Phylogeography and population structure of the global, wide host-range hybrid pathogen Phytophthora × cambivora.

Invasive, exotic plant pathogens pose a major threat to native and agricultural ecosystems. Phytophthora × cambivora is an invasive, destructive pathogen of forest and fruit trees causing severe damage worldwide to chestnuts (Castanea), apricots, peaches, plums, almonds and cherries (Prunus), apples (Malus), oaks (Quercus), and beech (Fagus). It was one of the first damaging invasive Phytophthora species to be introduced to Europe and North America, although its origin is unknown. We determined its population genetic history in Europe, North and South America, Australia and East Asia (mainly Japan) using genotyping-by-sequencing. Populations in Europe and Australia appear clonal, those in North America are highly clonal yet show some degree of sexual reproduction, and those in East Asia are partially sexual. Two clonal lineages, each of opposite mating type, and a hybrid lineage derived from these two lineages, dominated the populations in Europe and were predominantly found on fagaceous forest hosts (Castanea, Quercus, Fagus). Isolates from fruit trees (Prunus and Malus) belonged to a separate lineage found in Australia, North America, Europe and East Asia, indicating the disease on fruit trees could be caused by a distinct lineage of P. × cambivora, which may potentially be a separate sister species and has likely been moved with live plants. The highest genetic diversity was found in Japan, suggesting that East Asia is the centre of origin of the pathogen. Further surveys in unsampled, temperate regions of East Asia are needed to more precisely identify the location and range of the centre of diversity.

Phylogeography of the wide-host range panglobal plant pathogen Phytophthora cinnamomi.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen.

Transcriptome-Derived Amplicon Sequencing Markers Elucidate the U.S. Podosphaera macularis Population Structure Across Feral and Commercial Plantings of Humulus lupulus.

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. P. macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that P. macularis from U.S. commercial hop yards form one population closely related to P. macularis of the United Kingdom, while P. macularis from U.S. feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

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.

Population Diversity and Structure of Podosphaera macularis in the Pacific Northwestern United States and Other Populations.

Powdery mildew, caused by Podosphaera macularis, is one of the most important diseases of hop. The disease was first reported in the Pacific Northwestern United States, the primary hop-growing region in this country, in the mid-1990s. More recently, the disease has reemerged in newly planted hopyards of the eastern United States, as hop production has expanded to meet demands of local craft brewers. The spread of strains virulent on previously resistant cultivars, the paucity of available fungicides, and the potential introduction of the MAT1-2 mating type to the western United States, all threaten sustainability of hop production. We sequenced the transcriptome of 104 isolates of P. macularis collected throughout the western United States, eastern United States, and Europe to quantify genetic diversity of pathogen populations and elucidate the possible origins of pathogen populations in the western United States. Discriminant analysis of principal components grouped isolates within three to five geographic populations, dependent on stringency of grouping criteria. Isolates from the western United States were phenotyped and categorized into one of three pathogenic races based on disease symptoms generated on differential cultivars. Western U.S. populations were clonal, irrespective of pathogenic race, and grouped with isolates originating from Europe. Isolates originating from wild hop plants in the eastern United States were genetically differentiated from all other populations, whereas isolates from cultivated hop plants in the eastern United States mostly grouped with isolates originating from the west, consistent with origins from nursery sources. Mating types of isolates originating from cultivated western and eastern U.S. hop plants were entirely MAT1-1. In contrast, a 1:1 ratio of MAT1-1 and MAT1-2 was observed with isolates sampled from wild plants or Europe. Within the western United States a set of highly differentiated loci were identified in P. macularis isolates associated with virulence to the powdery mildew R-gene R6. The weight of genetic and phenotypic evidence suggests a European origin of the P. macularis populations in the western United States, followed by spread of the pathogen from the western United States to re-emergent production regions in the eastern United States. Furthermore, R6 compatibility appears to have been selected from an extant isolate within the western United States. Greater emphasis on sanitation measures during propagation and quarantine policies should be considered to limit further spread of novel genotypes of the pathogen, both between and within production areas.

Genetic Differentiation and Clonal Expansion of Chinese Botrytis cinerea Populations from Tomato and Other Crops in China.

