Genome-based discovery of polyketide-derived secondary metabolism pathways in the barley pathogen <i>Ramularia collo-cygni</i>

Ramularia collo-cygni causes Ramularia leaf spot (RLS) disease of barley. The fungus develops asymptomatically within its host until late in the growing season, when necrotic lesions become visible on upper leaves. Fungal secondary metabolites (SM) have been proposed as important factors in RLS lesion formation but the biosynthetic pathways involved remain largely unknown. Mining the R. collo-cygni genome revealed the presence of 10 polyketide synthases (PKS), 10 nonribosomal peptide synthetases (NRPS), and 3 hybrid PKS-NRPS (HPS) identified within clusters of genes with predicted functions associated with secondary metabolism. SM core genes along with their predicted transcriptional regulators exhibited transcriptional coexpression during infection of barley plants. Moreover, their expression peaked during early stages of host colonization and preceded or overlapped with the appearance of disease symptoms, suggesting that SM may manipulate the host to promote colonization or protect R. collo-cygni from competing organisms. Accordingly, R. collo-cygni inhibited the growth of several fungi in vitro, indicating that it synthesized and excreted antifungal agents. Taken together, these findings demonstrate that the R. collo-cygni genome contains the genetic architecture to synthesize a wide range of SM and suggests that coexpression of PKS and HPS is associated with competitive colonization of the host and early symptom development. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license . </jats:p

The genome of the emerging barley pathogen Ramularia collo-cygni

Background Ramularia collo-cygni is a newly important, foliar fungal pathogen of barley that causes the disease Ramularia leaf spot. The fungus exhibits a prolonged endophytic growth stage before switching life habit to become an aggressive, necrotrophic pathogen that causes significant losses to green leaf area and hence grain yield and quality. Results The R. collo-cygni genome was sequenced using a combination of Illumina and Roche 454 technologies. The draft assembly of 30.3 Mb contained 11,617 predicted gene models. Our phylogenomic analysis confirmed the classification of this ascomycete fungus within the family Mycosphaerellaceae, order Capnodiales of the class Dothideomycetes. A predicted secretome comprising 1053 proteins included redox-related enzymes and carbohydrate-modifying enzymes and proteases. The relative paucity of plant cell wall degrading enzyme genes may be associated with the stealth pathogenesis characteristic of plant pathogens from the Mycosphaerellaceae. A large number of genes associated with secondary metabolite production, including homologs of toxin biosynthesis genes found in other Dothideomycete plant pathogens, were identified. Conclusions The genome sequence of R. collo-cygni provides a framework for understanding the genetic basis of pathogenesis in this important emerging pathogen. The reduced complement of carbohydrate-degrading enzyme genes is likely to reflect a strategy to avoid detection by host defences during its prolonged asymptomatic growth. Of particular interest will be the analysis of R. collo-cygni gene expression during interactions with the host barley, to understand what triggers this fungus to switch from being a benign endophyte to an aggressive necrotroph

Confusions in Fungal Systematics

The focus of this thesis has been on fungi with corticioid, polyporoid or stipitate stereoid sporocarps in the Agaricomycetes, but included are also two papers which are methodologically oriented. The family Podoscyphaceae, which is comprised of stipitate stereoid fungi, is studied with regard to repre sentatives of five genera utilizing molecular marker LSU. Species in the family Podoscyphaceae are recovered in orders Agaricales, Hymenochaetales, Polyporales, Atheliales, and in one new order. The new order Stereopsidales, and the family Stereopsidaceae are described after molecular phylogenetic analyses of the nuclear genes rpb2, tef1, nLSU and nSSU, incorporating Stereopsis radicans and the new combination Stereopsis globosum, formerly Clavulicium globosum. Clavulicium macounii, which shares morphological traits with the Stereopsidales, is recovered as the sister lineage to the Stereopsidales or as sister to the Phallomycetidae, and is left incertae sedis. Polyporales is studied with genomic data and multi-locus phylogenies, but the species relationships are still difficult to resolve, especially with regard to corticioid and resupinate species. Steccherinaceae, which is comprised of resupinate polypores and resupinate hydnoid species, is studied in more detail. Genera Antrodiella, Steccherinum and Junghunia, are highly polyphyletic, showing once more that the morphological characters used to classify fungi have been misleading. Adding unidentified sequences to phylogenetic studies on any fungal group has an effect on the phylogenetic interpretation which can not be ignored, and it is recommended that significant BLAST hits are included in the phylogeny. Single copy genes inferred from proteomes of 50 Agaricomycotina species have slightly different evolutionary histories, and many splits can not be resolved

