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Gray mold is a common and severe disease that affects strawberry production and storage. To clarify the occurrence and mechanism of Botrytis cinerea in strawberry resistance to boscalid [2-Chloro-N-(4'-chlorobiphenyl-2-yl) nicotinamide], a widely used fungicide, 137 isolates of Botrytis cinerea in strawberry were collected from Beibei, Xiema, and Changshou of Chongqing by random sampling method in 2021–2022 and monitored for resistance in the field by the discriminatory dosage method. The succinate dehydrogenase (SDH) related gene of 55 resistant strains was sequenced, and molecular docking was used to predict the binding mode of boscalid to wild-type and mutant SDH of B. cinerea. The results showed that the resistance frequency of B. cinerea to boscalid in Chongqing was 93.43%. Mutation sites on the B subunit of SDH were found in 45 of the 55 resistant strains, with three primary mutation types, P225F, N230I, and H272R, responsible for 43.6%, 16.4%, and 21.8% of mutations, respectively. Out of the 55 resistant strains, five were found to have mutation sites on the C subunit of SDH. Only one mutation type, G85A+I93V+M158V+V168I, was observed in these strains, where four amino acids were mutated simultaneously. However, no mutation sites were detected in the remaining five resistant strains. Molecular docking results predicted that the P225F mutation prevented boscalid from entering the binding pocket, which affects the formation of hydrogen bonds and other interaction forces and ultimately hinders the ability of boscalid to bind to SDH. The H272R mutation narrows the cavity bottom of the binding pocket and rotates part of the boscalid chemical bond, while the N230I mutation has no effect on boscalid binding. This study clarified the occurrence and mechanism of boscalid resistance in B. cinerea in the Chongqing region, and the results provide an experimental data for formulating strawberry gray mold management strategies and also provide some insights for the development of succinic dehydrogenase inhibitors (SDHIs) fungicides. [ABSTRACT FROM AUTHOR]

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Succinate dehydrogenase inhibitors (SDHIs) are a class of broadspectrum 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. [ABSTRACT FROM AUTHOR]

Copyright of Erwerbs-Obstbau is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Erwerbs-Obstbau is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Shift of sensitivity to boscalid in Botrytis cinerea collected from plastic tunnels growing fruits and vegetables in Zhejiang Province was monitored by the mycelial growth rate assay based on our previously reported sensitivity baseline, (1.07 ± 0.11) mg/L for the wild sensitive isolates collected from 2004 to 2006. The results showed that resistance to boscalid in B. cinerea was developed rapidly. The mean EC50 was (5.23 ± 7.79) and (24.30 ± 49.33) mg/L, respectively for isolates collected during 2012 to 2013 and during 2017 to 2018. The total resistance frequency during 2012 to 2013 was 15.3% and all resistant isolates were low-level resistant (LR). For the isolates collected from 2017 to 2018, the total resistance frequency was increased to 53.2%, including 7.5% MR isolates and 1.3% HR isolates. There was no significant difference in mycelial growth, sporulation, sclerotial production, and pathogenicity between boscalid resistant and sensitive isolates. No point mutation was detected in SDH A and SDH D. However, a single point mutation, H272R, P225, or N230I, was found in SDH B of all boscalid-resistant B. cinerea. Among them, 88.5% of resistant isolates were H272R. And no obvious association was found between boscalid-resistance and mutations (G85A + I93V + M158V + V168I) in SDH C. [ABSTRACT FROM AUTHOR]

In order to evaluate the sensitivity and the resistant mechanism of Botrytis cinerea Pers. (the causal agent of gray mold disease) from strawberry to boscalid, the sensitivity of 195 isolates in 6 districts in Shanghai were tested with petri plate. And the succinate dehydrogenase subunits genes of 20 isolates with different sensitivity were sequenced. The results showed that the lowest EC50 value of mycelial growth inhibition was 0.15 μg/mL, and the highest value was over 110 μg/mL. The lowest EC50 value of conidial germination was 0.19 μg/mL, while the highest value was over 50 μg/mL. In Shanghai, the resistance frequency was 29.74% (resistance factor >10), and the frequency of high resistance was 20.51% (resistance factor >100). Resistance mechanism analysis indicated that the resistance to boscalid could be attributed to point mutations at codon 272 or 225, which would lead to the substitution of histidine to arginine (H272R), or proline to phenylalanine (P225F) in SdhB subunit of succinate dehydrogenase. And the H272R substitution was more common. This study showed the high resistance level and frequency to boscalid of B. cinerea from strawberry in Shanghai, and revealed that the H272R substitution in SdhB could be the main cause of the resistance. [ABSTRACT FROM AUTHOR]

