Contrasting demographic histories revealed in two invasive populations of the dry rot fungus Serpula lacrymans.

Globalization and international trade have impacted organisms around the world leading to a considerable number of species establishing in new geographic areas. Many organisms have taken advantage of human-made environments, including buildings. One such species is the dry rot fungus Serpula lacrymans, which is the most aggressive wood-decay fungus in indoor environments in temperate regions. Using population genomic analyses of 36 full genome sequenced isolates, we demonstrated that European and Japanese isolates are highly divergent and the populations split 3000-19,000 generations ago, probably predating human influence. Approximately 250 generations ago, the European population went through a tight bottleneck, probably corresponding to the fungus colonization of the built environment in Europe. The demographic history of these populations, probably lead to low adaptive potential. Only two loci under selection were identified using a Fst outlier approach, and selective sweep analyses identified three loci with extended haplotype homozygosity. The selective sweep analyses found signals in genes possibly related to decay of various substrates in Japan and in genes involved DNA replication and protein modification in Europe. Our results suggest that the dry rot fungus independently established in indoor environments in Europe and Japan and that invasive species can potentially establish large populations in new habitats based on a few colonizing individuals.

Identifying the Active Microbiome Associated with Roots and Rhizosphere Soil of Oilseed Rape.

RNA stable isotope probing and high-throughput sequencing were used to characterize the active microbiomes of bacteria and fungi colonizing the roots and rhizosphere soil of oilseed rape to identify taxa assimilating plant-derived carbon following 13CO2 labeling. Root- and rhizosphere soil-associated communities of both bacteria and fungi differed from each other, and there were highly significant differences between their DNA- and RNA-based community profiles. Verrucomicrobia, Proteobacteria, Planctomycetes, Acidobacteria, Gemmatimonadetes, Actinobacteria, and Chloroflexi were the most active bacterial phyla in the rhizosphere soil. Bacteroidetes were more active in roots. The most abundant bacterial genera were well represented in both the 13C- and 12C-RNA fractions, while the fungal taxa were more differentiated. Streptomyces, Rhizobium, and Flavobacterium were dominant in roots, whereas Rhodoplanes and Sphingomonas (Kaistobacter) were dominant in rhizosphere soil. "Candidatus Nitrososphaera" was enriched in 13C in rhizosphere soil. Olpidium and Dendryphion were abundant in the 12C-RNA fraction of roots; Clonostachys was abundant in both roots and rhizosphere soil and heavily 13C enriched. Cryptococcus was dominant in rhizosphere soil and less abundant, but was 13C enriched in roots. The patterns of colonization and C acquisition revealed in this study assist in identifying microbial taxa that may be superior competitors for plant-derived carbon in the rhizosphere of Brassica napusIMPORTANCE This microbiome study characterizes the active bacteria and fungi colonizing the roots and rhizosphere soil of Brassica napus using high-throughput sequencing and RNA-stable isotope probing. It identifies taxa assimilating plant-derived carbon following 13CO2 labeling and compares these with other less active groups not incorporating a plant assimilate. Brassica napus is an economically and globally important oilseed crop, cultivated for edible oil, biofuel production, and phytoextraction of heavy metals; however, it is susceptible to several diseases. The identification of the fungal and bacterial species successfully competing for plant-derived carbon, enabling them to colonize the roots and rhizosphere soil of this plant, should enable the identification of microorganisms that can be evaluated in more detailed functional studies and ultimately be used to improve plant health and productivity in sustainable agriculture.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BACKGROUND: Improved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq. RESULTS: Approximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10% of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation. CONCLUSIONS: This RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.

Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen.

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Niche differentiation and evolution of the wood decay machinery in the invasive fungus Serpula lacrymans.

Ecological niche breadth and the mechanisms facilitating its evolution are fundamental to understanding adaptation to changing environments, persistence of generalist and specialist lineages and the formation of new species. Woody substrates are structurally complex resources utilized by organisms with specialized decay machinery. Wood-decaying fungi represent ideal model systems to study evolution of niche breadth, as they vary greatly in their host range and preferred decay stage of the substrate. In order to dissect the genetic basis for niche specialization in the invasive brown rot fungus Serpula lacrymans, we used phenotyping and integrative analysis of phylogenomic and transcriptomic data to compare this species to wild relatives in the Serpulaceae with a range of specialist to generalist decay strategies. Our results indicate specialist species have rewired regulatory networks active during wood decay towards decreased reliance on enzymatic machinery, and therefore nitrogen-intensive decay components. This shift was likely accompanied with adaptation to a narrow tree line habitat and switch to a pioneer decomposer strategy, both requiring rapid colonization of a nitrogen-limited substrate. Among substrate specialists with narrow niches, we also found evidence for pathways facilitating reversal to generalism, highlighting how evolution may move along different axes of niche space.

