Mikael Brandström Durling

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.

Production of ectomycorrhizal mycelium peaks during canopy closure in Norway spruce forests

*Here, species composition and biomass production of actively growing ectomycorrhizal (EM) mycelia were studied over the rotation period of managed Norway spruce (Picea abies) stands in south-western Sweden. *The EM mycelia were collected using ingrowth mesh bags incubated in the forest soil during one growing season. Fungal biomass was estimated by ergosterol analysis and the EM species were identified by 454 sequencing of internal transcribed spacer (ITS) amplicons. Nutrient availability and the fungal biomass in soil samples were also estimated. *Biomass production peaked in young stands (10-30 yr old) before the first thinning phase. Tylospora fibrillosa dominated the EM community, especially in these young stands, where it constituted 80% of the EM amplicons derived from the mesh bags. Species richness increased in older stands. *The establishment of EM mycelial networks in young Norway spruce stands requires large amounts of carbon, while much less is needed to sustain the EM community in older stands. The variation in EM biomass production over the rotation period has implications for carbon sequestration rates in forest soils.

Ancient DNA reveals traces of Iberian Neolithic and Bronze Age lineages in modern Iberian horses.

Multiple geographical regions have been proposed for the domestication of Equus caballus. It has been suggested, based on zooarchaeological and genetic analyses that wild horses from the Iberian Peninsula were involved in the process, and the overrepresentation of mitochondrial D1 cluster in modern Iberian horses supports this suggestion. To test this hypothesis, we analysed mitochondrial DNA from 22 ancient Iberian horse remains belonging to the Neolithic, the Bronze Age and the Middle Ages, against previously published sequences. Only the medieval Iberian sequence appeared in the D1 group. Neolithic and Bronze Age sequences grouped in other clusters, one of which (Lusitano group C) is exclusively represented by modern horses of Iberian origin. Moreover, Bronze Age Iberian sequences displayed the lowest nucleotide diversity values when compared with modern horses, ancient wild horses and other ancient domesticates using nonparametric bootstrapping analyses. We conclude that the excessive clustering of Bronze Age horses in the Lusitano group C, the observed nucleotide diversity and the local continuity from wild Neolithic Iberian to modern Iberian horses, could be explained by the use of local wild mares during an early Iberian domestication or restocking event, whereas the D1 group probably was introduced into Iberia in later historical times.

Insights on the Evolution of Mycoparasitism from the Genome of Clonostachys rosea

Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. rosea clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/saprotrophic Trichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Trichoderma virens. In contrast with mycoparasitic Trichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusarium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chlororaphis. The data suggest that tolerance toward secondary metabolites is a prominent feature in the biology of C. rosea.

Transcriptional Responses Associated with Virulence and Defence in the Interaction between Heterobasidion annosum s.s. and Norway Spruce

Heterobasidion annosum sensu lato is a serious pathogen causing root and stem rot to conifers in the northern hemisphere and rendering the timber defective for sawing and pulping. In this study we applied next-generation sequencing to i) identify transcriptional responses unique to Heterobasidion-inoculated Norway spruce and ii) investigate the H. annosum transcripts to identify putative virulence factors. To address these objectives we wounded or inoculated 30-year-old Norway spruce clones with H. annosum and 454-sequenced the transcriptome of the interaction at 0, 5 and 15 days post inoculation. The 491860 high-quality reads were de novo assembled and the relative expression was analysed. Overall, very few H. annosum transcripts were represented in our dataset. Three delta-12 fatty acid desaturase transcripts and one Clavaminate synthase-like transcript, both associated with virulence in other pathosystems, were found among the significantly induced transcripts. The analysis of the Norway spruce transcriptional responses produced a handful of differentially expressed transcripts. Most of these transcripts originated from genes known to respond to H. annosum. However, three genes that had not previously been reported to respond to H. annosum showed specific induction to inoculation: an oxophytodienoic acid–reductase (OPR), a beta–glucosidase and a germin-like protein (GLP2) gene. Even in a small data set like ours, five novel highly expressed Norway spruce transcripts without significant alignment to any previously annotated protein in Genbank but present in the P. abies (v1.0) gene catalogue were identified. Their expression pattern suggests a role in defence. Therefore a more complete survey of the transcriptional responses in the interactions between Norway spruce and its major pathogen H. annosum would probably provide a better understanding of gymnosperm defence than accumulated until now.

Finding the founder of Stockholm - a kinship study based on Y-chromosomal, autosomal and mitochondrial DNA.

