Genetic architecture of disease resistance and tolerance in Douglas-fir trees.

Understanding the genetic basis of how plants defend against pathogens is important to monitor and maintain resilient tree populations. Swiss needle cast (SNC) and Rhabdocline needle cast (RNC) epidemics are responsible for major damage of forest ecosystems in North America. Here we investigate the genetic architecture of tolerance and resistance to needle cast diseases in Douglas-fir (Pseudotsuga menziesii) caused by two fungal pathogens: SNC caused by Nothophaeocryptopus gaeumannii, and RNC caused by Rhabdocline pseudotsugae. We performed case-control genome-wide association analyses and found disease resistance and tolerance in Douglas-fir to be polygenic and under strong selection. We show that stomatal regulation as well as ethylene and jasmonic acid pathways are important for resisting SNC infection, and secondary metabolite pathways play a role in tolerating SNC once the plant is infected. We identify a major transcriptional regulator of plant defense, ERF1, as the top candidate for RNC resistance. Our findings shed light on the highly polygenic architectures underlying fungal disease resistance and tolerance and have important implications for forestry and conservation as the climate changes.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Evidence of Coevolution Between Cronartium harknessii Lineages and Their Corresponding Hosts, Lodgepole Pine and Jack Pine.

Variation in rate of infection and susceptibility of Pinus spp. to the fungus Cronartium harknessii (syn. Endocronartium harknessii), the causative agent of western gall rust, has been well documented. To test the hypothesis that there is a coevolutionary relationship between C. harknessii and its hosts, we examined genetic structure and virulence of C. harknessii associated with lodgepole pine (P. contorta var. latifolia), jack pine (P. banksiana), and their hybrids. A secondary objective was to improve assessment and diagnosis of infection in hosts. Using 18 microsatellites, we assessed genetic structure of C. harknessii from 90 sites within the ranges of lodgepole pine and jack pine. We identified two lineages (East and West, FST = 0.677) associated with host genetic structure (r = 0.81, P = 0.001), with East comprising three sublineages. In parallel, we conducted a factorial experiment in which lodgepole pine, jack pine, and hybrid seedlings were inoculated with spores from the two primary genetic lineages. With this experiment, we refined the phenotypic categories associated with infection and demonstrated that stem width can be used as a quantitative measure of host response to infection. Overall, each host responded differentially to the fungal lineages, with jack pine exhibiting more resiliency to infection than lodgepole pine and hybrids exhibiting intermediate resiliency. Taken together, the shared genetic structure between fungus and host species, and the differential interaction of the fungal species with the hosts, supports a coevolutionary relationship between host and pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative Gene Expression Analysis Reveals Mechanism of Pinus contorta Response to the Fungal Pathogen Dothistroma septosporum.

Many conifers have distributions that span wide ranges in both biotic and abiotic conditions, but the basis of response to biotic stress has received much less attention than response to abiotic stress. In this study, we investigated the gene expression response of lodgepole pine (Pinus contorta) to attack by the fungal pathogen Dothistroma septosporum, which causes Dothistroma needle blight, a disease that has caused severe climate-related outbreaks in northwestern British Columbia. We inoculated tolerant and susceptible pines with two D. septosporum isolates and analyzed the differentially expressed genes (DEGs), differential exon usage, and coexpressed gene modules using RNA-sequencing data. We found a rapid and strong transcriptomic response in tolerant lodgepole pine samples inoculated with one D. septosporum isolate, and a late and weak response in susceptible samples inoculated with another isolate. We mapped 43 of the DEG- or gene module-identified genes to the reference plant-pathogen interaction pathway deposited in the Kyoto Encyclopedia of Genes and Genomes database. These genes are present in PAMP-triggered and effector-triggered immunity pathways. Genes comprising pathways and gene modules had signatures of strong selective constraint, while the highly expressed genes in tolerant samples appear to have been favored by selection to counterattack the pathogen. We identified candidate resistance genes that may respond to D. septosporum effectors. Taken together, our results show that gene expression response to D. septosporum infection in lodgepole pine varies both among tree genotypes and pathogen strains and involves both known candidate genes and a number of genes with previously unknown functions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Evolution and Adaptation of Forest and Crop Pathogens in the Anthropocene.

Anthropocene marks the era when human activity is making a significant impact on earth, its ecological and biogeographical systems. The domestication and intensification of agricultural and forest production systems have had a large impact on plant and tree health. Some pathogens benefitted from these human activities and have evolved and adapted in response to the expansion of crop and forest systems, resulting in global outbreaks. Global pathogen genomics data including population genomics and high-quality reference assemblies are crucial for understanding the evolution and adaptation of pathogens. Crops and forest trees have remarkably different characteristics, such as reproductive time and the level of domestication. They also have different production systems for disease management with more intensive management in crops than forest trees. By comparing and contrasting results from pathogen population genomic studies done on widely different agricultural and forest production systems, we can improve our understanding of pathogen evolution and adaptation to different selection pressures. We find that in spite of these differences, similar processes such as hybridization, host jumps, selection, specialization, and clonal expansion are shaping the pathogen populations in both crops and forest trees. We propose some solutions to reduce these impacts and lower the probability of global pathogen outbreaks so that we can envision better management strategies to sustain global food production as well as ecosystem services.