Two invasive populations of the dry rot fungus Serpula lacrymans show divergent population genetic structures.

The dry rot fungus Serpula lacrymans is a devastating basidiomycete occurring in wooden constructions in temperate regions worldwide. In this study, we compare the genetic structures of two invasive populations from Europe and Japan. Microsatellite data from 14 loci and DNA sequences from four loci demonstrated that the two populations were highly differentiated. Significant isolation by distance effect was observed in Europe and Japan. Higher genetic variation was observed within the Japanese population than within the European population, corresponding with the observed higher richness of vegetative compatibility types in Japan, indicating that there has been a higher level of gene flow from the Asian source populations to Japan than to Europe. The European population is genetically more homogenous with only six detected vegetative compatibility types. Various tests indicate that both the European and the Japanese populations have gone through population bottlenecks prior to population expansion. No identical multi-locus genotypes were observed within Japan and very few within Europe, indicating limited clonal dispersal. Deviations from Hardy Weinberg expectations were observed both in Europe and Japan and heterozygote excess were observed at several loci, especially in Europe. Possible explanations for this pattern are discussed.

Draft genome of the brown-rot fungus Fomitopsis pinicola GR9-4.

Basidiomycete brown-rot fungi have a huge importance for wood decomposition and thus the global carbon cycle. Here, we present the genome sequence of Fomitopsis pinicola GR9-4 which represent different F. pinicola clade than the previously sequenced North American isolate FP-58527 SS1. The genome was sequenced by using a paired-end sequence library of Illumina and a 2.5k and 5k mate-pair library (ABI SOLiD). The final assembly adds up to a size of 45 Mb (including gaps between contigs), with a GC-content of 56%. The gene prediction resulted in 13,888 gene models. The genome sequence will be used as a basis for understanding population genomics, genome-wide association studies and wood decay mechanisms of this brown-rot fungus.

Transcriptional responses of the bacterial antagonist Serratia plymuthica to the fungal phytopathogen Rhizoctonia solani.

Rhizobacteria with biocontrol ability exploit a range of mechanisms to compete successfully with other microorganisms and to ensure their growth and survival in the rhizosphere, ultimately promoting plant growth. The rhizobacterium Serratia plymuthica AS13 is able to promote oilseed rape growth and improve seedling survival in the presence of the fungal pathogen, Rhizoctonia solani AG 2-1; however, our understanding of the mechanisms underlying the antagonism of Serratia is limited. To elucidate possible mechanisms, genome-wide gene expression profiling of S. plymuthica AS13 was carried out in the presence or absence of R. solani. We used RNA sequencing methodology to obtain a comprehensive overview of Serratia gene expression in response to R. solani. The differential gene expression profiles of S. plymuthica AS13 revealed significantly increased expression of genes related to the biosynthesis of the antibiotic pyrrolnitrin (prnABCD), protease production and transporters. The results presented here provide evidence that antibiosis is a major functional mechanism underlying the antagonistic behaviour of S. plymuthica AS13.

Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists.

To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes.

Genotypic diversity and migration patterns of Phytophthora infestans in the Nordic countries.

In this study we investigated the genotypic diversity and the migration patterns of Phytophthora infestans in the Nordic countries. Isolates of P. infestans from outbreaks in 43 fields sampled in 2008 were collected using stratified sampling with country, field, and disease foci as the different strata. Microsatellites were used as markers to determine the genotypic variation in the sampled material. The results show a high genotypic variation of P. infestans in the Nordic countries with most of the genotypes found only once among the collected isolates. The major part of the genotypic variation was observed within the fields, with low differentiation between the fields. The observed low association of alleles among loci is consistent with frequent sexual reproduction of P. infestans in the Nordic countries. Coalescence analyses did not support a single common population for the four countries, thus indicating some degree of geographic differentiation. The analyses of migration patterns showed differing levels of gene flow among the Nordic countries. No correlation between migration rates and geographical distance could be seen. This could be explained by different degrees of genetic similarity between the pathogen populations in the different countries.

