Analysis of the emerging situation of resistance to succinate dehydrogenase inhibitors in Pyrenophora teres and Zymoseptoria tritici in Europe

Phytopathogenic fungi such as Pyrenophora teres and Zymoseptoria tritici cause destructive diseases of barley and wheat in all major cereal production areas worldwide. The control of net blotch of barley caused by P. teres and Septoria tritici blotch (STB) of wheat caused by Z. tritici mainly relies on the usage of fungicides. Thereby, three single-site inhibiting fungicide classes, the quinone outside inhibitors (QoIs), the demethylation inhibitors (DMIs) and the succinate dehydrogenase inhibitors (SDHIs) have the highest relevance. The class of SDHIs is the most newly introduced fungicide class and inhibits the fungal succinate dehydrogenase complex (SDH) which is a critical enzyme of the respiratory chain and the tricarboxylic cycle. The upcoming SDHI resistance in European populations of P. teres and Z. tritici was investigated in the present study and resistance mechanisms underlying SDHI resistance were characterised. SDHI resistant isolates of both pathogens were collected in intensive monitoring programmes which covered the major barley and wheat growing areas in Europe. SDHI resistant isolates showed point mutations in the genes SdhB, SdhC and SdhD which cause amino acid alteration in the subunits B, C and D of the SDH complex. First SDHI resistant isolates of both pathogens were detected in 2012 and showed amino acid alteration, histidine to tyrosine at position 277 in SDH B (B-H277Y) in the case of P. teres and a threonine to asparagine exchange at position 79 in SDH-C (C-T79N) in the case of Z. tritici. In P. teres, a significant increase of SDHI resistant isolates from 2012 to 2015 was observed, particularly in countries such as France and Germany. Several target-site mutations leading to amino acid exchanges, namely B-H277Y, C-S73P, C-N75S, C-G79R, C-H134R, C-S135R, D-D124N/E, D-H134R, D-G138V, D-D145G and D-E178K, were identified in those isolates. Sequencing of SdhB, SdhC and SdhD genes of several isolates confirmed that each isolate carried one mutation in the Sdh genes, and not two or more in combination. In vitro and in planta sensitivity tests were performed and revealed that each SDH-variant causes a distinct resistance phenotype towards SDHIs. Commercially available SDHIs were compared and isolates showed cross-resistance towards all SDHIs tested, although some minor differences in the response to different mutations were observed. Most of the SDHI resistant P. teres isolates carried C-G79R substitution which was shown to exhibit one of the strongest effects of all detected alterations. In addition to C-G79R, other substitutions, such as C-N75S and D-D145G, were frequently found in the field. These SDH-variants were shown to confer low to moderate levels of resistance. In contrast to the rapid build-up of resistant isolates in the population of P. teres in countries such as France and Germany, the emergence of SDHI resistance in Z. tritici did not evolve as fast as observed in net blotch. Here, only a few resistant isolates have been sampled so far (42 resistent of 3431 investigated isolates, 1.2%). An increase of resistant isolates of Z. tritici was observed mainly in Ireland, the United Kingdom and the Netherlands, however, still at low levels. SDH variants B-N225I, B-T268I/A, C-N86S/A, C-T79N/I, C-W80S, C-H152R and C-V166M were detected in SDHI resistant isolates collected in these and other countries such as France and Germany. Four isolates showed two mutations in the Sdh genes in combination. In vitro and in planta sensitivity measurements demonstrated that C-H152R mutants showed the highest resistance level of all investigated SDH variants collected in the field. C-T79N and C-N86S exchanges which have been detected more frequently in the field than C-H152R, were shown to confer lower levels of resistance compared to C-H152R. Both phytopathogenic species were shown to evolve a range of diverse target-site mutations, which led to different alterations in both pathogen species with exception of C-N75S in P. teres and the homologous variant, C-N86S, in Z. tritici. This can be explained by species-specific variation of the SDH enzyme, a different nature of the pathogens (e.g. host plants and disease geographical spread) as well as a different fungicide use pattern (e.g. mode of action diversity and fungicide application intensity). The absence of a dominant major target-site mutation in the case of SDHI resistance in both pathogens is thought to allow SDHIs as effective control agent against both pathogen species also in the future. Nevertheless, anti-resistance management strategies are highly recommended for the usage of SDHIs. These strategies should not only be based on the use of mixtures and alternations of fungicides but should also implement integrated disease control measurements (e.g. resistant host cultivars).Phytopathogene Pilze wie Pyrenophora teres und Zymoseptoria tritici verursachen ertragsrelevante Krankheiten an Gerste und Weizen und sind in allen Getreide-anbaugebieten weltweit verbreitet. Die Bekämpfung der Netzfleckenkrankheit an Gerste, ausgelöst durch den Erreger P. teres, und der Septoria-Blattdürre an Weizen, ausgelöst durch den Erreger Z. tritici, wird größtenteils durch den Einsatz von Fungiziden gewährleistet. Dabei finden die drei single-site-Fungizid-Klassen der Quinone-outside-Inhibitoren (QoIs), der Demethylase-Inhibitoren (DMIs) und der Succinat-Dehydrogenase-Inhibitoren (SDHIs) den größten Einsatz. Die SDHIs repräsentieren die neuste der drei Fungizid-Klassen und inhibieren die pilzliche Succinat-Dehydrogenase (SDH), die ein wichtiger Bestandteil der Atmungskette und des Citrat-Zyklus in Lebewesen darstellt. Die Entstehung von Fungizid-Resistenzen in den Pflanzenpathogenen P. teres und Z. tritici gegenüber der Klasse der SDHIs und deren Charakterisierung war Bestandteil der vorliegenden Arbeit. Isolate beider Pathogene wurden in großangelegten Monitoring-Studien gesammelt und umfassten alle wichtigen Gersten- und Weizenanbaugebiete Europas. SDHI-resistente Isolate zeigten Punktmutationen in den Genen SdhB, SdhC und SdhD, die zu Aminosäure-Substitutionen in den SDH-B, SDH-C und SDH-D-Untereinheiten des SDH-Komplexes führen. Die ersten resistenten Isolate wurden im Jahr 2012 gesammelt und führten zu der Aminosäure-Substitution, Histidin zu Tyrosin an Position 277 der SDH B Untereinheit in P. teres (B-H277Y) und Threonin zu Asparagin an Position 79 der SDH-C Untereinheit in Z. tritici (C-T79N). Im Falle von P. teres wurde ein starker Anstieg der resistenten Isolate in den folgenden Jahren hauptsächlich in Deutschland und in Frankreich festgestellt. Die detektierten Punktmutationen führten zu den Aminosäure-Substitutionen B-H277Y, C-S73P, C-N75S, C-G79R, C-H134R, C-S135R, D-D124N/E, D-H134R, D-G138V, D-D145G und D-E178K. Die Sequenzierung der resistenten Isolate zeigte, dass jedes Isolat nur einen Austausch in der SDH aufwies, nie jedoch zwei oder mehr Substitutionen in einem Isolat aufzufinden waren. In vitro- und in planta-Sensitivitätsstudien wurden durchgeführt und zeigten, dass jede Substitution einen spezifischen Einfluss auf die Sensitivität der SDHIs hatte. Verschiedene SDHIs, die auf dem Markt erhältlich sind, wurden verglichen und es zeigte sich, dass sich alle SDHIs in Bezug auf die Resistenzstärke der einzelnen Mutationen ähnlich verhielten. Die meisten SDHI-resistenten Isolate von P. teres hatten den C-G79R-Austausch. Diese Substitution führte zu einem der stärksten Wirkungsverluste aller SDH-Varianten. Substitutionen, die einen schwächerem Wirkungsverlust der SDHIs aufwiesen, wie z.B. die C-N75S- und D-D145G-Substitution, wurden zusätzlich zur C-G79R häufig in der Feldpopulation gefunden. Im Vergleich zu dem raschen Aufkommen von SDHI-resistenten Isolaten von P. teres in Ländern, wie z.B. Deutschland und Frankreich, scheint die Entwicklung von SDHI-Resistenzen bei Z. tritici langsamer vonstattenzugehen. Bisher wurden nur wenige SDHI-resistente Z. tritici Isolate überhaupt gesammelt (42 resistente von 3431 untersuchten Isolaten, 1,2%). In Ländern wie Irland, Großbritannien und in den Niederlanden wurde auch bei Z. tritici ein Anstieg der resistenten Isolate beobachtet, jedoch nur in geringem Ausmaß. Die Aminosäure-Substitutionen B-N225I, B-T268I/A, C-N86S/A, C-T79N/I, C-W80S, C-H152R und C-V166M wurden in den resistenten Isolaten aus diesen und weiteren Ländern, wie Frankreich und Deutschland, über die Jahre gefunden. Der höchste Wirkungsverlust aller SDHIs wurde bei Isolaten mit der C-H152R-Substitution beobachtet. Die am häufigsten gefundenen SDH-Varianten, C-T79N und C-N86S, wiesen dagegen deutlich geringere Wirkungsverluste, verglichen mit der C-H152R-Substitution, auf. Es wurde gezeigt, dass beide phytopathogene Pilzarten eine hohe Anzahl an verschiedenen Target-site-Mutationen im Falle der SDHI-Resistenz entwickeln können und diese sich stark zwischen den beiden Pathogenen unterscheiden. Das kann mit der spezies-abhängigen Variation des SDH-Enzyms zusammenhängen, aber auch in der unterschiedlichen Biologie der Pathogene (z.B. Wirtpflanze, geographisches Vorkommen der Krankheiten) und einem unterschiedlichen Fungizid-Einsatz (z.B. Intensität in Gerste und Weizen) begründet liegen. Die Abwesenheit von einer dominanten Target-site-Mutation in beiden Pathogenen, wie z.B. im Falle der QoI-Resistenz durch G143A in Z. tritici, deutet darauf hin, dass SDHIs in der Zukunft immer noch effektiv zur Kontrolle beider Pflanzenkrankheiten eingesetzt werden können. Trotzdem ist ein Anti-Resistenz-Management für den Einsatz von SDHIs essentiell und sollte nicht nur auf Mischungen und Alternierung von Fungiziden beruhen, sondern auch integrierte Bekämpfungsstrategien stärker mit einbinden

Das Bistum Konstanz 3. Das Zisterzienserinnenkloster Wald

Maren Kuhn-Rehfus, Das Bistum Konstanz 3: Das Zisterzienserinnenkloster Wald (Germania Sacra N. F. 30), Berlin/New York 1992

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51

Pyrenophora teres f. sp. teres is the cause of net form of net blotch, an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of net form of net blotch in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modelling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localised constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2 and Cyp51A1 was shown to be 1.6-, 3- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use

“What About Your Friends?”: How a Collaborative Transdisciplinary Training Approach Supports FAIR Data Sharing Principles in Federally Funded Research

Objective: This paper details a pilot project to establish a baseline for current data management planning activities and offers more targeted data management planning training to researchers.Methods: The authors incorporated a collaborative transdisciplinary approach, leading to the development and delivery of a series of surveys to gain accurate feedback about current workflows, policy adherence, and identifying knowledge gaps.Results: Using formal survey results and informal feedback from researcher interactions to inform targeted training sessions and materials results in a more productive and collaborative experience for researchers and leads to more complete and structured data management plans.Conclusions: Understanding researchers’ current practices and needs is crucial to developing effective training and resources to help improve data management planning and workflows

Non‐target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici

BACKGROUND A new generation of more active succinate dehydrogenase (Sdh) inhibitors (SDHIs) is currently widely used to control Septoria leaf blotch in northwest Europe. Detailed studies were conducted on Zymoseptoria tritici field isolates with reduced sensitivity to fluopyram and isofetamid; SDHIs which have only just or not been introduced for cereal disease control, respectively. RESULTS Strong cross‐resistance between fluopyram and isofetamid, but not with other SDHIs, was confirmed through sensitivity tests using laboratory mutants and field isolates with and without Sdh mutations. The sensitivity profiles of most field isolates resistant to fluopyram and isofetamid were very similar to a lab mutant carrying SdhC‐A84V, but no alterations were found in SdhB, C and D. Inhibition of mitochondrial Sdh enzyme activity and control efficacy in planta for those isolates was severely impaired by fluopyram and isofetamid, but not by bixafen. Isolates with similar phenotypes were not only detected in northwest Europe but also in New Zealand before the widely use of SDHIs. CONCLUSION This is the first report of SDHI‐specific non‐target site resistance in Z. tritici. Monitoring studies show that this resistance mechanism is present and can be selected from standing genetic variation in field populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry