Shifting sensitivity of septoria tritici blotch compromises field performance and yield of main fungicides in Europe.

Septoria tritici blotch (STB; Zymoseptoria tritici) is a severe leaf disease on wheat in Northern Europe. Fungicide resistance in the populations of Z. tritici is increasingly challenging future control options. Twenty-five field trials were carried out in nine countries across Europe from 2019 to 2021 to investigate the efficacy of specific DMI and SDHI fungicides against STB. During the test period, two single DMIs (prothioconazole and mefentrifluconazole) and four different SDHIs (fluxapyroxad, bixafen, benzovindiflupyr and fluopyram) along with different co-formulations of DMIs and SDHIs applied at flag leaf emergence were tested. Across all countries, significant differences in azole performances against STB were seen; prothioconazole was outperformed in all countries by mefentrifluconazole. The effects also varied substantially between the SDHIs, with fluxapyroxad providing the best efficacy overall, while the performance of fluopyram was inferior to other SDHIs. In Ireland and the UK, the efficacy of SDHIs was significantly lower compared with results from continental Europe. This reduction in performances from both DMIs and SDHIs was reflected in yield responses and also linked to decreased sensitivity of Z. tritici isolates measured as EC50 values. A clear and significant gradient in EC50 values was seen across Europe. The lower sensitivity to SDHIs in Ireland and the UK was coincident with the prevalence of SDH-C-alterations T79N, N86S, and sporadically of H152R. The isolates' sensitivity to SDHIs showed a clear cross-resistance between fluxapyroxad, bixafen, benzovindiflupyr and fluopyram, although the links with the latter were less apparent. Co-formulations of DMIs + SDHIs performed well in all trials conducted in 2021. Only minor differences were seen between fluxapyroxad + mefentrifluconazole and bixafen + fluopyram + prothioconazole; the combination of benzovindiflupyr + prothioconazole gave an inferior performance at some sites. Fenpicoxamid performed in line with the most effective co-formulations. This investigation shows a clear link between reduced field efficacy by solo SDHIs as a result of increasing problems with sensitivity shifting and the selection of several SDH-C mutations. The presented data stress the need to practice anti-resistance strategies to delay further erosion of fungicide efficacy.

Evidence for the association of target-site resistance in cyp51 with reduced DMI sensitivity in European Cercospora beticola field isolates.

BACKGROUND: Cercospora leaf spot caused by Cercospora beticola is the most relevant foliar disease in sugar beet cultivation. In the last decade a decreasing sensitivity of C. beticola towards demethylation inhibitors (DMIs) occurred. Different mechanisms mediating a reduced sensitivity towards DMIs have been identified in different plant pathogens to date, such as target site mutations, over-expression or active excretion of the fungicide. RESULTS: A sequencing of the cytochrome P450-dependent sterol 14α-demethylase gene sequence (cyp51) of diverse C. beticola isolates collected in different European countries with reduced DMI sensitivity was performed in order to find a possible correlation of mutations with higher EC50 values. The amino acid alterations Y464S, L144F and I309T combined with L144F were found to be associated with a reduced sensitivity. Furthermore, mutations I387M, M145W and M145W with E460Q were found uniquely. Additionally, constitutive and fungicide triggered expression of cyp51 was assayed by means of RT-qPCR. A very strong induction of cyp51 mRNA expression in sensitive isolates suggests that the fungal cells upregulate expression to maintain ergosterol biosynthesis in DMI presence. The less intensive cyp51 induction in isolates with higher EC50 values underlines the possible correlation of the found target-site mutations with reduced sensitivity. CONCLUSION: This study provides new results about possible alterations in the target gene mediating reduced sensitivity of C. beticola towards DMIs and hypothesized a fungicide induced over-expression of the target enzyme CYP51 as natural reaction of the fungus to fungicide application. © 2020 Society of Chemical Industry.

Sensitivity of the Pyrenophora teres Population in Algeria to Quinone outside Inhibitors, Succinate Dehydrogenase Inhibitors and Demethylation Inhibitors.