Non-contiguous finished genome sequence of plant-growth promoting Serratia proteamaculans S4.

Serratia proteamaculans S4 (previously Serratia sp. S4), isolated from the rhizosphere of wild Equisetum sp., has the ability to stimulate plant growth and to suppress the growth of several soil-borne fungal pathogens of economically important crops. Here we present the non-contiguous, finished genome sequence of S. proteamaculans S4, which consists of a 5,324,944 bp circular chromosome and a 129,797 bp circular plasmid. The chromosome contains 5,008 predicted genes while the plasmid comprises 134 predicted genes. In total, 4,993 genes are assigned as protein-coding genes. The genome consists of 22 rRNA genes, 82 tRNA genes and 58 pseudogenes. This genome is a part of the project "Genomics of four rapeseed plant growth-promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI's Community Sequencing Program.

Complete genome sequence of Serratia plymuthica strain AS12.

A plant-associated member of the family Enterobacteriaceae, Serratia plymuthica strain AS12 was isolated from rapeseed roots. It is of scientific interest because it promotes plant growth and inhibits plant pathogens. The genome of S. plymuthica AS12 comprises a 5,443,009 bp long circular chromosome, which consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome was sequenced within the 2010 DOE-JGI Community Sequencing Program (CSP2010) as part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens".

Complete genome sequence of the plant-associated Serratia plymuthica strain AS13.

Serratia plymuthica AS13 is a plant-associated Gammaproteobacteria, isolated from rapeseed roots. It is of special interest because of its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The complete genome of S. plymuthica AS13 consists of a 5,442,549 bp circular chromosome. The chromosome contains 4,951 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome was sequenced as part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens" within the 2010 DOE-JGI Community Sequencing Program (CSP2010).

Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9.

Serratia plymuthica are plant-associated, plant beneficial species belonging to the family Enterobacteriaceae. The members of the genus Serratia are ubiquitous in nature and their life style varies from endophytic to free-living. S. plymuthica AS9 is of special interest for its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The genome of S. plymuthica AS9 comprises a 5,442,880 bp long circular chromosome that consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome is part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI Community Sequencing Program (CSP2010).

The plant cell wall-decomposing machinery underlies the functional diversity of forest fungi.

Brown rot decay removes cellulose and hemicellulose from wood--residual lignin contributing up to 30% of forest soil carbon--and is derived from an ancestral white rot saprotrophy in which both lignin and cellulose are decomposed. Comparative and functional genomics of the "dry rot" fungus Serpula lacrymans, derived from forest ancestors, demonstrated that the evolution of both ectomycorrhizal biotrophy and brown rot saprotrophy were accompanied by reductions and losses in specific protein families, suggesting adaptation to an intercellular interaction with plant tissue. Transcriptome and proteome analysis also identified differences in wood decomposition in S. lacrymans relative to the brown rot Postia placenta. Furthermore, fungal nutritional mode diversification suggests that the boreal forest biome originated via genetic coevolution of above- and below-ground biota.

Asian origin and rapid global spread of the destructive dry rot fungus Serpula lacrymans.

The dry rot fungus Serpula lacrymans (Basidiomycota) is the most damaging destroyer of wood construction materials in temperate regions. While being a widespread aggressive indoor biodeterioration agent, it is only found in a few natural environments. The geographical source of spread and colonization by this fungus in human environments is thus somewhat of an enigma. Employing genetic markers (amplified fragment length polymorphisms, DNA sequences and microsatellites) on a worldwide sample of specimens, we show that the dry rot fungus is divided into two main lineages; one nonaggressive residing naturally in North America and Asia (var. shastensis), and another aggressive lineage including specimens from all continents, both from natural environments and buildings (var. lacrymans). Our genetic analyses indicate that the two lineages represent well-differentiated cryptic species. Genetic analyses pinpoint mainland Asia as the origin of the aggressive form var. lacrymans. A few aggressive genotypes have migrated worldwide from Asia to Europe, North and South America and Oceania followed by local population expansions. The very low genetic variation in the founder populations indicate that they have established through recent founder events, for example by infected wood materials transported over land or sea. A separate colonization has happened from mainland Asia to Japan. Our data also indicate that independent immigration events have happened to Oceania from different continents followed by admixture.