Botrytis cinerea is an important pathogen of vegetable and fruit crops but little is known about its population structure and genetics in China. We hypothesized that the geographic populations of B. cinerea in China would be genetically differentiated by host, geographic location, and/or year. In this study, we collected 393 B. cinerea isolates representing 28 populations from tomato, cherry, and nectarine from 2006 to 2014 in China. The isolates were analyzed using 14 microsatellite markers, including six new markers that provided more genotyping power than the eight previously published loci. We also investigated the B. cinerea population structure and inferred its mode of reproduction and dispersal based on genotype data. High genotypic diversity was detected in all populations, and clonal reproduction was dominant. Southern China populations harbored more genotypes than northern populations. Differentiation by host plant was evident. Between 2011 and 2012, genotypes changed only slightly among years for Liaoning populations, but they changed substantially among years for the Shanghai and Fujian populations. Clonal dispersal was detected and the farthest dispersal distance was estimated to be about 1,717 km. Two high-frequency genotypes were widely distributed in more than 10 populations and across several years. Our results provide useful, novel information for plant breeding programs and control of B. cinerea in China.

Genetic Diversity of Verticillium dahliae Isolates From Mint Detected with Genotyping by Sequencing.

Verticillium wilt is the most important disease threatening the commercial production of mint grown for essential oil. An important long-term goal for mint breeders is the production of cultivars with resistance to Verticillium wilt. Before that can be accomplished, a better understanding of the genetic variation within and among populations of Verticillium dahliae is needed. We characterized the extent of phenotypic and genetic diversity present in contemporary and archival populations of V. dahliae from mint fields in Oregon and other production regions of the United States using genotyping by sequencing, PCR assays for mating type and pathogenic race, vegetative compatibility group (VCG) tests, and aggressiveness assays. We report that the population in the Pacific Northwest can be described as one common genetic group and four relatively rare genetic groups. Eighty-three percent of the isolates belonged to VCG2B, and all isolates possessed the MAT1-2 idiomorph and were characterized as pathogenic race 2. These results indicate low levels of genetic diversity and a negligible risk of sexual recombination in populations of this host-adapted pathogen population. Knowledge of the genetic structure of V. dahliae in the Pacific Northwest will inform breeders about the diversity of pathogenicity factors that may need to be considered in their breeding programs.

Genotyping-by-Sequencing Reveals Fine-Scale Differentiation in Populations of Pseudoperonospora humuli.

Pseudoperonospora humuli is the causal agent of downy mildew of hop, one of the most important diseases of this plant and a limiting factor for production of susceptible cultivars in certain environments. The degree of genetic diversity and population differentiation within and among P. humuli populations at multiple spatial scales was quantified using genotyping-by-sequencing to test the hypothesis that populations of P. humuli have limited genetic diversity but are differentiated at the scale of individual hop yards. Hierarchical sampling was conducted to collect isolates from three hop yards in Oregon, plants within these yards, and infected shoots within heavily diseased plants. Additional isolates also were collected broadly from other geographic regions and from the two previously described clades of the sister species, P. cubensis. Genotyping of these 240 isolates produced a final quality-filtered data set of 216 isolates possessing 25,227 variants. Plots of G'ST values indicated that the majority of variants had G'ST values near 0 and were scattered randomly across contig positions. However, there was a subset of variants that were highly differentiated (G'ST > 0.3) and reproducible when genotyped independently. Within P. humuli, there was evidence of genetic differentiation at the level of hop yards and plants within yards; 19.8% of the genetic variance was associated with differences among yards and 20.3% of the variance was associated with plants within the yard. Isolates of P. humuli were well differentiated from two isolates of P. cubensis representative of the two clades of this organism. There was strong evidence of linkage disequilibrium in variant loci, consistent with nonrandom assortment of alleles expected from inbreeding and/or asexual recombination. Mantel tests found evidence that the genetic distance between isolates collected from heavily diseased plants within a hop yard was associated with the physical distance of the plants from which the isolates were collected. The sum of the data presented here indicates that populations of P. humuli are consistent with a clonal or highly inbred genetic structure with a small, yet significant differentiation of populations among yards and plants within yards. Fine-scale genetic differentiation at the yard and plant scales may point to persistence of founder genotypes associated with planting material, and chronic, systemic infection of hop plants by P. humuli. More broadly, genotyping-by-sequencing appears to have sufficient resolution to identify rare variants that differentiate subpopulations within organisms with limited genetic variability.

Population Genetic Structure and Cryptic Species of Plasmopara viticola in Australia.

Downy mildew of grape caused by Plasmopara viticola is a global pathogen of economic importance to commercial viticulture. In contrast to populations in the northern hemisphere, few studies have investigated the population biology, genetic diversity, and origin of the pathogen in Australian production systems. DNA was extracted from 381 P. viticola samples from Vitis vinifera and alternate hosts collected via fresh and herbarium leaves from populations within Australia and Whatman FTA cards from North America, Brazil, and Uruguay. A total of 32 DNA samples were provided from a French population. The populations were genotyped using 16 polymorphic microsatellite markers. Representative samples from within Australia, Brazil, and Uruguay were also genotyped to determine which of the cryptic species (clades) within the P. viticola species complex were present. Our findings suggest the Australian and South American populations of P. viticola are more closely related to the European population than the North American population, the reported source of origin of the pathogen. The Western Australian population had similarities to the South Australian population, and the tight clustering of samples suggests a single introduction into Western Australia. P. viticola clade aestivalis was the only clade detected in Australian and South American populations. Analysis of the Western Australian population suggests that it is reproducing clonally, but additional research is required to determine the mechanism as to how this is occurring.

