, by contrast, led to higher adenosine triphosphate levels in flight muscles and a higher wingbeat frequency. Consuming the two compounds together increased wingbeat frequency, demonstrating a hitherto unrecognized mechanism by which dietary phytochemicals may act to ameliorate toxic effects of pesticides to promote honeybee health. In carrying out this work, we also introduce two methodological improvements for use in testing for pesticide effects on flight capacity-a 'force-feeding' to standardize flight fuel supply and a novel indoor flight treadmill.