Characterization of fungal and bacterial communities associated with mat-forming ectomycorrhizal fungi from old-growth stands in the H.J. Andrews Experimental Forest

Mat-forming ectomycorrhizal (EcM) fungi represent a prevalent constituent of many temperate forest ecosystems and create dramatic changes in soil structure and chemistry. EcM mat soil have been shown to have increased microbial respiration rates and have been hypothesized to harbor unique assemblages of fungi and bacteria. The objectives of this dissertation were to characterize and examine the fungal and bacterial communities associated with EcM mats in old-growth forests of the H.J. Andrews Experimental Forest located in the Oregon Cascades. Additionally, this work assessed the application of traditional, emerging, and novel molecular sampling techniques for determining microbial communities of environmental samples. This research investigated the microbial communities associated with two common EcM mat genera found in old-growth Douglas fir stands in the Pacific Northwest; Piloderma (Atheliales, Basidiomycota) and Ramaria (Gomphales, Basidiomycota). Soil samples were collected from Piloderma and Ramaria mats and surrounding non- mat soil for molecular analysis of nucleic acids. First, a comparative study was conducted to determine the most appropriate rDNA molecular sampling technique for microbial community characterization. Two next-generation sequencing methods, Roche 454 pyrosequencing and Illumina-based environmental sequencing, the latter developed by the author, were compared to a more traditional sequencing approach, i.e., Sanger sequencing of clone libraries. These findings informed the subsequent sampling of the fungal ITS and bacterial 16S rDNA fragment with 454 pyrosequencing to determine the microbial communities within mat and non-mat soils. Second, this work utilized a pyrosequencing approach to explore fungal community structure in EcM mat and non-mat soils. This work concluded that differences in microbial communities do exist between Piloderma mat, Ramaria mat, and non-mat soils, but the differences are largely quantitative with relatively few distinct taxonomic shifts in microbial constituents. Piloderma, Ramaria and Russula, in addition to being the dominant taxa found on mycorrhizal root tips, were found to be the most abundant taxa in bulk soils within their respective mat types or non-mat sample. The background fungal communities within the EcM mats in this study exhibited considerable taxonomic overlap with the exception of Piloderma vs. non-mat comparisons; Russula species dominated nonmat soils but tended to be excluded or significantly underrepresented in Piloderma mats. Lastly, this study explored the bacterial communities associated with Piloderma and Ramaria mats using lower- coverage 454-Jr pyrosequencing. Bacterial communities exhibited significant structure as a function of mat-type, soil horizon and pH, but this finding should be interpreted with respect to the nonrandom distribution of Piloderma-mats in the O- horizon and the Ramaria-mats in the A-horizon, and the tendancy for EcM mats to be more acidic than surrounding soils. Nonetheless, the total microbial (bacterial and fungal) community was typically dominated by the mat-forming taxa, or Russula, in the case of non-mat soils. While the presence of Piloderma mats did enrich or restrict some bacterial groups, soil pH was also found to be a significant driver of bacterial richness and taxonomic diversity. Fungal and bacterial richness were also found to be positively related to one another, regardless of soil horizon or EcM mat type. This work, taken together, contributes to the understanding of hyperdiversity and heterogeneity of microbial communities of temperate forest soils and highlights the potential for fungal and bacterial communities to be influenced by the presence of EcM mats

The Rare Codon AGA Is Involved in Regulation of Pyoluteorin Biosynthesis in Pseudomonas protegens Pf-5

The soil bacterium Pseudomonas protegens Pf-5 can colonize root and seed surfaces of many plants, protecting them from infection by plant pathogenic fungi and oomycetes. The capacity to suppress disease is attributed to Pf-5's production of a large spectrum of antibiotics, which is controlled by complex regulatory circuits operating at the transcriptional and post-transcriptional levels. In this study, we analyzed the genomic sequence of Pf-5 for codon usage patterns and observed that the six rarest codons in the genome are present in all seven known antibiotic biosynthesis gene clusters. In particular, there is an abundance of rare codons in pltR, which encodes a member of the LysR transcriptional regulator family that controls the expression of pyoluteorin biosynthetic genes. To test the hypothesis that rare codons in pltR influence pyoluteorin production, we generated a derivative of Pf-5 in which 23 types of rare codons in pltR were substituted with synonymous preferred codons. The resultant mutant produced pyoluteorin at levels 15 times higher than that of the wild-type Pf-5. Accordingly, the promoter activity of the pyoluteorin biosynthetic gene pltL was 20 times higher in the codon-modified stain than in the wild-type. pltR has six AGA codons, which is the rarest codon in the Pf-5 genome. Substitution of all six AGA codons with preferred Arg codons resulted in a variant of pltR that conferred increased pyoluteorin production and pltL promoter activity. Furthermore, overexpression of tRNAUCUArg, the cognate tRNA for the AGA codon, significantly increased pyoluteorin production by Pf-5. A bias in codon usage has been linked to the regulation of many phenotypes in eukaryotes and prokaryotes but, to our knowledge, this is the first example of the role of a rare codon in the regulation of antibiotic production by a Gram-negative bacterium.Keywords: Pseudomonas protegens, pyoluteorin, rare codon, AGA codon, regulatio

Metagenome sequence of Elaphomyces granulatus from sporocarp tissue reveals Ascomycota ectomycorrhizal fingerprints of genome expansion and a Proteobacteria-rich microbiome

Many obligate symbiotic fungi are difficult to maintain in culture, and there is a growing need for alternative approaches to obtaining tissue and subsequent genomic assemblies from such species. In this study, the genome of Elaphomyces granulatus was sequenced from sporocarp tissue. The genome assembly remains on many contigs, but gene space is estimated to be mostly complete. Phylogenetic analyses revealed that the Elaphomyces lineage is most closely related to Talaromyces and Trichocomaceae s.s. The genome of E. granulatus is reduced in carbohydrate-active enzymes, despite a large expansion in genome size, both of which are consistent with what is seen in Tuber melanosporum, the other sequenced ectomycorrhizal ascomycete. A large number of transposable elements are predicted in the E. granulatus genome, especially Gypsy-like long terminal repeats, and there has also been an expansion in helicases. The metagenome is a complex community dominated by bacteria in Bradyrhizobiaceae, and there is evidence to suggest that the community may be reduced in functional capacity as estimated by KEGG pathways. Through the sequencing of sporocarp tissue, this study has provided insights into Elaphomyces phylogenetics, genomics, metagenomics and the evolution of the ectomycorrhizal association.This is the publisher’s final pdf. The article is copyrighted by the Society for Applied Microbiology and John Wiley & Sons, Ltd. It is published by John Wiley & Sons, Ltd. and can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291462-292

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis.

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode

Botryosphaeria dothidea : a latent pathogen of global importance to woody plant health

Botryosphaeria dothidea is the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales). Fungi residing in this order are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea one of the most common species on a large number of hosts. Its taxonomic circumscription has undergone substantial change in the past decade, making it difficult to interpret the large volume of literature linked to the name B. dothidea. This pathogen profile synthesises the current understanding of B. dothidea pertaining to its distribution, host associations and its role as a pathogen in managed and natural woody environments. The prolonged latent infection or endophytic phase is of particular importance as it implies that the fungus can easily pass undetected by quarantine systems in traded living plants, fruits and other plant parts. Infections typically become obvious only under conditions of host stress, when disease symptoms develop. This study also considers the knowledge emerging from the recently sequenced B. dothidea genome elucidating previously unknown aspects of the species, including mating and hostinfection strategies. Despite more than 150 years of research on B. dothidea, there is clearly much to be learned regarding this global tree pathogen. This is increasingly important given the stresses imposed on various woody hosts due to climate change.The National Research Foundation (NRF) of South Africa and members of the Tree Protection Co-operative Programme (TPCP).http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1364-37032018-05-31hb2017GeneticsMicrobiology and Plant Patholog

The genome of the xerotolerant mold Wallemia sebi reveals adaptations to osmotic stress and suggests cryptic sexual reproduction

Wallemia (Wallemiales, Wallemiomycetes) is a genus of xerophilic Fungi of uncertain phylogenetic position within Basidiomycota. Most commonly found as food contaminants, species of Wallemia have also been isolated from hypersaline environments. The ability to tolerate environments with reduced water activity is rare in Basidiomycota. We sequenced the genome of W. sebi in order to understand its adaptations for surviving in osmotically challenging environments, and we performed phylogenomic and ultrastructural analyses to address its systematic placement and reproductive biology. W. sebi has a compact genome (9.8 Mb), with few repeats and the largest fraction of genes with functional domains compared with other Basidiomycota. We applied several approaches to searching for osmotic stress-related proteins. In silico analyses identified 93 putative osmotic stress proteins; homology searches showed the HOG (High Osmolarity Glycerol) pathway to be mostly conserved. Despite the seemingly reduced genome, several gene family expansions and a high number of transporters (549) were found that also provide clues to the ability of W. sebi to colonize harsh environments. Phylogenetic analyses of a 71-protein dataset support the position of Wallemia as the earliest diverging lineage of Agaricomycotina, which is confirmed by septal pore ultrastructure that shows the septal pore apparatus as a variant of the Tremella-type. Mating type gene homologs were identified although we found no evidence of meiosis during conidiogenesis, suggesting there may be aspects of the life cycle of W. sebi that remain cryptic.Keywords: Ion homeostasis, Aqua(glycero)porins, Solute accumulation, Electron microscopy, Xerophile, Halophil

Appunti sul movimento antifascista sloveno della Venezia Giulia

<div><p>The class <em>Dothideomycetes</em> is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The <em>Dothideomycetes</em> most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several <em>Dothideomycetes</em> contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 <em>Dothideomycetes</em> offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the <em>Dothideomycetes</em>, the <em>Capnodiales</em> and <em>Pleosporales</em>, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most <em>Dothideomycetes</em> and upregulated during infection in <em>L. maculans</em>, suggesting a possible function in response to oxidative stress.</p> </div

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

Correction 5 Mar 2013: Ohm RA, Feau N, Henrissat B, Schoch CL, Horwitz BA, et al. (2013) Correction: Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi. PLOS Pathogens 9(3): 10.1371/annotation/fcca88ac-d684-46e0-a483-62af67e777bdThe class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress

The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non–lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period