Historical records claim that Birger Magnusson (died 1266), famous regent of Sweden and the founder of Stockholm, was buried in Varnhem Abbey in Västergötland. After being lost for centuries, his putative grave was rediscovered during restoration work in the 1920s. Morphological analyses of the three individuals in the grave concluded that the older male, the female and the younger male found in the grave were likely to be Birger, his second wife Mechtild of Holstein and his son Erik from a previous marriage. More recent evaluations of the data from the 1920s seriously questioned these conclusions, ultimately leading to the reopening and reexamination of the grave in 2002. Ancient DNA-analyses were performed to investigate if the relationship between the three individuals matched what we would expect if the individuals were Birger, Erik and Mechtild. We used pyrosequencing of Y-chromosomal and autosomal SNPs and compared the results with haplogroup frequencies of modern Swedes to investigate paternal relations. Possible maternal kinship was investigated by deep FLX-sequencing of overlapping mtDNA amplicons. The authenticity of the sequences was examined using data from independent extractions, massive clonal data, the c-statistics, and real-time quantitative data. We show that the males carry the same Y-chromosomal haplogroup and thus we cannot reject a father-son type of relation. Further, as shown by the mtDNA analyses, none of the individuals are maternally related. We conclude that the graves indeed belong to Birger, Erik and Mechtild, or to three individuals with the exact same kind of biological relatedness.

Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

Fungal species participate in vast numbers of processes in the landscape around us. However, their often cryptic growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Recent technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enable identification and relative quantification of fungal community members across well-replicated experimental settings. Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, that is, the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon mix to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile.

Genus-Specific Primers for Study of Fusarium Communities in Field Samples.

ABSTRACT Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

Comparative proteomic analysis of hyphae and germinating cysts of Phytophthora pisi and Phytophthora sojae.

UNLABELLED The recently described oomycete pathogen Phytophthora pisi causes root rot on pea and faba bean, while the closely related Phytophthora sojae is the causal agent of soybean root and stem rot. Differences in the pathogenicity factor repertoires that enable the two species to have distinct host specificity towards pea and soybean, were studied using tandem mass spectrometry in a global proteome study of hyphae and germinating cysts in P. pisi and P. sojae. In total 2775 proteins from P. pisi and 2891 proteins from P. sojae were identified. Fifty-eight orthologous proteins were more abundant in germinated cysts of both pathogens and thus identified as candidate proteins for the infective stage. Several of these proteins were associated with lipid transport and metabolism, and energy production. Twenty-three orthologous proteins were more abundant in hyphae of both pathogens and thus identified as candidate proteins for vegetative growth. Proteins uniquely present in germinating cysts of either P. pisi or P. sojae were considered as candidates for species-specific pathogenicity factors that may be involved in host specificity. Among these proteins were serine proteases, membrane transporters and a berberine-like protein. These results significantly expand the knowledge of the expressed proteome in P. pisi and P. sojae. BIOLOGICAL SIGNIFICANCE P. sojae and P. pisi are closely related species that specifically cause root rot on soybean and pea, respectively. The pathogenicity factors contributing to their host specificity remained unknown. We carried out a comparative large-scale proteome analysis of vegetative (hyphae) and infective (germinating cysts) life stages in P. pisi and P. sojae. This study provides knowledge of the common factors and mechanism involved in initiation of infection and species-specific proteins that may contribute to the host specificity of these pathogens. This knowledge will lead to a better understanding of the infection biology of these pathogens, allowing new possibilities towards developing alternative and effective plant protection measures.

The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey

Clonostachys rosea is a necrotrophic mycoparasitic fungus, used for biological control of plant pathogenic fungi. A better understanding of the underlying mechanisms resulting in successful biocontrol is important for knowledge‐based improvements of the application and use of biocontrol in agricultural production systems. Transcriptomic analyses revealed that C. rosea responded with both common and specific gene expression during interactions with the fungal prey species Botrytis cinerea and Fusarium graminearum. Genes predicted to encode proteins involved in membrane transport, biosynthesis of secondary metabolites and carbohydrate‐active enzymes were induced during the mycoparasitic attack. Predicted major facilitator superfamily (MFS) transporters constituted 54% of the induced genes, and detailed phylogenetic and evolutionary analyses showed that a majority of these genes belonged to MFS gene families evolving under selection for increased paralog numbers, with predicted functions in drug resistance and transport of carbohydrates and small organic compounds. Sequence analysis of MFS transporters from family 2.A.1.3.65 identified rapidly evolving loop regions forming the entry to the transport tunnel, indicating changes in substrate specificity as a target for selection. Deletion of the MFS transporter gene mfs464 resulted in mutants with increased growth inhibitory activity against F. graminearum, providing evidence for a function in interspecific fungal interactions. In summary, we show that the mycoparasite C. rosea can distinguish between fungal prey species and modulate its transcriptomic responses accordingly. Gene expression data emphasize the importance of secondary metabolites in mycoparasitic interactions.