Signatures of Post-Glacial Genetic Isolation and Human-Driven Migration in the Dothistroma Needle Blight Pathogen in Western Canada.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

Chemical, Bioactivity, and Biosynthetic Screening of Epiphytic Fungus Zasmidium pseudotsugae.

We report the first secondary metabolite, 8,8'-bijuglone, obtained from pure cultures of the slow growing Douglas fir- (Pseudotsuga menziesii var. menziesii) foliage-associated fungus Zasmidium pseudotsugae. The quinone was characterized using extensive LC/MS and NMR-based spectroscopic methods. 8,8'-Bijuglone exhibited moderate antibiotic activity against Gram-positive pathogens and weak cytotoxic activity in the NCI-60 cell line panel and in our in-house human colon carcinoma (HCT-116) cell line. An analysis of the fungal genome sequence to assess its metabolic potential was implemented using the bioinformatic tool antiSMASH. In total, 36 putative biosynthetic gene clusters were found with a majority encoding for polyketides (17), followed by non-ribosomal peptides (14), terpenes (2), ribosomal peptides (1), and compounds with mixed biosynthetic origin (2). This study demonstrates that foliage associated fungi of conifers produce antimicrobial metabolites and suggests this guild of fungi may present a rich source of novel molecules.

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

Genetic and genomic evidence of niche partitioning and adaptive radiation in mountain pine beetle fungal symbionts.

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetle's life cycle by providing nutritional supplementation, overcoming tree defences and modifying host tissues to favour brood development. The maintenance of stable multipartite symbioses with seemingly less competitive symbionts in similar habitats is of fundamental interest to ecology and evolution. We tested the hypothesis that the coexistence of three fungal species associated with the mountain pine beetle is the result of niche partitioning and adaptive radiation using SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate under different temperatures. We found that genetic variation and population structure within each species is best explained by distinct spatial and environmental variables. We observed both common (temperature seasonality and the host species) and distinct (drought, cold stress, precipitation) environmental and spatial factors that shaped the genomes of these fungi resulting in contrasting outcomes. Phenotypic intraspecific variations in Grosmannia clavigera and Leptographium longiclavatum, together with high heritability, suggest potential for adaptive selection in these species. By contrast, Ophiostoma montium displayed narrower intraspecific variation but greater tolerance to extreme high temperatures. Our study highlights unique phenotypic and genotypic characteristics in these symbionts that are consistent with our hypothesis. By maintaining this multipartite relationship, the bark beetles have a greater likelihood of obtaining the benefits afforded by the fungi and reduce the risk of being left aposymbiotic. Complementarity among species could facilitate colonization of new habitats and survival under adverse conditions.

Further Support of Conspecificity of Oak and Mango Powdery Mildew and First Report of Erysiphe quercicola and Erysiphe alphitoides on Mango in Mainland Europe.

Mango leaves and inflorescences infected by powdery mildew in southern Spain were analyzed using multigene sequencing (ITS + 4 single-copy coding genes) to identify the causal agent. Erysiphe quercicola was detected in 97% out of 140 samples, collected in six different orchards in the Malaga region. Among these, a small proportion also yielded E. alphitoides (8% of all samples) and E. alphitoides was found alone in 3% of samples. A phylogenetic approach was completed by cross inoculations between oak and mango, which led to typical symptoms, supporting the conspecificity of oak and mango powdery mildews. To our knowledge, this is the first report of E. quercicola and E. alphitoides causing powdery mildew on mango trees in mainland Spain, and thus mainland Europe, based on unequivocal phylogenetic and biological evidence. Our study thus confirmed the broad host range of both E. quercicola and E. alphitoides. These results have practical implications in terms of the demonstrated ability for host range expansion in powdery mildews. They also open interesting prospects to the elucidation of molecular mechanisms underlying the ability to infect single versus multiple and unrelated host plants since these two closely related powdery mildew species belong to a small clade with both generalist and specialist powdery mildews.

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.

Comparative genomics of the pine pathogens and beetle symbionts in the genus Grosmannia.

Studies on beetle/tree fungal symbionts typically characterize the ecological and geographic distributions of the fungal populations. There is limited understanding of the genome-wide evolutionary processes that act within and between species as such fungi adapt to different environments, leading to physiological differences and reproductive isolation. Here, we assess genomic evidence for such evolutionary processes by extending our recent work on Grosmannia clavigera, which is vectored by the mountain pine beetle and jeffrey pine beetle. We report the genome sequences of an additional 11 G. clavigera (Gc) sensu lato strains from the two known sibling species, Grosmannia sp. (Gs) and Gc. The 12 fungal genomes are structurally similar, showing large-scale synteny within and between species. We identified 103,430 single-nucleotide variations that separated the Grosmannia strains into divergent Gs and Gc clades, and further divided each of these clades into two subclades, one of which may represent an additional species. Comparing variable genes between these lineages, we identified truncated genes and potential pseudogenes, as well as seven genes that show evidence of positive selection. As these variable genes are involved in secondary metabolism and in detoxifying or utilizing host-tree defense chemicals (e.g., polyketide synthases, oxidoreductases, and mono-oxygenases), their variants may reflect adaptation to the specific chemistries of the host trees Pinus contorta, P. ponderosa, and P. jeffreyi. This work provides a comprehensive resource for developing informative markers for landscape population genomics of these ecologically and economically important fungi, and an approach that could be extended to other beetle-tree-associated fungi.

Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi.

The 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.

Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen.

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera (Gc), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc, MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

Obligate biotrophy features unraveled by the genomic analysis of rust fungi.

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Ophiostoma haidaense, sp. nov., a new member of the O. piceae species complex associated with yellow-cedar, Callitropsis nootkatensis.

Ophiostoma haidanensis is described as a new species of the Ophiostoma piceae complex isolated from yellow-cedar (Callitropsis nootkatensis (D. Don) Oerst. ex D.P. Little) sapwood in the Haida Gwaii island archipelago and the North Coast of British Columbia, Canada. The fungus is characterized by the production of a typical sporothrix-like asexual morph but is distinguished morphologically from other members of the O. piceae species complex by its large, multiseptate primary conidia. Phylogenetic analysis of DNA sequences from the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the ß-tubulin (BTUB) and translation elongation factor 1-α (TEF1) genes supports the inclusion of O. haidensis as a distinct member within the O. piceae complex. To our knowledge, this is the first report of a blue stain fungus infecting yellow-cedar, an ecologically, culturally, and economically important conifer naturally distributed along the coastal forests of the Pacific Northwest in North America.

Genome sequences of three genetic lineages of the fungus Nothophaeocryptopus gaeumannii, the causal agent of Swiss needle cast on Douglas-fir trees.

Here, we present the nearly complete genome sequences of the three main genetic lineages of Nothophaeocryptopus gaeumannii, an endophytic ascomycete fungus responsible for Swiss needle cast, a foliar disease that is emerging as a significant threat to the Douglas-fir tree in its natural distribution range.

Genome sequence of Heterobasidion occidentale, a fungus that causes annosus root and butt rot among conifer trees in North America.

We report an annotated draft genome of Heterobasidion occidentale, a fungus (Basidiomycota, Agaricomycetes) that has pathogenic and saprophytic lifestyles. This fungus belongs to the H. annosum (Fr.) Bref. sensu lato species complex that comprises several root rot pathogens. Heterobasidion occidentale causes annosus root and butt rot primarily in true fir (Abies spp.) and spruce (Picea spp.) species throughout western North America.

Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi.

Fungal species belonging to the genus Melampsora (Basidiomycota, Pucciniales) comprise rust pathogens that alternate between Salicaceae and other plant hosts. Species delineation and identification are difficult within this group due to the paucity of observable morphological features. Several Melampsora rusts are highly host-specific and this feature has been used for identification at the species level. However, this criterion is not always reliable since different Melampsora rust species can overlap on one host but specialize on a different one. To date, two different species recognition methods are used to recognize and define species within the Melampsora genus: (i) morphological species recognition, which is based solely on morphological criteria; and (ii) ecological species recognition, which combines morphological criteria with host range to recognize and define species. In order to clarify species recognition within the Melampsora genus, we applied phylogenetic species recognition to Melampsora poplar rusts by conducting molecular phylogenetic analyses on 15 Melampsora taxa using six nuclear and mitochondrial loci. By assessing the genealogical concordance between phylogenies, we identified 12 lineages that evolved independently, corresponding to distinct phylogenetic species. All 12 lineages were concordant with host specialization, but only three belonged to strictly defined morphological species. The estimation of the species tree obtained with Bayesian concordance analysis highlighted a potential co-evolutionary history between Melampsora species and their reciprocal aecial host plants. Within the Melampsora speciation process, aecial host may have had a strong effect on ancestral evolution, whereas telial host specificity seems to have evolved more recently. The morphological characters initially used to define species boundaries in the Melampsora genus are not reflective of the evolutionary and genetic relationships among poplar rusts. In order to construct a more meaningful taxonomy, host specificity must be considered an important criterion for delineating and describing species within the genus Melampsora as previously suggested by ecological species recognition.

DNA-Barcoding Identification of Plant Pathogens for Disease Diagnostics.

The accurate identification of plant pathogens is a critical step to prevent their spread and attenuate their impact. Among the wide range of methods available, DNA-barcoding, i.e., the identification of an organism through the PCR amplification and sequencing of a single locus, remains one of the most straightforward and accurate plant-pathogen identification techniques that can be used in a generic molecular biology lab. This chapter provides a detailed protocol for the isolation of genomic DNA of fungal and oomycete pathogens from fresh field samples and the amplification and sequencing of the internal transcribed spacer (ITS) locus for DNA-barcoding purpose. Amendments to the protocol are provided to help in resolving issues related to the analysis of complicated samples and to the lack of species resolution that can be encountered with ITS barcodes.