The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola.

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC50 < 0.83 µg/mL), moderately resistant isolates (MR, 1.18 < EC50 < 55.67 µg/mL), and highly resistant isolates (HR, EC50 > 56.98 µg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola.

Residual analysis of QoI fungicides in multiple (six) types of aquatic organisms by UPLC-MS/MS under acutely toxic conditions.

In view of the significance of food safety and the possible relationship between residual enrichment and acute toxicity for pesticides in different aquatic organisms, it is essential to establish a sensitive and reliable determination method for pesticides in different aquatic organisms to analyze the enrichment levels. Quinone outside inhibitor fungicides (QoIs) are lipophilic fungicides that pose environmental threats to aquatic organisms. Previous research has mainly focused on QoI residues in aquatic organisms under chronic toxicity, whereas less is known about how pesticide residues differ among aquatic organism under acutely toxic conditions. In the present study, the residues of QoIs in aquatic organisms (Danio rerio, Rana pipiens, Cherax quadricarinatus, Misgurnus anguillicaudatus, Corbicula fluminea, and Ampullaria gigas) were analyzed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with a proposed QuEChERS method. The proposed method was validated in terms of linearity (coefficients of determination of 0.9980-0.9999), the limits of quantification (0.01 µg·kg-1), the relative standard deviation (0.6-4.4%), and recovery (70.12-118.15%). The results demonstrated that the proposed method fulfilled the requirements for pesticide analysis in all tested aquatic organisms. The residues of QoIs in the same aquatic organism exposed to QoI concentrations of 5 and 500 µg L-1 decreased in the order pyraoxystrobin > pyraclostrobin > triclopyricarb > picoxystrobin > azoxystrobin > fluoxastrobin. Furthermore, the acute toxicity was strongly correlated with the enrichment level of the QoIs in aquatic organisms. This study provides the first documentation of a correlation between the enrichment level of QoIs and acute toxicity in aquatic organisms, which provides a basis for the management of agrochemicals considering aquatic ecological risks.

Toxicological risks of SDHIs and QoIs to zebrafish (Danio rerio) and the corresponding poisoning mechanism.

Quinone outside inhibitor fungicides (QoIs) and succinate dehydrogenase inhibitor fungicides (SDHIs) were classified as highly or moderately toxic to nontarget aquatic organisms, which deterred their application in paddy scenario. Currently, the mechanism of toxicity regarding which factors govern their risk ranking in fish species are not fully explored. In this study, adult zebrafish were exposed to four QoIs (pyraclostrobin, trifloxystrobin, kresoxim-methyl, and azoxystrobin) and three SDHIs (isopyrazam, thifluzamide, and boscalid) to assess its acute toxicity and effects on tissue accumulation and gill injury. The results showed that the overall toxicity level was in the order of QoIs > SDHIs, whereas the order of accumulation capacity was SDHIs > QoIs. Seven mitochondrial respiratory inhibitors exposure induced serious histological damage in the gills, including aneurism, curling, telangiectasia and swelling, and caused mitochondrial dysfunction and weaker complex II and III activities. The correlation between their acute toxicities and in vitro gill cytotoxicity was significant (R = 0.868), whereas the bioaccumulation level was not markedly associated with their 96h-LC50 values in zebrafish (R = -0.686), indicating the degree of target organ (gill) injury may be the decisive factor that governs the risk grade of respiratory inhibitors in fish. Additionally, the docking positions and binding energies of fungicides with the target proteins may be responsible for their differential branchial damage. These results offer a point of reference and theoretical support for the design of fungicides and appropriate formulations with improved environmental safety that could broaden their application scenario.

A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR.

Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins' efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease.

Synergy between Cu-NPs and fungicides against Botrytis cinerea.

Combating drug-resistance is a daunting task, especially due to the shortage of available drug alternatives with multisite modes of action. In this study, the potential of copper nanoparticles (Cu-NPs) to suppress 15 Botrytis cinerea isolates, which are sensitive or resistant to fungicides, alone or in combination with conventional fungicides, was tested in vitro and in vivo. Sensitivity screening in vitro revealed two fungicide resistance phenotypes, resulting from target site mutations. DNA sequencing revealed three B. cinerea isolates highly resistant to benzimidazoles (BEN-R), thiophanare methyl (TM), and carbendazim, bearing the E198A resistance mutation in the ß-tubulin gene, and four isolates highly resistant to the QoI pyraclostrobin (PYR-R) with a G143A mutation in the cytb gene. Cu-NPs were equally effective against sensitive and resistant isolates. An additive/synergistic effect was observed between Cu-NPs and TM in the case of BEN-S isolates both in vitro and when applied in apple fruit. A positive correlation was observed between TM and TM + Cu-NPs treatments, suggesting that an increased TM availability in the target site could be related with the observed additive/synergistic action. No correlation between Cu(OH)2 and Cu-NPs sensitivity was found, indicating that different mechanisms govern the fungitoxic activity between nano and bulk counterparts. A synergistic profile was observed between Cu-NPs and fluazinam (FM) - an oxidative phosphorylation inhibitor - in all isolates regardless of resistance phenotype, suggesting that ATP metabolism could be involved in the mode of action of Cu-NPs. Furthermore, the observed cross sensitivity and antagonistic action between Cu-NPs and NaCl also provided evidence for copper ions contribution to the fungitoxic action of Cu-NPs. The results suggested that Cu-NPs in combination with conventional fungicides can provide the means for an environmentally safe, sustainable resistance management strategy by reducing fungicide use and combating resistance against B. cinerea.

Spatiotemporal dynamics of fungicide resistance in the wheat pathogen Zymoseptoria tritici in France.

BACKGROUND: Management of pesticide resistance is a major issue in modern agricultural systems, particularly in the context of the broader challenge of reducing pesticide use. However, such management must be adapted to resistance dynamics, which remains challenging to predict due to its dependence on many biological traits of pests, interactions with the environment and pesticide use. We retrospectively studied the evolution of reported resistances to four modes of action (benzimidazoles, quinone outside inhibitors, sterol demethylation inhibitors and succinate dehydrogenase inhibitors), in French populations of the wheat pathogen Zymoseptoria tritici. RESULTS: We used statistical models to analyse the Performance trial network data set (2004-2017; ∼ 70 locations in France yearly). They highlighted contrasting behaviours between phenotypes, for example: (i) stable spatial distributions and colonization front structures over time, and (ii) different frequency growth rates at the national scale and between regions. CONCLUSION: We provide a quantitative description of the spatiotemporal patterns of resistance evolution for fungicides with several modes of action. Moreover, we highlight some unexpected resistance dynamics in France, with major differences between the north and south. This complex pattern of resistance evolution in French populations is consistent with previous descriptions of dynamics at the European scale. These results should make it easier to anticipate evolution locally and to improve the management of resistance. © 2019 Society of Chemical Industry.

Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum of Botrytis cinerea.

A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould.