Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross-resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51(I381V Y461H) or CYP51(I381V ΔY459/) (G460) , respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.

Ramularia collo-cygni is the biotic factor responsible for the disease Ramularia leaf spot (RLS) of barley (Hordeum vulgare). Despite having been described over 100 years ago and being considered a minor disease in some countries, the fungus is attracting interest in the scientific community as a result of the increasing number of recorded economically damaging disease epidemics. New reports of disease spread and fungal identification using molecular diagnostics have helped redefine RLS as a global disease. This review describes recent developments in our understanding of the biology and epidemiology of the fungus, outlines advances made in the field of the genetics of both the fungus and host, and summarizes the control strategies currently available.

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Ramularia leaf spot is becoming an ever-increasing problem in main barley-growing regions since the 1980s, causing up to 70% yield loss in extreme cases. Yet, the causal agent Ramularia collo-cygni remains poorly studied. The diversity of the pathogen in the field thus far remains unknown. Furthermore, it is unknown to what extent the pathogen has a sexual reproductive cycle. The teleomorph of R. collo-cygni has not been observed. To study the genetic diversity of R. collo-cygni and get more insights in its biology, we sequenced the genomes of 19 R. collo-cygni isolates from multiple geographic locations and diverse hosts. Nucleotide polymorphism analyses of all isolates shows that R. collo-cygni is genetically diverse worldwide, with little geographic or host specific differentiation. Next, we used two different methods to detect signals of recombination in our sample set. Both methods find putative recombination events, which indicate that sexual reproduction happens or has happened in the global R. collo-cygni population. Lastly, we used these data on recombination to perform historic population size analyses. These suggest that the effective population size of R. collo-cygni decreased during the domestication of barley and subsequently grew with the rise of agriculture. Our findings deepen our understanding of R. collo-cygni biology and can help us to understand the current epidemic. We discuss how our findings support possible global spread through seed transfer, and we highlight how recombination, clonal spreading, and lack of host specificity could amplify global epidemics of this increasingly important disease and suggest specific approaches to combat the pathogen.

Ramularia leaf spot has recently emerged as a major threat to barley production world-wide, causing 25% yield loss in many barley growing regions. Here, we provide a new reference genome of the causal agent, the Dothideomycete Ramularia collo-cygni. The assembly of 32 Mb consists of 78 scaffolds. We used RNA-seq to identify 11,622 genes of which 1,303 and 282 are coding for predicted secreted proteins and putative effectors respectively.The pathogen separated from its nearest sequenced relative, Zymoseptoria tritici ∼27 Ma. We calculated the divergence of the two species on protein level and see remarkably high synonymous and nonsynonymous divergence. Unlike in many other plant pathogens, the comparisons of transposable elements and gene distributions, show a very homogeneous genome for R. collo-cygni. We see no evidence for higher selective pressure on putative effectors or other secreted proteins and repetitive sequences are spread evenly across the scaffolds. These findings could be associated to the predominantly endophytic life-style of the pathogen. We hypothesize that R. collo-cygni only recently became pathogenic and that therefore its genome does not yet show the typical pathogen characteristics. Because of its high scaffold length and improved CDS annotations, our new reference sequence provides a valuable resource for the community for future comparative genomics and population genetics studies.

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Ramularia Leaf Spot (RLS) has emerged as a threat for barley production in many regions of the world. Late appearance of unspecific symptoms caused that Ramularia collo-cygni could only by molecular diagnostics be detected as the causal agent of RLS. Although recent research has shed more light on the biology and genomics of the pathogen, the cause of the recent global spread remains unclear.To address urgent questions, especially on the emergence to a major disease, life-cycle, transmission, and quick adaptation to control measures, we de-novo sequenced the genome of R. collo-cygni (urug2 isolate). Additionally, we sequenced fungal RNA from 6 different conditions, which allowed for an improved genome annotation. This resulted in a high quality draft assembly of about 32 Mb, with only 78 scaffolds with an N50 of 2.1 Mb. The overall annotation enabled the prediction of 12.346 high confidence genes. Genomic comparison revealed that R. collo-cygni has significantly diverged from related Dothidiomycetes, including gain and loss of putative effectors, however without obtaining species-specific genome features. To evaluate the species-wide genetic diversity, we sequenced the genomes of 19 R. collo-cygni isolates from multiple geographic locations and diverse hosts and mapped sequences to our reference genome. Admixture analyses show that R. collo-cygni is world-wide genetically uniform and that samples do not show a strong clustering on either geographical location or host species. To date, the teleomorph of R. collo-cygni has not been observed. Analysis of linkage disequilibrium shows that in the world-wide sample set there are clear signals of recombination and thus sexual reproduction, however these signals largely disappear when excluding three outliers samples, suggesting that the main global expansion of R. collo-cygni comes from mixed or clonally propagating populations. We further analysed the historic population size (Ne) of R. collo-cygni using Bayesian simulations.We discuss how our genomic data and population genetics analysis can help understand the current R. collocygni epidemic and provide different hypothesis that are supported by our data. We specifically highlight how recombination, clonal spreading and lack of host-specificity could further support global epidemics of this increasingly recognized plant disease and suggest specific approaches to combat this pathogen.

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