Search Results (23)

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used in Australia for the control of blackleg disease (caused by Leptosphaeria maculans, also called Plenodomus lingam). Populations of L. maculans are highly variable and therefore at risk of evolving fungicide resistance. The baseline sensitivities of L. maculans isolates towards the SDHI fungicides pydiflumetofen and bixafen were determined through in vitro mycelial growth assays, and the mean EC₅₀s were found to be 4.89 and 2.71 ng mL⁻¹, respectively. L. maculans populations were also screened against three commercial SDHI fungicides, Saltro^®, ILeVO^®, and Aviator^®, using an in planta assay to reveal very low levels of resistance. Nineteen of these ascospore populations from 2022 were analysed in a deep amplicon sequencing (DAS) assay and showed no mutations in the genes likely to be associated with resistance to SDHI chemistries. This study establishes baseline sensitivities of L. maculans isolates towards commonly used SDHI fungicides, importantly before and during the introduction of these new chemistries for blackleg control, and outlines monitoring techniques to allow timely identification of resistance if it evolves. Full article

Venturia inaequalis, the cause of apple scab, readily develops resistance to fungicides with specific modes of action. Knowledge of the spatial and temporal pattern of resistance development is therefore relevant to fruit producers and their consultants. In the Lower Elbe region of Northern Germany, a two-year survey based on a conidial germination test was conducted, examining fungicide resistance in 35 orchards under Integrated Pest Management (IPM), 16 orchards of susceptible cultivars as well as a further 12 orchards of scab-resistant (Vf) cultivars under organic management, and 34 abandoned or unmanaged sites. No evidence of resistance to SDHI compounds (fluopyram, fluxapyroxad) was found after >5 yr of their regular use. Resistance to anilinopyrimidines (cyprodinil, pyrimethanil) had disappeared 15 yr after its widespread occurrence. Isolates from a few IPM orchards showed a reduced sensitivity to dodine. Double resistance to the MBC compound thiophanate-methyl and the QoI trifloxystrobin was rare in V. inaequalis strains that had achieved breakage of Vf-resistance, but very common (>50%) on scab-susceptible cultivars in IPM, organic and abandoned orchards in the ‘Altes Land’ core area of the Lower Elbe region, and in IPM orchards in the periphery. We conclude that resistance to QoI and MBC fungicides is persistent even decades after their last use, and that the core area harbours a uniform population adapted to intensive crop protection, whereas isolated orchards in the periphery are colonised by discrete populations of V. inaequalis. Full article

Microdochium species are harmful pathogens of winter cereals, causing snow mould and stem base diseases such as root rot. With changing climatic conditions, including prolonged wet autumns and mild winters, addressing pathogens that thrive at low positive temperatures has become increasingly important. Integrated strategies, including optimized sowing times, resistant cultivars, and the use of seed treatment fungicides have been suggested as effective approaches to mitigate Microdochium-induced damage. Field trials were conducted between 2021 and 2024 using five winter wheat cultivars treated with different seed treatment fungicides and sown at either optimal or delayed sowing times. Laboratory analyses identified Microdochium spp. as the dominant pathogens on the stem base across all trial years. Disease severity assessments indicated that seed treatment fungicides were generally effective against root rot, with products containing fludioxonil and SDHI group fungicides delivering the best performance. While disease pressure varied between optimal and late sowing experiments, late-sown winter wheat exhibited slightly reduced damage in most years. Additionally, some of the tested winter wheat cultivars demonstrated better performance against Microdochium spp. damage compared to others, highlighting the importance of selecting resistant cultivars. This study provides valuable insights into the control of Microdochium spp. under changing climatic conditions, particularly during the early growth stages of winter wheat. Full article

