Genome Typing and Epidemiology of Human Listeriosis in New Zealand, 1999 to 2018.

This study describes the epidemiology of listeriosis in New Zealand between 1999 and 2018 as well as the retrospective whole-genome sequencing (WGS) of 453 Listeria monocytogenes isolates corresponding to 95% of the human cases within this period. The average notified rate of listeriosis was 0.5 cases per 100,000 population, and non-pregnancy-associated cases were more prevalent than pregnancy-associated cases (averages of 19 and 5 cases per annum, respectively). WGS data was assessed using multilocus sequencing typing (MLST), including core-genome and whole-genome MLST (cgMLST and wgMLST, respectively) and single-nucleotide polymorphism (SNP) analysis. Thirty-nine sequence types (STs) were identified, with the most common being ST1 (21.9%), ST4 (13.2%), ST2 (11.3%), ST120 (6.1%), and ST155 (6.4%). A total of 291 different cgMLST types were identified, with the majority (n = 243) of types observed as a single isolate, consistent with the observation that listeriosis is predominately sporadic. Among the 49 cgMLST types containing two or more isolates, 18 cgMLST types were found with 2 to 4 isolates each (50 isolates in total, including three outbreak-associated isolates) that shared low genetic diversity (0 to 2 whole-genome alleles), some of which were dispersed in time or geographical regions. SNP analysis also produced results comparable to those from wgMLST. The low genetic diversity within these clusters suggests a potential common source, but incomplete epidemiological data impaired retrospective epidemiological investigations. Prospective use of WGS analysis together with thorough exposure information from cases could potentially identify future outbreaks more rapidly, including those that may have been undetected for some time over different geographical regions.

Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine.

The detrimental effect of fungal pathogens on forest trees is an increasingly important problem that has implications for the health of our planet. Despite this, the study of molecular plant-microbe interactions in forest trees is in its infancy, and very little is known about the roles of effector molecules from forest pathogens. Dothistroma septosporum causes a devastating needle blight disease of pines, and intriguingly, is closely related to Cladosporium fulvum, a tomato pathogen in which pioneering effector biology studies have been carried out. Here, we studied D. septosporum effectors that are shared with C. fulvum, by comparing gene sequences from global isolates of D. septosporum and assessing effector function in both host and non-host plants. Many of the effectors were predicted to be non-functional in D. septosporum due to their pseudogenization or low expression in planta, suggesting adaptation to lifestyle and host. Effector sequences were polymorphic among a global collection of D. septosporum isolates, but there was no evidence for positive selection. The DsEcp2-1 effector elicited cell death in the non-host plant Nicotiana tabacum, whilst D. septosporum DsEcp2-1 mutants showed increased colonization of pine needles. Together these results suggest that DsEcp2-1 might be recognized by an immune receptor in both angiosperm and gymnosperm plants. This work may lead to the identification of plant targets for DsEcp2-1 that will provide much needed information on the molecular basis of gymnosperm-pathogen interactions in forests, and may also lead to novel methods of disease control.

Chromatin-level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum.

Genes required for fungal secondary metabolite production are usually clustered, co-regulated and expressed in stationary growth phase. Chromatin modification has an important role in co-regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP-qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin-level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.

Complex epigenetic regulation of alkaloid biosynthesis and host interaction by heterochromatin protein I in a fungal endophyte-plant symbiosis.

Epichloë festucae forms mutualistic symbiotic interactions with grasses of the Lolium and Festuca genera. Protection from insect and mammalian herbivory are the best-documented host benefits of these associations. The two main classes of anti-mammalian alkaloids synthesized by E. festucae are the ergot alkaloids and indole diterpenes, of which ergovaline and lolitrems are the principal terminal products. Synthesis of both metabolites require multiple gene products encoded by clusters of 11 genes located at the subtelomeric regions of chromosomes I and III respectively. These loci are essentially unexpressed in axenic culture but among the most highly expressed genes in planta. We show here that heterochromatin 1 protein (HepA) is an important component of the regulatory machinery that maintains these loci in a silent state in culture. Deletion of this gene led to derepression of eas and ltm gene expression under non-symbiotic culture conditions. Although there was no obvious culture phenotype, RNAseq analysis revealed that around 1000 genes were differentially expressed in the ΔhepA mutant compared to wild type with just one-third upregulated. Inoculation of the ΔhepA mutants into seedlings of Lolium perenne led to a severe host interaction phenotype characterized by a reduction in tiller length but an increase in tiller number. Hyphae within the leaves of these associations were much more abundant in the intercellular spaces of the leaves and aberrantly colonized the vascular bundles. This physiological change was accompanied by a dramatic change in the transcriptome with around 900 genes differentially expressed, with two thirds of these upregulated. This major physiological change was accompanied by a decrease in ltm gene expression and loss of the ability to synthesize lolitrems. These results show that HepA has an important role in controlling the chromatin state of these sub-telomeric secondary metabolite genes, including their symbiosis-specific regulation.

Host Tissue Environment Directs Activities of an Epichloë Endophyte, While It Induces Systemic Hormone and Defense Responses in Its Native Perennial Ryegrass Host.

Increased resilience of pasture grasses mediated by fungal Epichloë endophytes is crucial to pastoral industries. The underlying mechanisms are only partially understood and likely involve very different activities of the endophyte in different plant tissues and responses of the plant to these. We analyzed the transcriptomes of Epichloë festucae and its host, Lolium perenne, in host tissues of different function and developmental stages. The endophyte contributed approximately 10× more to the transcriptomes than to the biomass of infected tissues. Proliferating mycelium in growing host tissues highly expressed genes involved in hyphal growth. Nonproliferating mycelium in mature plant tissues, transcriptionally equally active, highly expressed genes involved in synthesizing antiherbivore compounds. Transcripts from the latter accounted for 4% of fungal transcripts. Endophyte infection systemically but moderately increased transcription of L. perenne genes with roles in hormone biosynthesis and perception as well as stress and pathogen resistance while reducing expression of genes involved in photosynthesis. There was a good correlation between transcriptome-based observations and physiological observations. Our data indicate that the fitness-enhancing effects of the endophyte are based both on its biosynthetic activities, predominantly in mature host tissues, and also on systemic alteration of the host's hormonal responses and induction of stress response genes. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Evolution of polyketide synthesis in a Dothideomycete forest pathogen.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

HyLiTE: accurate and flexible analysis of gene expression in hybrid and allopolyploid species.

BACKGROUND: Forming a new species through the merger of two or more divergent parent species is increasingly seen as a key phenomenon in the evolution of many biological systems. However, little is known about how expression of parental gene copies (homeologs) responds following genome merger. High throughput RNA sequencing now makes this analysis technically feasible, but tools to determine homeolog expression are still in their infancy. RESULTS: Here we present HyLiTE - a single-step analysis to obtain tables of homeolog expression in a hybrid or allopolyploid and its parent species directly from raw mRNA sequence files. By implementing on-the-fly detection of diagnostic parental polymorphisms, HyLiTE can perform SNP calling and read classification simultaneously, thus allowing HyLiTE to be run as parallelized code. HyLiTE accommodates any number of parent species, multiple data sources (including genomic DNA reads to improve SNP detection), and implements a statistical framework optimized for genes with low to moderate expression. CONCLUSIONS: HyLiTE is a flexible and easy-to-use program designed for bench biologists to explore patterns of gene expression following genome merger. HyLiTE offers practical advantages over manual methods and existing programs, has been designed to accommodate a wide range of genome merger systems, can identify SNPs that arose following genome merger, and offers accurate performance on non-model organisms.

A Core Gene Set Describes the Molecular Basis of Mutualism and Antagonism in Epichloë spp.

Beneficial plant-fungal interactions play an important role in the ability of plants to survive changing environmental conditions. In contrast, phytopathogenic fungi fall at the opposite end of the symbiotic spectrum, causing reduced host growth or even death. In order to exploit beneficial interactions and prevent pathogenic ones, it is essential to understand the molecular differences underlying these alternative states. The association between the endophyte Epichloë festucae and Lolium perenne (perennial ryegrass) is an excellent system for studying these molecular patterns due to the existence of several fungal mutants that have an antagonistic rather than a mutualistic interaction with the host plant. By comparing gene expression in a wild-type beneficial association with three mutant antagonistic associations disrupted in key signaling genes, we identified a core set of 182 genes that show common differential expression patterns between these two states. These gene expression changes are indicative of a nutrient-starvation response, as supported by the upregulation of genes encoding degradative enzymes, transporters, and primary metabolism, and downregulation of genes encoding putative small-secreted proteins and secondary metabolism. These results suggest that disruption of a mutualistic symbiotic interaction may lead to an elevated uptake and degradation of host-derived nutrients and cell-wall components, reminiscent of phytopathogenic interactions.

Fungal endophyte infection of ryegrass reprograms host metabolism and alters development.

Beneficial associations between plants and microbes play an important role in both natural and agricultural ecosystems. For example, associations between fungi of the genus Epichloë, and cool-season grasses are known for their ability to increase resistance to insect pests, fungal pathogens and drought. However, little is known about the molecular changes induced by endophyte infection. To study the impact of endophyte infection, we compared the expression profiles, based on RNA sequencing, of perennial ryegrass infected with Epichloë festucae with noninfected plants. We show that infection causes dramatic changes in the expression of over one third of host genes. This is in stark contrast to mycorrhizal associations, where substantially fewer changes in host gene expression are observed, and is more similar to pathogenic interactions. We reveal that endophyte infection triggers reprogramming of host metabolism, favouring secondary metabolism at a cost to primary metabolism. Infection also induces changes in host development, particularly trichome formation and cell wall biogenesis. Importantly, this work sheds light on the mechanisms underlying enhanced resistance to drought and super-infection by fungal pathogens provided by fungal endophyte infection. Finally, our study reveals that not all beneficial plant-microbe associations behave the same in terms of their effects on the host.

Exploring the Bacterial Community in Aged Fecal Sources from Dairy Cows: Impacts on Fecal Source Tracking.

(1) Background: This paper discusses the impact of agricultural activities on stream health, particularly in relation to dairy cow fecal pollution. The study explores the fecal microbiome of cattle and the potential ecological implications of aging fecal pollution on waterways. (2) Methods: The study examines changes in the bacterial community available for mobilization from in-situ decomposing cowpats and the effects of simulated rainfall. The microbiome of individual cowpats was monitored over 5.5 months. We used 16S rRNA metagenomics and machine learning software, FEAST (Fast Expectation-mAximization for microbial Source Tracking), for bacterial and fecal source assignments. (3) Results: The phyla Bacillota and Bacteroidota are dominant in the fecal microbiota of fresh cow feces but shift to Pseudomonodota, Actinomycetota, and environmental Bacteroidota in aged cowpats. Potential impacts of these bacterial community shifts on inputs to local agricultural streams are discussed in relation to water quality monitoring and aging sources of fecal contamination. We identified taxon orders that are potential indicators of fresh cattle sources (Oscillospirales and Bacteroidales) and aged sources (Peptostreptococcales-Tissierellales) in water bodies. (4) The paper highlights that bacterial metagenomic profiling can inform our understanding of the ecology of microbial communities in aquatic environments and the potential impacts of agricultural activities on ecosystem health.

Computational identification of transcriptionally co-regulated genes, validation with the four ANT isoform genes.

BACKGROUND: The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. RESULTS: From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases. CONCLUSIONS: Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.

Mobilization of Escherichia coli and fecal source markers from decomposing cowpats.

In rural environments, the sources of fecal contamination in freshwater environments are often diffuse and a mix of fresh and aged fecal sources. It is important for water monitoring purposes, therefore, to understand the impacts of weathering on detection of the fecal source markers available for mobilization from livestock sources. This study targets the impacts of rainfall events on the mobilization of fecal source tracking (FST) markers from simulated cowpats decomposing in situ for five-and-a-half-months. The FST markers analysed were Escherichia coli, microbial source tracking (MST) markers, fecal steroids and a fecal ageing ratio based on the ratio between counts of river microflora and total coliforms. There was a substantial concentration of E. coli (104/100 mL) released from the ageing cowpats suggesting a long-term reservoir of E. coli in the cowpat. Mobilization of fecal markers from rainfall-impacted cowpats, however, was markedly reduced compared with fecal markers in the cowpat. Overall, the Bacteroidales bovine-associated MST markers were less persistent than E. coli in the cowpat and rainfall runoff. The ten fecal steroids, including the major herbivore steroid, 24-ethylcoprostanol, are shown to be stable markers of bovine pollution due to statistically similar degradation rates among all steroids. The mobilizable fraction for each FST marker in the rainfall runoff allowed generation of mobilization decline curves and the derived decline rate constants can be incorporated into source attribution models for agricultural contaminants. Findings from this study of aged bovine pollution sources will enable water managers to improve attribution of elevated E. coli to the appropriate fecal source in rural environments.

Prevalence and Genotyping of Campylobacter jejuni and Campylobacter coli from Ovine Carcasses in New Zealand.

ABSTRACT: A pilot survey was performed to determine the prevalence of Campylobacter jejuni and Campylobacter coli on three age classes (lamb, hogget, and mutton) of ovine carcass trim postdressing and prechill. Sampling of hogget carcasses was undertaken 6 months before sampling of lamb and mutton carcasses. A total of 120 trim samples were collected from 11 processing plants across New Zealand. All samples were enriched and screened using PCR for the presence of C. jejuni and C. coli, and isolation was attempted for all screen-positive samples. Enumeration of Campylobacter from lamb trim samples showed that Campylobacter bacteria were present in very low numbers (<10 CFU/g). The overall prevalence of Campylobacter for ovine trim based on PCR detection was 33% (39 of 120 samples), with prevalences for hogget, lamb, and mutton carcass trim of 56% (28 of 50), 11% (4 of 35), and 20% (7 of 35), respectively. Whole genome sequencing was performed on a selection of C. jejuni and C. coli isolates, and the data were used to subtype using multilocus sequence typing (MLST) and whole genome MLST. Twenty-five MLST sequence types (STs) were identified among 44 isolates, including ST42, ST50, ST3222, and ST3072, which have been previously reported to be associated with ruminant sources. Four novel STs were also identified. Whole genome MLST analysis further discriminated isolates within a single ST type and demonstrated a genetic diversity among the ovine isolates collected. Genes associated with the oxacillinase class of ß-lactamase enzymes were identified in 41 of 44 Campylobacter isolates. This study provides preliminary data that can be incorporated into existing source attribution models to assist in determining the potential contribution of ovine sources to the burden of campylobacteriosis in New Zealand.

Description and assessment of a model for GSK-3beta database virtual screening.

Molecular docking was used in order to prioritize organic syntheses or experimental evaluations. Different GSK-3beta protein models were generated in silico from a known X-ray structure. A set of 42 known inhibitors were then flexibly docked into each rigid model and re-scored with various functions, which led to different rankings. The biological activities of the chemicals were then compared to each set of results and one of the rigid models emerged in combination with two scoring functions as giving the best correlation. This methodology constitutes an easy and accurate way to generate reliable models for virtual database screening.

Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida.

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant-pathogen interactions in gymnosperm forest trees, including kauri.

Use of Sonication for Enhanced Sampling of Attached Microbes from Groundwater Systems.

The vast majority of microorganisms in aquifers live as biofilms on sediment surfaces, which presents significant challenges for sampling as only the suspended microbes will be sampled through normal pumping. The use of a down-well low frequency sonicator has been suggested as a method of detaching microbes from the biofilm and allowing rapid sampling of this community. We developed a portable, easy to use, low-frequency electric sonicator and evaluated its performance for a range of well depths (tested up to 42 m below ground level) and casing types. Three sonicators were characterized in laboratory experiments using a 1 m long tank filled with pea gravel. These included a commercially available pneumatic sonicator, a rotating flexible shaft sonicator, and the prototype electric sonicator. The electric sonicator detached between 56 and 74% of microbes grown on gravel-containing biobags at distances ranging between 2 and 50 cm from the sonicator. The field testing comprises of a total of 55 sampling events from 48 wells located in 4 regions throughout New Zealand. Pre- and post-sonication samples showed an average 33 times increase in bacterial counts. Microbial sequence data showed that the same classes are present in pre- and post-sonicated samples and only slight differences were seen in the proportions present. The sampling process was rapid and the significant increases in bacterial counts mean that microbial samples can be quickly obtained from wells, which permits more detailed analysis than previously possible.

Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum.

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.

Reduced Virulence of an Introduced Forest Pathogen over 50 Years.

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys.

Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains.

Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.