Population-level immunologic variation in wild threespine stickleback (Gasterosteusaculeatus).

Wild organisms are regularly exposed to a wide range of parasites, requiring the management of an effective immune response while avoiding immunopathology. Currently, our knowledge of immunoparasitology primarily derives from controlled laboratory studies, neglecting the genetic and environmental diversity that contribute to immune phenotypes observed in wild populations. To gain insight into the immunologic variability in natural settings, we examined differences in immune gene expression of two Alaskan stickleback (Gasterosteus aculeatus) populations with varying susceptibility to infection by the cestode Schistocephalus solidus. Between these two populations, we found distinct immune gene expression patterns at the population level in response to infection with fish from the high-infection population displaying signs of parasite-driven immune manipulation. Further, we found significant differences in baseline immune gene profiles between the populations, with uninfected low-infection population fish showing signatures of inflammation compared to uninfected high-infection population fish. These results shed light on divergent responses of wild populations to the same parasite, providing valuable insights into host-parasite interactions in natural ecosystems.

Herptile gut microbiomes: a natural system to study multi-kingdom interactions between filamentous fungi and bacteria.

Reptiles and amphibians (herptiles) are some of the most endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the gut microbiome of wild herptiles and how it relates to the health of these populations. Here, we report results from the gut microbiome characterization of both a broad survey of herptiles, and the correlation between the fungus Basidiobolus, and the bacterial community supported by a deeper, more intensive sampling of Plethodon glutinosus, known as slimy salamanders. We demonstrate that bacterial communities sampled from frogs, lizards, and salamanders are structured by the host taxonomy and that Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus operational taxonomic units present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity support a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides.IMPORTANCEThis work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Rapid New Diagnostic LAMP (Loop-Mediated Isothermal Amplification) Assays to Distinguish Among the Four Lineages of Phytophthora ramorum.

Sudden oak death (SOD) is caused by Phytophthora ramorum, an invasive oomycete pathogen. This pathogen is of major regulatory concern for nurseries, horticulture, and forestry in the United States and around the world. Three of the 12 identified lineages of P. ramorum currently occur in the United States (NA1, NA2, and EU1) affecting wildland forests and nurseries. Rapid identification and lineage determination is essential to accelerate management decisions, detect introductions of new lineages, and control the spread of SOD. The objective of this study was to develop and validate diagnostic tools to rapidly identify P. ramorum and distinguish among the four common lineages of the pathogen and to accelarate management decision making. The loop-mediated isothermal amplification (LAMP) assays developed here are species specific with no cross reaction to common Phytophthora species found in Oregon, California, and Washington. The lineage-specific assays unambiguously distinguish among the four common clonal lineages. These assays are sensitive and able to detect P. ramorum DNA ranging in concentration from 30 to 0.03 ng/µl depending on the assay. These assays work effectively on a variety of sample types including plant tissue, cultures, and DNA. They have been integrated into the SOD diagnostic process in the forest pathology lab at Oregon State University. To date, 190 samples have been correctly identified from over 200 field samples tested for lineage determination. The development of these assays will help managers in forestry and horticulture identify and rapidly respond to new outbreaks of P. ramorum.

The Dictyostelium discoideum genome lacks significant DNA methylation and uncovers palindromic sequences as a source of false positives in bisulfite sequencing.

DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in Dictyostelium before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing.

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.

Genome Sequence of the Estuarine Synechococcus sp. Strain NB0720_010.

Marine Synechococcus spp. are unicellular cyanobacteria widely distributed in the world's oceans. We report the complete genome sequence of Synechococcus sp. strain NB0720_010, isolated from Narragansett Bay, Rhode Island. NB0702_10 has several large (>3,000-amino acid) protein-coding genes that may be important in its interactions with other cells, including grazers in estuarine habitats.

Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Genetic Diversity and Aggressiveness of Fusarium virguliforme Isolates Across the Midwestern United States.

Sudden death syndrome (SDS) of soybean is a damaging disease caused by the fungus Fusarium virguliforme. Since this pathogen was first reported in the southern U.S. state of Arkansas in 1971, it has spread throughout the midwestern United States. The SDS pathogen primarily colonizes roots but also produces toxins that translocate to and damage leaves. Previous studies have detected little to no genetic differentiation among isolates, suggesting F. virguliforme in North America has limited genetic diversity and a clonal population structure. Yet, isolates vary in virulence to roots and leaves. We characterized a set of F. virguliforme isolates from the midwestern United States, representing a south to north latitudinal gradient from Arkansas to Minnesota. Ten previously tested microsatellite loci were used to genotype isolates, and plant assays were conducted to assess virulence. Three distinct population clusters were differentiated across isolates. Although isolates ranged in virulence classes from low to very high, little correlation was found between virulence phenotype and cluster membership. Similarly, population structure and geographic location were not highly correlated. However, the earliest diverging cluster had the lowest genetic diversity and was detected only in southern states, whereas the two other clusters were distributed across the Midwest and were predominant in Minnesota. One of the midwestern clusters had the greatest genetic diversity and was found along the northern edge of the known distribution. The results support three genetically distinct population clusters of F. virguliforme in the United States, with two clusters contributing most to spread of this fungus across the Midwest.

Identity and Pathogenicity of Fungi Associated with Root, Crown, and Vascular Symptoms Related to Winter Squash Yield Decline.

Winter squash (Cucurbita maxima cultivar Golden Delicious) produced in Oregon's Willamette Valley for edible seed production has experienced significant yield losses because of a soilborne disease. The symptoms associated with this disease problem include root rot, crown rot, and vascular discoloration in the stems, leading to a severe late season wilt and plant collapse. Through field surveys, Fusarium oxysporum, F. solani, F. culmorum-like fungi, Plectosphaerella cucumerina, and Setophoma terrestris were identified to be associated with diseased tissues, and each produced symptoms of root rot, crown rot, or stem discoloration in preliminary pathogenicity trials. In this study, 219 isolates of these species were characterized by molecular identity analyses using BLAST of the internal transcribed spacer and translation elongation factor 1 alpha genomic regions and by pathogenicity testing in outdoor, large-container trials. Molecular identity analyses confirmed the identity of isolates at 99 to 100% similarity to reference isolates in the database. In pathogenicity experiments, F. solani produced the most severe symptoms, followed by F. culmorum-like fungi, F. oxysporum, P. cucumerina, and S. terrestris. Some treatments of mixed-species inoculum produced symptom severity greater than what was expected from individual species. In particular, the mixture of F. culmorum-like fungi, F. oxysporum, and P. cucumerina and the mixture of F. culmorum-like fungi, F. solani, and S. terrestris had symptom ratings as high as that of F. solani by itself. Results indicate that this soilborne disease is caused primarily by Fusarium solani, but interactions between the complex of F. solani, F. culmorum-like fungi, F. oxysporum, and P. cucumerina can exacerbate disease severity.

QTL Analysis of Adult Plant Resistance to Stripe Rust in a Winter Wheat Recombinant Inbred Population.

Stripe rust, caused by the fungus Puccinia striiformis f. sp. tritici, is a worldwide disease of wheat that causes devastating crop losses. Resistant cultivars have been developed over the last 40 years that have significantly reduced the economic impact of the disease on growers, but in heavy infection years it is mostly controlled through the intensive application of fungicides. The Pacific Northwest of the United States has an ideal climate for stripe rust and has one of the most diverse race compositions in the country. This has resulted in many waves of epidemics that have overcome most of the resistance genes traditionally used in elite germplasm. The best way to prevent high yield losses, reduce production costs to growers, and reduce the heavy application of fungicides is to pyramid multiple stripe rust resistance genes into new cultivars. Using genotyping-by-sequencing, we identified 4662 high quality variant positions in a recombinant inbred line population of 196 individuals derived from a cross between Skiles, a highly resistant winter wheat cultivar, and Goetze, a moderately to highly susceptible winter wheat cultivar, both developed at Oregon State University. A subsequent genome wide association study identified two quantitative trait loci (QTL) on chromosomes 3B and 3D within the predicted locations of stripe rust resistance genes. Resistance QTL, when combined together, conferred high levels of stripe rust resistance above the level of Skiles in some locations, indicating that these QTL would be important additions to future breeding efforts of Pacific Northwest winter wheat cultivars.

Molecular Phylogenomics and Population Structure of Phytophthora pluvialis.

Phytophthora pluvialis is an oomycete that was first isolated from soil, water, and tree foliage in mixed Douglas-fir-tanoak forests of the U.S. Pacific Northwest (PNW). It was then identified as the causal agent of red needle cast of radiata pine (Pinus radiata) in New Zealand (NZ). Genotyping-by-sequencing was used to obtain 1,543 single nucleotide polymorphisms across 145 P. pluvialis isolates to characterize the population structure in the PNW and NZ. We tested the hypothesis that P. pluvialis was introduced to NZ from the PNW using genetic distance measurements and population structure analyses among locations between countries. The low genetic distance, population heterozygosity, and lack of geographic structure in NZ suggest a single colonization event from the United States followed by clonal expansion in NZ. The PNW Coast Range was proposed as a presumptive center of origin of the currently known distribution of P. pluvialis based on its geographic range and position as the central cluster in a minimum spanning network. The Coastal cluster of isolates were located at the root of every U.S. cluster and emerged earlier than all NZ clusters. The Coastal cluster had the highest degree of heterozygosity (Hs = 0.254) and median pairwise genetic distance (0.093) relative to any other cluster. Finally, the rapid host diversification between closely related isolates of P. pluvialis in NZ indicate that this pathogen has the potential to infect a broader range of hosts than is currently recognized.

Phylogenomic Analyses of Non-Dikarya Fungi Supports Horizontal Gene Transfer Driving Diversification of Secondary Metabolism in the Amphibian Gastrointestinal Symbiont, Basidiobolus.

Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. The fungi rich in SM production are taxonomically concentrated in the subkingdom Dikarya, which comprises the phyla Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus BasidiobolusBasidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays also confirm that Basidiobolus has significant siderophore activity. The expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

Genome-Wide Increased Copy Number is Associated with Emergence of Dominant Clones of the Irish Potato Famine Pathogen Phytophthora infestans.

The plant pathogen that caused the Irish potato famine, Phytophthora infestans, continues to reemerge globally. These modern epidemics are caused by clonally reproducing lineages. In contrast, a sexual mode of reproduction is observed at its center of origin in Mexico. We conducted a comparative genomic analysis of 47 high-coverage genomes to infer changes in genic copy number. We included samples from sexual populations at the center of origin as well as several dominant clonal lineages sampled worldwide. We conclude that sexual populations at the center of origin are diploid, as was the lineage that caused the famine, while modern clonal lineages showed increased copy number (3×). Copy number variation (CNV) was found genome-wide and did not to adhere to the two-speed genome hypothesis. Although previously reported, tetraploidy was not found in any of the genomes evaluated. We propose a model of dominant clone emergence supported by the epidemiological record (e.g., EU_13_A2, US-11, US-23) whereby a higher copy number provides fitness, leading to replacement of prior clonal lineages.IMPORTANCE The plant pathogen implicated in the Irish potato famine, Phytophthora infestans, continues to reemerge globally. Understanding changes in the genome during emergence can provide insights useful for managing this pathogen. Previous work has relied on studying individuals from the United States, South America, Europe, and China reporting that these can occur as diploids, triploids, or tetraploids and are clonal. We studied variation in sexual populations at the pathogen's center of origin, in Mexico, where it has been reported to reproduce sexually as well as within clonally reproducing, dominant clones from the United States and Europe. Our results newly show that sexual populations at the center of origin are diploid, whereas populations elsewhere are more variable and show genome-wide variation in gene copy number. We propose a model of evolution whereby new pathogen clones emerge predominantly by increasing the gene copy number genome-wide.

Genomic Investigation of the Strawberry Pathogen Phytophthora fragariae Indicates Pathogenicity Is Associated With Transcriptional Variation in Three Key Races.

The oomycete Phytophthora fragariae is a highly destructive pathogen of cultivated strawberry (Fragaria × ananassa), causing the root rotting disease, "red core". The host-pathogen interaction has a well described gene-for-gene resistance relationship, but to date neither candidate avirulence nor resistance genes have been identified. We sequenced a set of American, Canadian, and United Kingdom isolates of known race type, along with three representatives of the closely related pathogen of the raspberry (Rubus idaeus), P. rubi, and found a clear population structure, with a high degree of nucleotide divergence seen between some race types and abundant private variation associated with race types 4 and 5. In contrast, between isolates defined as United Kingdom races 1, 2, and 3 (UK1-2-3) there was no evidence of gene loss or gain; or the presence of insertions/deletions (INDELs) or Single Nucleotide Polymorphisms (SNPs) within or in proximity to putative pathogenicity genes could be found associated with race variation. Transcriptomic analysis of representative UK1-2-3 isolates revealed abundant expression variation in key effector family genes associated with pathogen race; however, further long read sequencing did not reveal any long range polymorphisms to be associated with avirulence to race UK2 or UK3 resistance, suggesting either control in trans or other stable forms of epigenetic modification modulating gene expression. This work reveals the combined power of population resequencing to uncover race structure in pathosystems and in planta transcriptomic analysis to identify candidate avirulence genes. This work has implications for the identification of putative avirulence genes in the absence of associated expression data and points toward the need for detailed molecular characterisation of mechanisms of effector regulation and silencing in oomycete plant pathogens.

effectR: An Expandable R Package to Predict Candidate RxLR and CRN Effectors in Oomycetes Using Motif Searches.

Effectors are small, secreted proteins that facilitate infection of host plants by all major groups of plant pathogens. Effector protein identification in oomycetes relies on identification of open reading frames with certain amino acid motifs among additional minor criteria. To date, identification of effectors relies on custom scripts to identify motifs in candidate open reading frames. Here, we developed the R package effectR, which provides a convenient tool for rapid prediction of effectors in oomycete genomes, or with custom scripts for any genome, in a reproducible way. The effectR package relies on a combination of regular expressions statements and hidden Markov model approaches to predict candidate RxLR and crinkler effectors. Other custom motifs for novel effectors can easily be implemented and added to package updates. The effectR package has been validated with published oomycete genomes. This package provides a convenient tool for wet lab researchers interested in reproducible identification of candidate effectors in oomycete genomes.

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.

Evolution of the U.S. Biological Select Agent Rathayibacter toxicus.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Populations of Phytophthora rubi Show Little Differentiation and High Rates of Migration Among States in the Western United States.

Population genetics is a powerful tool to understand patterns and evolutionary processes that are involved in plant-pathogen emergence and adaptation to agricultural ecosystems. We are interested in studying the population dynamics of Phytophthora rubi, the causal agent of Phytophthora root rot in raspberry. P. rubi is found in the western United States, where most of the fresh and processed raspberries are produced. We used genotyping-by-sequencing to characterize genetic diversity in populations of P. rubi sampled in the United States and other countries. Our results confirm that P. rubi is a monophyletic species with complete lineage sorting from its sister taxon P. fragariae. Overall, populations of P. rubi show low genetic diversity across the western United States. Demographic analyses suggest that populations of P. rubi from the western United States are the source of pathogen migration to Europe. We found no evidence for population differentiation at a global or regional (western United States) level. Finally, our results provide evidence of migration from California and Oregon into Washington. This report provides new insights into the evolution and structure of global and western United States populations of the raspberry pathogen P. rubi, indicating that human activity might be involved in moving the pathogen among regions and fields.

Whole Genome Sequences of the Raspberry and Strawberry Pathogens Phytophthora rubi and P. fragariae.

Phytophthora rubi and P. fragariae are two closely related oomycete plant pathogens that exhibit strong morphological and physiological similarities but are specialized to infect different hosts of economic importance, namely, raspberry and strawberry. Here, we report the draft genome sequences of these two Phytophthora species as a first step toward understanding the genomic processes underlying plant host adaptation in these pathogens.