Net blotch of barley caused by Pyrenophora teres (Died.) Drechsler, is one of the most destructive diseases on barley in Algeria. It occurs in two forms: P. teres f. teres and P. teres f. maculata. A total of 212 isolates, obtained from 58 fields sampled in several barley growing areas, were assessed for fungicide sensitivity by target gene analysis. F129L and G137R mitochondrial cytochrome b substitution associated with quinone outside inhibitors (QoIs) resistance, and succinate dehydrogenase inhibitors (SDHIs) related mutations (B-H277, C-N75S, C-G79R, C-H134R, and C-S135R), were analyzed by pyrosequencing. In vitro sensitivity of 45 isolates, towards six fungicides belonging to three chemical groups (QoI, demethylase inhibitor, and SDHI) was tested by microtiter technique. Additionally, sensitivity towards three fungicides (azoxystrobin, fluxapyroxad, and epoxiconazole) was assessed in planta under glasshouse conditions. All tested isolates were QoI-sensitive and SDHI-sensitive, no mutation that confers resistance was identified. EC50 values showed that pyraclostrobin and azoxystrobin are the most efficient fungicides in vitro, whereas fluxapyroxad displayed the best disease inhibition in planta (81% inhibition at 1/9 of the full dose). The EC50 values recorded for each form of net blotch showed no significant difference in efficiency of QoI treatments and propiconazole on each form. However, in the case of fluxapyroxad, epoxiconazole and tebuconazole treatments, analysis showed significant differences in their efficiency. To our knowledge, this study is the first investigation related to mutations associated to QoI and SDHI fungicide resistance in Algerian P. teres population, as well as it is the first evaluation of the sensitivity of P. teres population towards these six fungicides.

Competitive Fitness of Phakopsora pachyrhizi Isolates with Mutations in the CYP51 and CYTB Genes.

Soybean rust (Phakopsora pachyrhizi) in Brazil is mainly controlled with applications of fungicides, including demethylation inhibitors (DMI) and quinone outside inhibitors (QoI). Isolates with less sensitivity to DMI and QoI have been reported, and these have been found to have mutations in the CYP51 and CYTB genes, respectively. There have been no reports of fitness costs in isolates with mutations in CYP51 and CYTB, and the aim of this work was to compare the competitive ability of isolates with lower DMI or QoI sensitivities with that of sensitive (wild-type) isolates. Urediniospores of sensitive wild-type isolates and isolates with different CYP51 or CYTB alleles were mixed and inoculated on detached soybean leaves. After 3 weeks, urediniospores were harvested and used as inoculum for the next disease cycle. Frequencies of relevant target site mutations were monitored using the pyrosequencing method over four disease cycles. Isolates with lower DMI sensitivity and different CYP51 alleles had competitive disadvantages compared with a DMI-sensitive, wild-type CYP51 isolate. In contrast, the isolate with the F129L mutation in the CYTB gene competed equally well with a QoI-sensitive, wild-type CYTB isolate under the conditions of this experiment. The CYP51 and CYTB alleles were stable in all isolates over four disease cycles when cultivated alone.

Proposal for a unified nomenclature for target-site mutations associated with resistance to fungicides.

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino acid substitutions is to cite the wild-type amino acid, the codon number and the new amino acid, using the one-letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the present paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied 'archetype' species. Orthologous amino acids in all species are then assigned numerical 'labels' based on the position of the amino acid in the archetype protein. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Emergence of succinate dehydrogenase inhibitor resistance of Pyrenophora teres in Europe.

BACKGROUND: Net blotch caused by Pyrenophora teres is an important disease of barley worldwide. In addition to strobilurins (quinone ouside inhibitors) and azoles (demethylation inhibitors), succinate dehydrogenase inhibitors (SDHIs) are very effective fungicides for net blotch control. Recently, SDHI-resistant isolates have been found in the field. Intensive sensitivity monitoring programmes across Europe were carried out to investigate the situation concerning SDHI resistance in P. teres. RESULTS: The first isolates with a lower sensitivity to SDHIs registered in barley were found in Germany in 2012 and carried the B-H277Y substitution in the succinate dehydrogenase enzyme. In 2013 and 2014, a significant increase in isolates with lower SDHI sensitivity was detected mainly in France and Germany, and the range of target-site mutations increased. Most of the resistant isolates carried the C-G79R substitution, which exhibits a strong impact on all SDHIs in microtitre tests. All SDHIs tested were shown to be cross-resistant. Other substitutions are gaining in importance, e.g. C-N75S in France and D-D145G in Germany. So far, no double mutants in SDH genes have been detected. Glasshouse tests showed that SDHI-resistant isolates were still controlled by the SDHI fluxapyroxad when applied preventively. To date, most isolates with C-G79R substitution have not simultaneously carried the F129L change in cytochrome b, which causes resistance towards QoI fungicides at low to moderate levels. CONCLUSION: Several target-site mutations in the genes of subunits SDH-B, SDH-C and SDH-D with different impact on SDHI fungicides were detected. The pattern of mutations varied from year to year and between different regions. Strict resistance management strategies are recommended to maintain SDHIs as effective tools for net blotch control, especially in areas with low frequencies of resistant isolates. © 2016 Society of Chemical Industry.

Detection of the F129L mutation in the cytochrome b gene in Phakopsora pachyrhizi.

BACKGROUND: Asian soybean rust, caused by Phakopsora pachyrhizi, is mostly controlled by demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides. Mutations in the cytochrome b (CYTB) gene can lead to pathogen resistance to QoIs. The occurrence of the mutations in codons 129, 137 and 143 in the CYTB gene was investigated, and a pyrosequencing assay was developed for rapid and quantitative detection of the F129L mutation. RESULTS: Molecular analysis of the CYTB gene showed the presence of the F129L mutation in field samples and monouredinial isolates, while other mutations (G143A and G137R) were not found. The pyrosequencing was an effective method for quantitative detection of the F129L mutation, and many of the P. pachyrhizi samples showed high frequency of F129L. CONCLUSION: This is the first report of the occurrence of the F129L mutation in P. pachyrhizi. The practical relevance of this mutation for field efficacy of QoIs needs further investigation. © 2015 Society of Chemical Industry.

Differences in the efficacy of carboxylic acid amide fungicides against less sensitive strains of Plasmopara viticola.

BACKGROUND: Plasmopara viticola is controlled by fungicides with different modes of action, including carboxylic acid amides (CAAs). The aim of this study was to evaluate differences in the response of CAA-resistant P. viticola strains towards CAAs. RESULTS: The G1105S mutation affects all four CAAs, but with different impacts. While this confirms that they have the same mode of action, it shows that differences between CAAs can occur. CONCLUSION: Further molecular modelling and docking studies are needed to gain a better understanding of the different behaviours reported here. © 2015 Society of Chemical Industry.

Binding of the respiratory chain inhibitor ametoctradin to the mitochondrial bc1 complex.

BACKGROUND: Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc1 complex of oomycetes. The bc1 complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross-resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. RESULTS: Cross-resistance analyses, the reduction of isolated Pythium sp. bc1 complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc1 complex similarly to stigmatellin. CONCLUSION: The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross-resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates.

A Botrytis cinerea Population from a Single Strawberry Field in Germany has a Complex Fungicide Resistance Pattern.