Stereopsidales - A new order of mushroom-forming fungi

One new order, one new family, and one new combination are presented, as the result of molecular phylogenetic analyses. The new order Stereopsidales and the new family Stereopsidaceae are described incorporating Stereopsis radicans and S. globosa, formerly Clavulicium globosum. We show that not only do these species represent an old overlooked lineage, but both species harbor cryptic diversity. In addition, a third species, C. macounii, appears as a plausible sister to the new lineage, but there is conflict in the data. All specimens of S. radicans and S. globosa analysed here are from the South and Central Americas; several records of S. radicans have been made also from tropical Asia. We expect the true diversity in this group to be a lot higher than presented in this paper. Stereopsis radicans was formerly included in Polyporales, but a placement within that order is rejected by our data through SH tests. The dataset consisted of four nuclear markers: rpb2, tef1, LSU and SSU, each of which was analysed separately using maximum likelihood and Bayesian inference. Recombination detection tests indicate no plausible recombinations. The potential of S. radicans, S. globosa and C. macounii being amphitallic is briefly discussed

Deciphering Molecular Host-Pathogen Interactions During Ramularia Collo-Cygni Infection on Barley

Ramularia collo-cygni is the causal agent of Ramularia leaf spot disease (RLS) on barley and became, during the recent decades, an increasing threat for farmers across the world. Here, we analyze morphological, transcriptional, and metabolic responses of two barley cultivars having contrasting tolerance to RLS, when infected by an aggressive or mild R. collo-cygni isolate. We found that fungal biomass in leaves of the two cultivars does not correlate with their tolerance to RLS, and both cultivars displayed cell wall reinforcement at the point of contact with the fungal hyphae. Comparative transcriptome analysis identified that the largest transcriptional differences between cultivars are at the early stages of fungal colonization with differential expression of kinases, calmodulins, and defense proteins. Weighted gene co-expression network analysis identified modules of co-expressed genes, and hub genes important for cultivar responses to the two R. collo-cygni isolates. Metabolite analyses of the same leaves identified defense compounds such as p-CHDA and serotonin, correlating with responses observed at transcriptome and morphological level. Together these all-round responses of barley to R. collo-cygni provide molecular tools for further development of genetic and physiological markers that may be tested for improving tolerance of barley to this fungal pathogen

SSU phylogeny.

<p>Maximum likelihood tree of nSSU with Bootstrap/Bayesian frequencies as a percentage shown above branches. Thick branches receive full support of both Bayesian frequencies and Bootstrap. The collapsed and colored groups represent current orders and subclasses of the Agaricomycetes.</p

Microscope images of hymenium structures.

<p>Hymenium structures in 3% KOH showing basidiospores and palisades of basidia. a) <i>Stereopsis radicans</i>, arrows indicating developing spore (subglobose) attached to sterigmata, and mature dried (angular) spore. b) <i>Stereopsis globosa</i>, arrows indicating basidia with developed sterigmata, and dried angular spores. c) <i>Clavulicium macounii</i> arrow indicating free floating mature spore (cylindrical).</p

Collection ID, Collection information and GenBank numbers of newly generated sequences.

<p>Collection ID, Collection information and GenBank numbers of newly generated sequences.</p

Results of the SH tests.

<p>The question of whether the tested hypothesis results in a significantly worse tree than the ML tree under a p value of 0.05 is answered by a yes or a no. Log Likelihood values for the best tree under each hypothesis is given as a negative value. H1: <i>Stereopsis radicans</i>, <i>C. macounii</i>, <i>C. globosum</i> monophyletic. H2: Phallomycetidae, <i>Stereopsis radicans</i>, <i>C. macounii</i>, <i>C. globosum</i> monophyletic. H3: Phallomycetidae monophyletic and <i>Stereopsis radicans</i>, <i>C. macounii</i>, <i>C. globosum</i> monophyletic. H4: Phallomycetidae, <i>C. macounii</i> monophyletic H5: Polyporales, Stereopsidales monophyletic.</p