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides. [ABSTRACT FROM AUTHOR]

[Objective] This study aimed to avoid and delay the generation of resistance in Botrytis cinerea to boscalid. [Method] The sensitivity of Botrytis cinerea to boscalid was tested by the mycelial growth rate method. The distribution of sensitivity variation of B. cinerea to boscalid in different regions was cleared, and the sensitivity baseline of B. cinerea from Liaoning Province to boscalid was established. [Result] The EC50 values of B. cinerea strains from Liaoning Province ranged from 0.080 0 to 7.787 2 μg/ml, and the highest EC50 value was 97.34 times higher than the minimum. The average EC50 value (1.973 μg/ml) of the 158 strains was treated as the baseline sensitivity of B. cinerea from Liaoning Province to boscalid. [Conclusion] It is essential to conduct a risk assessment of drug resistance in B. cinerea to boscalid. [ABSTRACT FROM AUTHOR]

During 2009–2013, 302 single-spore isolates of Botrytis cinerea were collected from vineyards located in the most important site of table grape production in Sicily, recognized by the European Community as Protected Geographical Indication (PGI) ‘Mazzarrone grape’. In preliminary studies, all isolates were tested in vitro for their sensitivity to six fungicides belonging to the following groups: benzimidazoles, dicarboximides, anilinopyrimidines, succinate dehydrogenase inhibitors, hydroxyanilides and phenylpyrroles. In these tests, 45.7% of the isolates were found to be resistant to at least one fungicide. Specific resistance to pyrimethanil was found in 30.8% of the isolates, whereas 13.9, 10.3 and 7.6% of the isolates exhibited resistance to carbendazim, iprodione and boscalid, respectively. No isolates resistant to fenhexamid and fludioxonil were detected within our dataset of B. cinerea isolates. However, 30 B. cinerea isolates possessed multiple resistance to two or more fungicides. In detail, 8 isolates were simultaneously resistant to four fungicides, whereas 5 and 17 isolates were resistant to three and two fungicides, respectively. For boscalid, 11/23 of isolates showing in vitro resistance possessed a mutation at the Sdh B gene, whereas all isolates resistant to carbendazim and iprodione possessed mutations at β-tubulin and BcOS1 histidine kinase genes, respectively. Accordingly, these fungicides failed to control gray mould infections caused by resistant or reduced sensitivity isolates on grape berries and grapevine leaves whereas the sensitive isolates were effectively managed by all fungicides applied at label rates. This study represents the first report of B. cinerea field isolates resistant and/or with simultaneous resistance to several botryticides from table grape vineyards in Sicily. Therefore, current strategies for fungicide resistance management of B. cinerea could be negatively affected in future. [ABSTRACT FROM AUTHOR]

In order to clarify the synergistic action of the mixtures of boscalid and pyrametostrobin against Botrytis cinerea, the inhibitory activity of the mixtures at different mixing ratios to isolate DZ3 was determined by mycelial growth rate method, and the synergistic radio was calculated according to the Wadley formula. The inhibitory activity of the best mixture to the other five isolates of B. cinerea was determined by inoculating fungicide-sprayed cucumber cotyledons with conidial suspension, and the control efficacy against tomato grey mould was determined in the field. The synergistic mechanism of the best synergistic combination was studied by detecting the content of dissolved oxygen, conductivity of the conidial suspension of isolate DZ3, and the content of malondialdehyde in the hyphae of isolate DZ3. The results showed that the mixtures of boscalid and pyrametostrobin (9 :1, 7 :1, 5 :1, 3 :1, 1 :1, 1 :3, 1 :5, 1 :7 and 1 :9,m/m) exhibited synergistic activity against the mycelial growth of isolate DZ3, and the 1 :3 mixture exhibited the most significant synergistic effects, with synergistic ratio up to 4.76. The 1 :3 mixture also showed synergistic inhibition to the other five isolates of B.cinerea. The control efficacy of the mixture (1 :3, m/m) against tomato gray mould in the field was 78.05%-93.96% at 200-300 g a.i./hm2, and the inhibitory activity of the mixture (1 :3, m/m) on the respiration of isolate DZ3 was similar to that of boscalid, but the impact on membrane permeability and mycelium membrane lipid peroxidation was more significant than that of individual boscalid and pyrametostrobin. The results of present study suggested that the mixture of boscalid and pyrametostrobin (1 :3, m/m) could be used in controling of tomato gray mould. [ABSTRACT FROM AUTHOR]

In a survey of Botrytis populations in Northern Germany in 2012-2014, an average of 67.7% and 73.8% of isolates from strawberry and raspberry fields (respectively) were resistant to QoI fungicides whilst 40.2% and 64.9% (respectively) were resistant to the SDHI boscalid. There was a sporadic occurrence (2.8% and 3.3%, respectively) of strains resistant to the new SDHI fungicide fluopyram, due to be used in German soft fruits from 2015 onwards. Both moderate (EC50 = 0.5-5.0 mg l-1) and high (EC50 > 10 mg l-1) levels of resistance to fluopyram were observed in a spore germination assay in which the baseline sensitivity was 0.02-0.2 mg l-1. Among several amino acid substitutions in the SDHI target site found in Botrytis isolates from fields of strawberries, raspberries and other horticultural crops, P225F and N230I, based on mutations in the sdhB gene, as well as a type of resistance of unknown basis, caused positive cross-resistance to boscalid and fluopyram. Further, N230I and the unknown type of resistance were detected in strains with multiple resistance to all other currently used fungicides, i.e. fenhexamid, cyprodinil, fludioxonil and QoIs. For resistance management purposes, it is suggested that the number of fluopyram applications should be limited to one per season. [ABSTRACT FROM AUTHOR]

A novel, high-resolution melting ( HRM) analysis was developed to detect single nucleotide polymorphisms ( SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty-six single-spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine ( F) with serine ( S) or valine ( V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdh B gene resulted in the replacement of histidine ( H) with arginine ( R) or tyrosine ( Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high-resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations. [ABSTRACT FROM AUTHOR]

BACKGROUND Succinate dehydrogenase inhibitors ( SDHIs), interfering with fungal respiration, are considered to be fungicides at medium to high risk of resistance. Boscalid was the first molecule belonging to the SDHIs that was introduced for the control of Botryotinia fuckeliana. A range of different target-site mutations leading to boscalid resistance have been found in field populations of the fungus. The different types of mutation confer different cross-resistance profiles towards novel SDHIs, such as the recently introduced fungicide fluopyram. This study combines the determination of cross-resistance profiles and the setting-up of methods for fast molecular detection of the mutations. RESULTS By means of in vitro tests, a range of SdhB mutations were characterised for resistance levels towards boscalid and fluopyram. SdhB mutations conferring P225L and P225F substitutions conferred high resistance to boscalid and high or moderate resistance to fluopyram respectively. Mutants carrying the N230I replacement were moderately resistant to both SDHIs. Substitutions at position H272 responsible for a high level of resistance to boscalid conferred sensitivity ( H272R), hypersensitivity ( H272Y) or moderate resistance ( H272V) to fluopyram. Allele-specific ( AS) PCR was developed and used for genotyping 135 B. fuckeliana isolates. The assay confirmed the strict association between resistance profiles and allelic variants of the SdhB gene. Real-time AS-PCR proved to be sensitive and specific for quantitative detection of different SDHI-resistant genotypes. CONCLUSION Fluopyram-resistant mutants are currently rarely detected in the field sprayed with boscalid, but this may change with intensive exposure of the fungal population to fluopyram. PCR assays/methods developed in the study provide tools for fast monitoring of field populations and observing possible changes in population composition following fluopyram introduction, useful for the setting-up of appropriate preventive measures. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The article presents a study on multiple resistance of the fungi Botrytis cinerea against chemical compounds including Succinate Dehydrogenase Inhibitor (SDHI)-based boscalid, the Qol pyraclostobin and anilinopyrimidine. It states that the study sought to determine fungicide sensitivity of Botrytis cinerea by tests on fungitoxicity conducted in vitro. It asserts that the fungi has displayed resistance against boscalid and pyraclostobin and may serve as a strong pathogen in kiwifruit.

This investigation was undertaken to test different control strategies against Botrytis cinerea vineyards. Two commercial vineyards, “Barbera” and “Moscato,” located in Piedmont (Northern Italy) were divided into seven plots and treated with different combinations of fungicides including fenhexamid, pyrimethanil, fludioxonil + cyprodinil, iprodione, and boscalid, a new carboxamide compound. An integrated strategy including a chemical (pyrimethanil) and a biocontrol agent (Trichoderma spp. t2/4ph1) was also included. At harvest, the percentage of bunches and berries attacked by B. cinerea and the concentration of the chemical fungicides were determined. All the pesticide residues at harvest were below the maximum residue level (MRL), except when two applications of pyrimethanil per season were applied. Boscalid was the most effective active ingredient against B. cinerea among the tested chemicals. When boscalid application was followed by a treatment with pyrimethanil, its efficacy was similar to that shown by two treatments of pyrimethanil. However, this second strategy was not feasible due to the risks of resistance development in the pathogen and to the residue accumulation as indicated by the analysis. [ABSTRACT FROM AUTHOR]

The pH-responsive controlled release of Boscalid with low toxicity. [Display omitted] • 18% Boscalid has been loaded in ZIF-67 nanocarriers with large pore size. • Release of Boscalid@ZIF-67 can be triggered in acid condition. • Boscalid@ZIF-67 exhibits remarkable antifungal effect over Botrytis cinerea. Pesticides have been always restricted by poor efficacy and inacceptable toxicity. In this work, a pH-sensitive controlled-release nano-capsule of fungicide, Boscalid@ZIF-67 (ZIF is a subclass of MOF), was prepared based on metal–organic framework (MOF) for higher antifungal effect and low bio-toxicity. The Boscalid@ZIF-67 was formed by physical adsorption method and it possesses uniform size and dodecahedral shape. The loading ratio is as high as 18%. In addition, the release of fungicide Boscalid showed pH sensitive response due to the weak acidic group in the MOF structure, in which the rate of release was more rapid under acid condition (＜7). Cumulative release rate release was higher about 25% and 38% adjusted from 7 to 3.5 and from 7 to 5.5 on the 6th day than that under neutral condition. At the early stage of infecting the host plant, Botrytis cinerea will produce more acidic substances such as oxalic acid, which are conducive to Boscalid@ZIF-67 disintegration for rapid release of Boscalid. Meanwhile, the antifungal effect of Boscalid@ZIF-67 was 4–6 times more of that commercially available product on Botrytis cinerea , while the bio-toxicity under the same condition is much lower than that of latter. Thus, this work not only proves that Boscalid@ZIF-67 could be used as nanoparticles platform for smart control over Botrytis cinerea , but also has great potential to improve traditional modalities by addressing the crop diseases in economic and ecological manner. [ABSTRACT FROM AUTHOR]

Grey mould, caused by the fungus Botrytis cinerea, is one of the most destructive diseases in greenhouses for which serious fungicide resistance has developed. Between 2003 and 2005, 213 isolates of B. cinerea from two geographical regions were characterised for baseline sensitivity to kresoxim-methyl. In the absence of salicylhydroxamic acid (SHAM), the mean 50% effective concentration (EC50) values were 6.67 ± 0.61 (mean ± SD) and 0.37 ± 0.10 mg L−1 during growth and germination, respectively. In the presence of 100 mg L−1 SHAM, baseline sensitivities were distributed as unimodal curves with mean EC50 values of 2.38 ± 0.21 and 0.28 ± 0.09 mg L−1 for inhibiting growth and inhibiting germination, respectively. The mixture of kresoxim-methyl and boscalid showed good control efficacy against strawberry grey mould disease. After the mixture was extensively used on strawberry for 2 years, 50 isolates were collected and determined for their sensitivity to kresoxim-methyl and boscalid, respectively. The mean EC50 of germination inhibition by boscalid was 0.39 ± 0.08 mg L−1. The mean EC50 of germination inhibition by kresoxim-methyl was 0.26 ± 0.07 mg L−1 in the presence of 100 mg L−1 SHAM. Sensitivities of B. cinerea to both kresoxim-methyl and boscalid did not show any significant decrease. These results suggest that their mixture is a satisfactory alternative candidate for management of grey mould disease in greenhouses. [ABSTRACT FROM AUTHOR]

In 2000, 137 strains of Botrytis cinerea were isolated from different plant hosts worldwide. The isolates had not been exposed to boscalid and were therefore suitable to establish a baseline sensitivity. In order to determine the sensitivity of these isolates, an appropriate microtiter assay was developed. YBA-medium was the medium of choice, as it allowed sufficient growth but did not lead to false positive results. ED50-values ranged from 0.01 to 0.21 ppm, the ED98-values from 0.1 to 2.21 ppm. No growth was observed at concentrations >3 ppm. [ABSTRACT FROM AUTHOR]