Gray mold, caused by the fungus Botrytis cinerea, is one of the most important diseases of strawberry in Germany. The application of site-specific fungicides remains the main strategy to reduce disease incidence and severity in the field. Isolates (n = 199) were collected from fungicide-treated strawberry fruit at a German research site with a long history of fungicide efficacy trials against gray mold. Sensitivities to the six site-specific botryticides registered in Germany were determined using microtiter assays. Values for the concentration of a fungicide at which fungal development is inhibited by 50% (EC50) ranged from 0.03 to ≥30 ppm for the succinate dehydrogenase inhibitor boscalid, 0.015 to ≥10 ppm for the hydroxyanilide fenhexamid, 0.009 to 0.739 ppm for the phenylpyrrole fludioxonil, 0.55 to 43.45 ppm for the dicarboximide iprodione, 0.021 to ≥3 ppm for the quinone outside inhibitor pyraclostrobin, and 0.106 to ≥30 ppm for the anilinopyrimidine pyrimethanil. Pyrosequencing revealed that amino acid substitutions in the target proteins Bos1 (I365S/N, V368F + Q369H), CytB (G143A), Erg27 (F412S), and SdhB (P225F, N230I, and H272R/Y) were associated with reduced sensitivity levels to the corresponding fungicide classes. In most cases, isolates with a decreased sensitivity to fludioxonil showed a reduced sensitivity to tolnaftate. This reduction is considered to be an indication of multidrug efflux pump activity. The amino acid change I365S, I365N, or V368F + Q369H in Bos1 and H272R in SdhB by itself showed EC50 values of 3.99 to 14.73 ppm, 3.87 to 5.37 ppm, 4.81 to 15.63 ppm, and 2.071 to ≥30 ppm, respectively. When isolates that contained one of these mutations were also multidrug resistant, the ranges of EC50 values shifted to 6.47 to 43.45 ppm for I365S, 7.28 to 29.84 ppm for I365N, 6.89 to 26.67 ppm for V368F + Q369H, and ≥30 ppm for H272R. The reported data suggest that the combination of multidrug resistance and an amino acid change in the target site may result in a lower sensitivity to the fungicides than one resistance mechanism by itself. Although 20% of the population analyzed was sensitive to all six different chemical classes, the majority showed reduced sensitivity to one (6%), two (13%), three (23%), four (17%), five (11%), and six (11%) different fungicides.

Sensitivity of Phakopsora pachyrhizi towards quinone-outside-inhibitors and demethylation-inhibitors, and corresponding resistance mechanisms.

BACKGROUND: Since the invasion of Phakopsora pachyrhizi (Syd. & P. Syd.) in Brazil, there have been detrimental yield losses of soybeans [Glycine max (L.) Merr.]. Disease management is mainly based on fungicide treatment. The sensitivity of single P. pachyhrizi isolates towards different demethylation-inhibitors (DMIs) and quinone-outside-inhibitors (QoI) was surveyed and the corresponding resistance mechanisms were analysed. RESULTS: The QoI-response remained stable, while a loss of sensitivity towards DMIs occurred. Molecular analyses of cytochrome b showed an intron after codon 143 which is reported to prevent the development of a G143A mutation. Analysis of cyp51 revealed that point mutations and overexpression are involved in the sensitivity reduction towards DMIs. Of the detected mutations, Y131F and Y131H, respectively, and K142R are likely homologous to mutations found in other pathogens. As suggested by modelling studies, these three mutations as well as additional mutations F120L, I145F and I475T correlate to increased effective doses of 50%, ED50 -values, towards all tested DMIs. Furthermore, a constitutive up-regulation of the cyp51-gene up to ten-fold was noticed in some of the DMI-adapted isolates, while all sensitive isolates responded as the wild type. CONCLUSION: The G143A mutation is thought to result in significant as well as stable resistance factors towards QoIs, while other mutations play only a minor role. Since G143A development is prevented in Phakopsora pachyhrizi, a stable control of soybean rust with QoIs in future is rather likely. In contrast, a shifting in sensitivity towards DMIs has been observed, which is due to multiple independent mechanisms.

A Two-Step Molecular Detection Method for Pyrenophora tritici-repentis Isolates Insensitive to QoI Fungicides.