Septoria tritici blotch (STB) ais one of the most damaging winter wheat diseases worldwide, presenting a significant threat to its yields. The causal STB agent, Zymoseptoria tritici, also presents a challenge to control due to its rapid adaptation to fungicides. This requires researchers to continuously monitor the pathogen and investigate and explore strategies to manage the spread of the disease and the development of resistance in the pathogen. Therefore, this study presents the current situation and describes changes in the sensitivity of Z. tritici isolates from Lithuania to quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) for the years 2019–2022. The isolates were tested at five different concentrations of two QoI fungicides (azoxystrobin and pyraclostrobin) and three SDHI fungicides (fluxapyroxad, benzovindiflupyr, and bixafen). During the test period, the EC50 values of the tested QoIs increased, while no clear changes were observed in the SDHIs. The most pronounced shift was observed for the active QoI substance pyraclostrobin. The distribution of the EC50 values of the SDHI fungicides showcased one isolate with an outstandingly high EC50 value of 2.6 mg L⁻¹. The results of this study did not reveal any strong patterns of cross-resistance between the fungicides tested. However, a significant positive, moderate correlation (r = 0.55) was found between fluxapyroxad and benzovindiflupyr. Overall, the results of this study contribute to the understanding of the fungicide-resistance situation of Z. tritici in Lithuania and may complement management strategies for the pathogen and its fungicide resistance. Full article

The Sigatoka disease complex (SDC), caused by Mycosphaerella fijiensis (Mf) and M. musicola (Mm), comprises the most destructive fungal leaf streak and spot diseases of commercial banana crops worldwide. In Brazil, the site-specific succinate dehydrogenase inhibitor (SDHI) fungicides labeled for SDC management since 2014 present a high risk for the emergence of resistance if deployed intensively and solo. Our study determined the levels of sensitivity to boscalid and fluxapyroxad in four populations of the SDC pathogens sampled in 2020 from three distinct geographical regions under contrasting fungicide programs. Resistance, defined as EC₅₀ values exceeding 20 µg mL⁻¹, was prevalent at 59.7% for fluxapyroxad and 94.0% for boscalid. Only 1.5% of isolates exhibited sensitivity to both fungicides. We also assessed the changes in the corresponding fungicide target protein-encoding genes (SdhB, C, and D). None of the target site alterations detected were associated with reduced sensitivity. A second SdhC paralog was also analyzed, but target alterations were not found. However, MDR (multidrug resistance) was detected in a selection of isolates. Further monitoring for Sdh target mutations will be important, but an important role for other resistance mechanisms such as the presence of additional Sdh paralogs and MDR cannot be ruled out. These results highlight the importance of implementing sound anti-resistance management strategies when SDHI fungicides are deployed for the management of SDC. Full article

Succinate dehydrogenase (SDH, EC 1.3.5.1) is one of the most promising targets for fungicide development and has attracted great attention worldwide. However, existing commercial fungicides targeting SDH have led to the increasingly prominent problem of pathogen resistance, so it is necessary to develop new fungicides. Herein, we used a structure-based molecular design strategy to design and synthesize a series of novel SDHI fungicides containing an N-(alkoxy)diphenyl ether carboxamide skeleton. The mycelial growth inhibition experiment showed that compound M15 exhibited a very good control effect against four plant pathogens, with inhibition rates of more than 60% at a dose of 50 μg/mL. A structure–activity relationship study found that N-O-benzyl-substituted derivatives showed better antifungal activity than others, especially the introduction of a halogen on the benzyl. Furthermore, the molecular docking results suggested that π–π interactions with Trp35 and hydrogen bonds with Tyr33 and Trp173 were crucial interaction sites when inhibitors bound to SDH. Morphological observation of mycelium revealed that M15 could inhibit the growth of mycelia. Moreover, in vivo and in vitro tests showed that M15 not only inhibited the enzyme activity of SDH but also effectively protected rice from damage due to R. solani infection, with a result close to that of the control at a concentration of 200 μg/mL. Thus, the N-(alkoxy)diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further investigation. Full article

Wheat blast, caused by the ascomycetous fungus Pyricularia oryzae Triticum lineage (PoTl), is mainly controlled by fungicide use, but resistance to the main fungicide groups—sterol demethylase (DMI), quinone outside (QoI), and succinate dehydrogenase inhibitors (SDHI)—has been reported in Brazil. In order to rationalize fungicide inputs (e.g., choice, timing, dose-rate, spray number, and mixing/alternation) for managing wheat blast, we describe a new monitoring tool, enabling the quantitative measurement of pathogen’s inoculum levels and detection of fungicide resistance alleles. Wheat blast airborne spores (aerosol populations) were monitored at Londrina in Paraná State, a major wheat cropping region in Brazil, using an automated high-volume cyclone coupled with a lab-based quantitative real-time PCR (qPCR) assay. The objectives of our study were as follows: (1) to monitor the amount of PoTl airborne conidia during 2019–2021 based on DNA detection, (2) to reveal the prevalence of QoI resistant (QoI-R) cytochrome b alleles in aerosol populations of wheat blast, and (3) to determine the impact of weather on the dynamics of wheat blast aerosol populations and spread of QoI resistant alleles. PoTl inoculum was consistently detected in aerosols during the wheat cropping seasons from 2019 to 2021, but amounts varied significantly between seasons, with highest amounts detected in 2019. High peaks of PoTl DNA were also continuously detected during the off-season in 2020 and 2021. The prevalence of QoI resistant (QoI-R) cytochrome b G143A alleles in aerosol populations was also determined for a subset of 10 PoTl positive DNA samples with frequencies varying between 10 and 91% using a combination of PCR-amplification and SNP detection pyrosequencing. Statistically significant but low correlations were found between the levels of pathogen and the weather variables. In conclusion, for wheat blast, this system provided prior detection of airborne spore levels of the pathogen and of the prevalence of fungicide resistance alleles. Full article

Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species. Full article

This study aimed at the development and validation of an accurate, more affordable, and precise digital imaging resazurin-based fungicide sensitivity colorimetric assay (COL-assay) for fungal plant pathogens from the genera Mycosphaerella and Pyricularia. This proposed digital imaging assay was based on colorimetric estimates of resazurin reduction, which was used as a metabolic indicator of fungal respiration activity on microplate cultures. As fungal model systems, we used the yellow and black Sigatoka pathogens [Mycosphaerella musicola (Mm) and M. fijiensis (Mf), respectively] and the wheat blast pathogen, Pyricularia oryzae Triticum lineage (PoTl), which were previously characterized for QoI, DMI, and SDHI fungicide sensitivity. We then compared the classical spectrophotometry detection assay (SPEC-assay) with the proposed COL-assay based on the analyses of digital images of the microplates’ cultures captured with mobile phone cameras on a handmade trans-illuminator built for poorly equipped labs. Qualitatively, in terms of accuracy, there was full correspondence between the SPEC-assay and the COL-assay according to the fungal EC₅₀ or the relative growth classes on QoI, SDHI, and DMI fungicides for both Mycosphaerella and Pyricularia pathogens. We also observed a strong to very strong correlation coefficient between the COL-assay and the SPEC-assay fungicide sensitivity values for the QoI azoxystrobin, the SDHI fluxapyroxad, and the DMI tebuconazole. Our conclusion was that the COL-assay had a similar accuracy as the SPEC-assay (i.e., resulted in similar fungicide-sensitivity categories for both resistant or sensitive fungal isolates) and high precision. By openly sharing here the COL-assay’s full methodology, and the blueprints of the handmade trans-illuminator, we foresee its adoption by poorly equipped labs throughout the country as an affordable venue for monitoring the fungicide resistance status of populations of important fungal plant pathogens such as M. fijiensis, M. musicola, and P. oryzae Triticum and Oryza lineages. Full article

Fusarium head blight (FHB), which is primarily caused by Fusarium graminearum, is a widespread and devastating disease of wheat. In the absence of resistant varieties, the control of FHB relies heavily on the application of fungicides, and the new generation SDHI fungicide, pydiflumetofen, has recently been registered in China for the control of FHB in wheat. The current study explored three genetically stable, highly resistant laboratory mutants (S2-4-2R, S27-3R, and S28-2R, with EC₅₀ values of 25.10, 28.57, and 19.22 μg/mL, respectively) to investigate the potential risks associated with pydiflumetofen resistance. Although the mycelial growth of the mutants differed little compared to their parental isolates, the study found that the resistant mutants exhibited significantly reduced (p < 0.05) levels of sporulation and pathogenicity, which suggests a significant fitness cost associated with pydiflumetofen resistance in F. graminearum. Sequence analysis of the Sdh target protein identified numerous amino acid substitutions in the predicted sequences of the four subunits: FgSdhA, FgSdhB, FgSdhC, and FgSdhD. Indeed, the mutants were found to have a series of substitution in multiple subunits such that all three exhibited five identical changes, including Y182F in the FgSdhA subunit; H53Q, C90S, and A94V in FgSdhB; and S31F in FgSdhC. In addition, gene expression analysis revealed that all of the FgSdh genes had significantly altered expression (p < 0.05), particularly FgSdhA and FgdhC, which exhibited remarkably low levels of expression. However, the study found no evidence of cross-resistance between pydiflumetofen and tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, or difenoconazole, which indicates that these fungicides, either in rotation or combination with pydiflumetofen, could mitigate the risk of resistance emerging and provide ongoing control of FHB to ensure high and stable wheat yields. Full article