Environmental fluctuations facilitate species co-existence and increase decomposition in communities of wood decay fungi.

A fluctuating environment may facilitate co-existence of species, and high species richness may be important for maintaining ecosystem processes under changing environmental conditions. A positive relationship has been found between species richness and primary production in many experiments, and there is now an increasing interest whether similar relationships also apply to microorganisms and decomposition. Basidiomycete fungi are the primary decomposers of wood with the functional groups brown and white rot fungi, which differ with respect to decay strategy. In this study, 16 species of boreal wood decay fungi, 8 brown rot fungi and 8 white rot fungi, were assembled in artificial communities. The aims were to study species persistence, wood decomposition and metabolic efficiency in fungal communities of increasing levels of species richness under constant and fluctuating temperature regimes. Species persistence was generally low, but temperature fluctuations facilitated co-existence of species. Decomposition was highest at intermediate diversity levels under the fluctuating temperature regime. Metabolic efficiency, estimated as the amount of fungal mycelium formed per amount of degraded wood, decreased with increasing community complexity under the fluctuating temperature regime. Brown and white rot fungi differed in decomposition rates and metabolic efficiency, but no synergistic effects were found where the two functional groups were mixed. This study demonstrates how niche differentiation in a variable environment may act to maintain diversity and function. In our experiment, differences in functional responses to the varying temperature rather than resource partitioning between brown and white rot fungi had significant effects. Niche differentiation is likely to be particularly important in maintaining species diversity in communities of wood decaying fungi, which are known from previous studies to be characterised by intense competition, and where otherwise metabolically costly interactions lead to species exclusion and dominance by highly competitive species.

Phylogenetic analysis of multiple loci reveal the population structure within Letharia in the Caucasus and Morocco.

The sequence variation within the genus Letharia in the Caucasus and Morocco was investigated. Twelve thalli from each area were sequenced at eight different loci. Phylogenetic analysis of the multiple loci data revealed the cryptic species Letharia 'lupina' in Morocco, previously known only from western North America. The two cryptic species L. vulpina and L. 'lupina' locally share the same ecology but are genetically isolated from each other. In the Caucasus, five different haplotypes of L. vulpina were detected, and in Morocco four L. vulpina haplotypes and six L. 'lupina' haplotypes were found. For L. vulpina, allelic differences were detected at five of the eight loci in the Caucasus and Morocco. The populations of L. vulpina in both the Caucasus and Morocco contain more genetic variation than those previously investigated in Europe, which indicates that the Caucasus and Morocco acted as refugia during quaternary glaciations, and that central and northern Europe may have been recolonised from one or both of these areas.

Extremely low AFLP variation in the european dry rot fungus (Serpula lacrymans): implications for self/nonself-recognition.

The devastating dry rot fungus, Serpula lacrymans, has a worldwide occurrence in buildings. We investigated the genetic variation in European isolates belonging to five vegetative compatibility groups (VCGs) by AFLP analysis. Our results indicate that S. lacrymans in Europe is genetically extremely homogenous; only five out of 308 scored AFLP fragments (1.6 %) were polymorphic. In contrast, S. himantioides, the closest relative of S. lacrymans, possessed 31.3 % polymorphic fragments (84 out of 268). AFLP polymorphisms observed in S. lacrymans were distributed independently of the VCG boundaries, indicating that the VCGs do not represent clones but that different genets of S. lacrymans frequently share similar vic alleles due to low genetic variation. Thus, although the European S. lacrymans is genetically extremely homogeneous, and our results suggest that the species reproduces and spreads mainly sexually and not by clones.

Reproductive mode and genetic variation suggest a North American origin of European Letharia vulpina.

Our data on the intercontinental population biology of Letharia vulpina show an unexpected shift from a recombining North American population with unique haplotypes to genetically depauperate Swedish and Italian populations, each with many representatives of a single repeated haplotype. Analysis of eight loci in 47 individuals supported recombination in North American populations and showed almost no variation among European populations. We infer that a genetic bottleneck caused by limited long-distance dispersal accounts for the lack of genetic variation found in marginal populations. This lack of variation in the European populations makes it impossible to use population genetic means to distinguish clonal reproduction from self-fertilization or even outcrossing, but phenotype indicates that reproduction in the marginal populations is by clonal spread, via soredia and isidioid soredia.