Tan spot, caused by Pyrenophora tritici-repentis, is an important disease of wheat worldwide. To manage tan spot, quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin have been applied in many countries. QoI fungicides target the cytochrome b (cyt b) site in complex III of mitochondria and, thus, pose a serious risk for resistance development. The resistance mechanism to QoI fungicides is mainly due to point mutations in the cyt b gene. The objective of this study was to develop a molecular detection method for the four currently known mutations responsible for shifts in sensitivity toward QoI fungicides in P. tritici-repentis. Twelve specific primers were designed based on sequences from the National Center for Biotechnology Information accessions AAXI01000704 and DQ919068 and used to generate a fragment of the cyt b gene which possesses four known single-nucleotide polymorphisms (SNPs). These mutant clones served as positive controls because QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis have not yet been reported in the United States. The partial cyt b gene clones were sequenced to identify the SNPs at sites G143A and F129L. Genomic DNA of the mutated partial cyt b gene clones and the European QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis possessing G143A (GCT) and F129L (TTA, TTG, and CTC) mutations were amplified by polymerase chain reaction (PCR) using two specific primer pairs and were further digested with three specific restriction enzymes (BsaJI, Fnu4HI, and MnlI). The results of the digested PCR product from genomic DNA of known QoI-insensitive and -reduced-sensitive isolates of P. tritici-repentis had DNA bands consistent with the mutation GCT at G143A and the mutations TTA, TTG, and CTC at F129L. The amplified region at the F129 site also had 99% sequence similarity with P. teres, the net blotch pathogen of barley. To validate mutations, we further tested two isolates of P. teres known to have reduced sensitivity to QoI fungicides possessing the mutations TTA and CTC at F129L. After PCR amplification and restriction digestion, DNA bands identical to those observed for the partial cyt b mutant clones were detected. These results suggest that this newly developed two-step molecular detection method is rapid, robust, and specific to monitor QoI-insensitive and -reduce-dsensitive isolates of P. tritici-repentis.

Generation and characterization of isolates of Peronophythora litchii resistant to carboxylic acid amide fungicides.

Four isolates of Peronophythora litchii with resistance to carboxylic acid amide (CAA) fungicides were selected on fungicide-amended agar. These isolates had various levels of resistance, as evidenced by their resistance factor (RF), which is the 50% effective concentration (EC(50)) value of a particular isolate divided by that of the wild-type parent. RF values to dimethomorph for the four isolates were 15, 24, 141, and >1,500. Resistance was stable for two isolates, while the EC(50) values decreased for the other two after repeated subculturing on fungicide-free medium. Cross-resistance occurred with all CAAs tested here (dimethomorph, mandipropamid, flumorph, and pyrimorph), but not with strobilurins (azoxystrobin and famoxadone) or other fungicides (metalaxyl, cymoxanil, and mancozeb). Studies on fitness parameters (mycelial growth, sporulation, spore germination, zoospore formation, aggressiveness, and temperature tolerance) in the parent wild-type and resistant isolates demonstrated that penalties in different parameters may be associated with CAA resistance, depending on the isolate. These studies show that Peronophythora litchii is able to express CAA resistance under laboratory conditions but it is not known if resistant strains could become established in the field and sensitivity monitoring studies are recommended.

Modulation of multidrug resistance in human ovarian cancer cell lines by inhibition of P-glycoprotein 170 and PKC isoenzymes with antisense oligonucleotides.

Multidrug resistance in human ovarian carcinoma cell lines is caused by the expression of several related proteins, namely P-glycoprotein 170 (Pgp-170), glutathione S-transferase-pi GST-pi), and thymidylate synthase (TS). These proteins seem to be regulated by a common mechanism in which the expression of protein kinase C (PKC) is involved. Additionally, the function of Pgp-170 is dependent on PKC phosphorylation. However, in ovarian carcinoma cell lines the role of different PKC enzymes responsible for resistance is not quite clear. In the present study we circumvented resistance in taxol resistant human ovarian carcinoma cell lines with antisense oligonucleotides to PKC alpha and PKC beta mRNA and compared the effects with those obtained by Pgp-170 antisense oligonucleotides. We found a significant inhibition of cell number after treatment with Pgp-170 antisense oligonucleotides in combination with taxol. Additionally, resistance could be reversed by treatment with taxol and antisense oligomers to PKC alpha and PKC beta. This shows that regulatory correlations between these proteins exist and that inhibition of the mRNA of PKC alpha and PKC beta isoforms and Pgp-170 can reverse multidrug resistance.