The resistance prevalence of chemical fungicides has caused increasingly serious agro-ecological environmental problems. However, there are few previous reports about resistance to succinate dehydrogenase (SDHI) or sterol demethylation inhibitor (DMI) in Rhizoctonia solani, one of the main agro-diseases. In this study, the fungicide resistance of 122 R. solani isolates in Sichuan Province was monitored by the mycelial growth rate method. Results showed that all isolates were susceptible to hexaconazole and most isolates were susceptible to thifluzamide, except for the field isolate MSRS-2-7 due to a moderate resistance to thifluzamide (16.43-fold resistance ratio, RR), compared to the sensitivity baseline of thifluzamide (0.042 μg/mL EC₅₀ values). On the contrary, many isolates showed moderate or high resistance to tebuconazole (10.59- to 60.78-fold RR), reaching EC₅₀ values of 0.54~3.10 μg/mL, especially for a highly resistant isolate LZHJ-1-8 displaying moderate resistance to epoxiconazole (35.40-fold RR due to a 3.54 μg/mL EC₅₀ value). The fitness determination found that the tebuconazole-resistant isolates showed higher fitness cost with these characteristics, including a lower growth rate, higher relative electric conductivity, an increased ability to tolerate tebuconazole, and high osmotic pressure. Four new mutations of cytochrome P450 sterol 14α-demethylase (CYP51), namely, S94A, N406S, H793R, and L750P, which is the target for DMI fungicides, was found in the tebuconazole-resistant isolates. Furthermore, the lowest binding energy with tebuconazole was also found in the LZHJ-1-8 isolate possessing all the mutations through analyses with Discovery Studio software. Therefore, these new mutation sites of CYP51 may be linked to the resistance against tebuconazole, and its application for controlling R. solani should be restricted in some areas. Full article

Widespread resistance to QoIs, DMI and SDHIs fungicides has been reported for Brazilian populations of the wheat blast pathogen Pyricularia oryzae Triticum lineage (PoTl). A pre-existing resistance mechanism not associated with target site mutations has been indicated for resistance to DMIs and SDHIs, with strong indication that PoTl has multidrugresistance (MDR). Therefore, the main objective of this study was to test the hypothesis that resistance to DMI and SDHI fungicides detected in PoTl was due to efflux pump mediated MDR mechanism(s) by characterizing the sensitivity to antifungal efflux pump substrates. Four antifungal substrates were tested: tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL). TPCL and RH6G were considered the most relevant indicators for enhanced MDR activity. Among the 16 PoTl isolates tested, 9 were insensitive to TPCL, 1 to TOL, 16 to RH6G and 1 to CHX. The PoTl isolates were grouped into four distinct multidrug resistance phenotypes (MDRPs) based on resistance to combinations of fungicides and antifungal efflux pump substrates. Insensitivity to TPCL, RH6G and or TOL correlated well with DMI insensitivity, but MDR was not associated with SDHI resistance. The identification of multiple MDRP phenotypes associated with DMI resistance in our study warrants further research aimed at revealing the exact mechanisms of multidrug resistance in the wheat blast pathogen, including efflux pumps overexpression via transcriptomic analyses of differentially expressed genes; identification and discovery of mutations associated with changes in promoter regions or transcription factors of efflux transporters associated with multidrug resistance. Full article

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. Full article

Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, has recently become widespread in Europe. Succinate dehydrogenase inhibitor (SDHI) and demethylation inhibitor (DMI) fungicides are mainly applied for disease control on barley fields, but pathogen isolates with a reduced sensitivity can cause difficulties. There is an urgent need for new spring barley cultivars that are more resistant to RLS development and can inhibit R. collo-cygni epidemics. Full article

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management. Full article