Forest health in the Anthropocene: the emergence of a novel tree disease is associated with poplar cultivation.

Plant domestication and movement are large contributors to the success of new diseases. The introduction of new host species can result in accelerated evolutionary changes in pathogens, affecting long-established coevolutionary dynamics. This has been observed in poplars where severe epidemics of pathogens that were innocuous in their natural pathosystems occurred following host domestication. The North American fungus Sphaerulina musiva is responsible for endemic leaf spots on Populus deltoides. We show that the expansion of poplar cultivation resulted in the emergence of a new lineage of this pathogen that causes stem infections on a new host, P. balsamifera. This suggests a host shift since this is not a known host. Genome analysis of this emerging lineage reveals a mosaic pattern with islands of diversity separated by fixed genome regions, which is consistent with a homoploid hybridization event between two individuals that produced a hybrid swarm. Genome regions of extreme divergence and low diversity are enriched in genes involved in host-pathogen interactions. The specialization of this emerging lineage to a new host and its clonal propagation represents a serious threat to poplars and could affect both natural and planted forests. This work provides a clear example of the changes created by the intensification of tree cultivation that facilitate the emergence of specialized pathogens, jeopardizing the natural equilibrium between hosts and pathogens. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Genomic biosurveillance detects a sexual hybrid in the sudden oak death pathogen.

Invasive exotic pathogens pose a threat to trees and forest ecosystems worldwide, hampering the provision of essential ecosystem services such as carbon sequestration and water purification. Hybridization is a major evolutionary force that can drive the emergence of pathogens. Phytophthora ramorum, an emergent pathogen that causes the sudden oak and larch death, spreads as reproductively isolated divergent clonal lineages. We use a genomic biosurveillance approach by sequencing genomes of P. ramorum from survey and inspection samples and report the discovery of variants of P. ramorum that are the result of hybridization via sexual recombination between North American and European lineages. We show that these hybrids are viable, can infect a host and produce spores for long-term survival and propagation. Genome sequencing revealed genotypic combinations at 54,515 single nucleotide polymorphism loci not present in parental lineages. More than 6,000 of those genotypes are predicted to have a functional impact in genes associated with host infection, including effectors, carbohydrate-active enzymes and proteases. We also observed post-meiotic mitotic recombination that could generate additional genotypic and phenotypic variation and contribute to homoploid hybrid speciation. Our study highlights the importance of plant pathogen biosurveillance to detect variants, including hybrids, and inform management and control.

Mitotic Recombination and Rapid Genome Evolution in the Invasive Forest Pathogen Phytophthora ramorum.

Invasive alien species often have reduced genetic diversity and must adapt to new environments. Given the success of many invasions, this is sometimes called the genetic paradox of invasion. Phytophthora ramorum is invasive, limited to asexual reproduction within four lineages, and presumed clonal. It is responsible for sudden oak death in the United States, sudden larch death in Europe, and ramorum blight in North America and Europe. We sequenced the genomes of 107 isolates to determine how this pathogen can overcome the invasion paradox. Mitotic recombination (MR) associated with transposons and low gene density has generated runs of homozygosity (ROH) affecting 2,698 genes, resulting in novel genotypic diversity within the lineages. One ROH enriched in effectors was fixed in the NA1 lineage. An independent ROH affected the same scaffold in the EU1 lineage, suggesting an MR hot spot and a selection target. Differences in host infection between EU1 isolates with and without the ROH suggest that they may differ in aggressiveness. Non-core regions (not shared by all lineages) had signatures of accelerated evolution and were enriched in putative pathogenicity genes and transposons. There was a striking pattern of gene loss, including all effectors, in the non-core EU2 genome. Positive selection was observed in 8.0% of RxLR and 18.8% of Crinkler effector genes compared with 0.9% of the core eukaryotic gene set. We conclude that the P. ramorum lineages are diverging via a rapidly evolving non-core genome and that the invasive asexual lineages are not clonal, but display genotypic diversity caused by MR.IMPORTANCE Alien species are often successful invaders in new environments, despite the introduction of a few isolates with a reduced genetic pool. This is called the genetic paradox of invasion. We found two mechanisms by which the invasive forest pathogen causing sudden oak and sudden larch death can evolve. Extensive mitotic recombination producing runs of homozygosity generates genotypic diversity even in the absence of sexual reproduction, and rapid turnover of genes in the non-core, or nonessential portion of genome not shared by all isolates, allows pathogenicity genes to evolve rapidly or be eliminated while retaining essential genes. Mitotic recombination events occur in genomic hot spots, resulting in similar ROH patterns in different isolates or groups; one ROH, independently generated in two different groups, was enriched in pathogenicity genes and may be a target for selection. This provides important insights into the evolution of invasive alien pathogens and their potential for adaptation and future persistence.

Genome-Enhanced Detection and Identification (GEDI) of plant pathogens.

Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed.

Development and Validation of Polymorphic Microsatellite Loci for the NA2 Lineage of Phytophthora ramorum from Whole Genome Sequence Data.

Phytophthora ramorum is the causal agent of sudden oak death and sudden larch death, and is also responsible for causing ramorum blight on woody ornamental plants. Many microsatellite markers are available to characterize the genetic diversity and population structure of P. ramorum. However, only two markers are polymorphic in the NA2 lineage, which is predominant in Canadian nurseries. Microsatellite motifs were mined from whole-genome sequence data of six P. ramorum NA2 isolates. Of the 43 microsatellite primer pairs selected, 13 loci displayed different allele sizes among the four P. ramorum lineages, 10 loci displayed intralineage variation in the EU1, EU2, and/or NA1 lineages, and 12 microsatellites displayed polymorphism in the NA2 lineage. Genotyping of 272 P. ramorum NA2 isolates collected in nurseries in British Columbia, Canada, from 2004 to 2013 revealed 12 multilocus genotypes (MLGs). One MLG was dominant when examined over time and across sampling locations, and only a few mutations separated the 12 MLGs. The NA2 population observed in Canadian nurseries also showed no signs of sexual recombination, similar to what has been observed in previous studies. The markers developed in this study can be used to assess P. ramorum inter- and intralineage genetic diversity and generate a better understanding of the population structure and migration patterns of this important plant pathogen, especially for the lesser-characterized NA2 lineage.

Genome sequences of six Phytophthora species threatening forest ecosystems.

The Phytophthora genus comprises of some of the most destructive plant pathogens and attack a wide range of hosts including economically valuable tree species, both angiosperm and gymnosperm. Many known species of Phytophthora are invasive and have been introduced through nursery and agricultural trade. As part of a larger project aimed at utilizing genomic data for forest disease diagnostics, pathogen detection and monitoring (The TAIGA project: Tree Aggressors Identification using Genomic Approaches; http://taigaforesthealth.com/), we sequenced the genomes of six important Phytophthora species that are important invasive pathogens of trees and a serious threat to the international trade of forest products. This genomic data was used to develop highly sensitive and specific detection assays and for genome comparisons and to make evolutionary inferences and will be useful to the broader plant and tree health community. These WGS data have been deposited in the International Nucleotide Sequence Database Collaboration (DDBJ/ENA/GenBank) under the accession numbers AUPN01000000, AUVH01000000, AUWJ02000000, AUUF02000000, AWVV02000000 and AWVW02000000.

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR.

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.