Stable predictive markers for Phytophthora sojae avirulence genes that impair infection of soybean uncovered by whole genome sequencing of 31 isolates.

BACKGROUND: The interaction between oomycete plant pathogen Phytophthora sojae and soybean is characterized by the presence of avirulence (Avr) genes in P. sojae, which encode for effectors that trigger immune responses and resistance in soybean via corresponding resistance genes (Rps). A recent survey highlighted a rapid diversification of P. sojae Avr genes in soybean fields and the need to deploy new Rps genes. However, the full genetic diversity of P. sojae isolates remains complex and dynamic and is mostly characterized on the basis of phenotypic associations with differential soybean lines. RESULTS: We sequenced the genomes of 31 isolates of P. sojae, representing a large spectrum of the pathotypes found in soybean fields, and compared all the genetic variations associated with seven Avr genes (1a, 1b, 1c, 1d, 1k, 3a, 6) and how the derived haplotypes matched reported phenotypes in 217 interactions. We discovered new variants, copy number variations and some discrepancies with the virulence of previously described isolates with Avr genes, notably with Avr1b and Avr1c. In addition, genomic signatures revealed 11.5% potentially erroneous phenotypes. When these interactions were re-phenotyped, and the Avr genes re-sequenced over time and analyzed for expression, our results showed that genomic signatures alone accurately predicted 99.5% of the interactions. CONCLUSIONS: This comprehensive genomic analysis of seven Avr genes of P. sojae in a population of 31 isolates highlights that genomic signatures can be used as accurate predictors of phenotypes for compatibility with Rps genes in soybean. Our findings also show that spontaneous mutations, often speculated as a source of aberrant phenotypes, did not occur within the confines of our experiments and further suggest that epigenesis or gene silencing do not account alone for previous discordance between genotypes and phenotypes. Furthermore, on the basis of newly identified virulence patterns within Avr1c, our results offer an explanation why Rps1c has failed more rapidly in the field than the reported information on virulence pathotypes.

Inferring Variation in Copy Number Using High Throughput Sequencing Data in R.

Inference of copy number variation presents a technical challenge because variant callers typically require the copy number of a genome or genomic region to be known a priori. Here we present a method to infer copy number that uses variant call format (VCF) data as input and is implemented in the R package vcfR. This method is based on the relative frequency of each allele (in both genic and non-genic regions) sequenced at heterozygous positions throughout a genome. These heterozygous positions are summarized by using arbitrarily sized windows of heterozygous positions, binning the allele frequencies, and selecting the bin with the greatest abundance of positions. This provides a non-parametric summary of the frequency that alleles were sequenced at. The method is applicable to organisms that have reference genomes that consist of full chromosomes or sub-chromosomal contigs. In contrast to other software designed to detect copy number variation, our method does not rely on an assumption of base ploidy, but instead infers it. We validated these approaches with the model system of Saccharomyces cerevisiae and applied it to the oomycete Phytophthora infestans, both known to vary in copy number. This functionality has been incorporated into the current release of the R package vcfR to provide modular and flexible methods to investigate copy number variation in genomic projects.

vcfr: a package to manipulate and visualize variant call format data in R.

Software to call single-nucleotide polymorphisms or related genetic variants has converged on the variant call format (VCF) as the output format of choice. This has created a need for tools to work with VCF files. While an increasing number of software exists to read VCF data, many only extract the genotypes without including the data associated with each genotype that describes its quality. We created the r package vcfr to address this issue. We developed a VCF file exploration tool implemented in the r language because r provides an interactive experience and an environment that is commonly used for genetic data analysis. Functions to read and write VCF files into r as well as functions to extract portions of the data and to plot summary statistics of the data are implemented. vcfr further provides the ability to visualize how various parameterizations of the data affect the results. Additional tools are included to integrate sequence (fasta) and annotation data (GFF) for visualization of genomic regions such as chromosomes. Conversion functions translate data from the vcfr data structure to formats used by other r genetics packages. Computationally intensive functions are implemented in C++ to improve performance. Use of these tools is intended to facilitate VCF data exploration, including intuitive methods for data quality control and easy export to other r packages for further analysis. vcfr thus provides essential, novel tools currently not available in r.

Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing, and Implications for Late Blight Epidemiology.

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.

SNP-Based Differentiation of Phytophthora infestans Clonal Lineages Using Locked Nucleic Acid Probes and High-Resolution Melt Analysis.

Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic differences can exist between lineages. Whole P. infestans genomes were aligned and single nucleotide polymorphisms (SNPs) identified as targets for the development of clonal-lineage-specific molecular diagnostic tools. Informative SNPs were used to develop high-resolution melt (HRM) assays and locked nucleic acid (LNA) probes to differentiate lineage US-23, the predominant lineage in the Eastern United States for the past several years, from three other U.S. lineages. Three different primer pairs targeting one to three SNPs were capable of separating lineage US-23 from lineages US-8, US-11, and US-24 using HRM analysis. A fourth HRM primer pair targeted a highly variable genomic region containing nine polymorphisms within 63 bp. These primers separated US-23, US-11, and US-8 plus US-24 into three separate groups following HRM analysis but did not separate US-8 from US-24. Additionally, two LNA probes were designed to target a portion of the P. infestans genome containing two SNPs diagnostic for US-23. A single multiplex quantitative polymerase chain reaction assay containing both differentially labeled LNA probes differentiated individuals belonging to lineage US-23 from those belonging to US-8, US-11, and US-24.

Simple sequence repeat markers that identify Claviceps species and strains.

BACKGROUND: Claviceps purpurea is a pathogen that infects most members of Pooideae, a subfamily of Poaceae, and causes ergot, a floral disease in which the ovary is replaced with a sclerotium. When the ergot body is accidently consumed by either man or animal in high enough quantities, there is extreme pain, limb loss and sometimes death. RESULTS: This study was initiated to develop simple sequence repeat (SSRs) markers for rapid identification of C. purpurea. SSRs were designed from sequence data stored at the National Center for Biotechnology Information database. The study consisted of 74 ergot isolates, from four different host species, Lolium perenne, Poa pratensis, Bromus inermis, and Secale cereale plus three additional Claviceps species, C. pusilla, C. paspali and C.fusiformis. Samples were collected from six different counties in Oregon and Washington over a 5-year period. Thirty-four SSR markers were selected, which enabled the differentiation of each isolate from one another based solely on their molecular fingerprints. Discriminant analysis of principle components was used to identify four isolate groups, CA Group 1, 2, 3, and 4, for subsequent cluster and molecular variance analyses. CA Group 1 consisting of eight isolates from the host species P. pratensis, was separated on the cluster analysis plot from the remaining three groups and this group was later identified as C. humidiphila. The other three groups were distinct from one another, but closely related. These three groups contained samples from all four of the host species. These SSRs are simple to use, reliable and allowed clear differentiation of C. humidiphila from C. purpurea. Isolates from the three separate species, C. pusilla, C. paspali and C.fusiformis, also amplified with these markers. CONCLUSIONS: The SSR markers developed in this study will be helpful in defining the population structure and genetics of Claviceps strains. They will also provide valuable tools for plant breeders needing to identify resistance in crops or for researchers examining fungal movements across environments.

Phytophthora community structure analyses in Oregon nurseries inform systems approaches to disease management.

Nursery plants are important vectors for plant pathogens. Understanding what pathogens occur in nurseries in different production stages can be useful to the development of integrated systems approaches. Four horticultural nurseries in Oregon were sampled every 2 months for 4 years to determine the identity and community structure of Phytophthora spp. associated with different sources and stages in the nursery production cycle. Plants, potting media, used containers, water, greenhouse soil, and container yard substrates were systematically sampled from propagation to the field. From 674 Phytophthora isolates recovered, 28 different species or taxa were identified. The most commonly isolated species from plants were Phytophthora plurivora (33%), P. cinnamomi (26%), P. syringae (19%), and P. citrophthora (11%). From soil and gravel substrates, P. plurivora accounted for 25% of the isolates, with P. taxon Pgchlamydo, P. cryptogea, and P. cinnamomi accounting for 18, 17, and 15%, respectively. Five species (P. plurivora, P. syringae, P. taxon Pgchlamydo, P. gonapodyides, and P. cryptogea) were found in all nurseries. The greatest diversity of taxa occurred in irrigation water reservoirs (20 taxa), with the majority of isolates belonging to internal transcribed spacer clade 6, typically including aquatic opportunists. Nurseries differed in composition of Phytophthora communities across years, seasons, and source within the nursery. These findings suggest likely contamination hazards and target critical control points for management of Phytophthora disease using a systems approach.

Multilocus analyses reveal little evidence for lineage-wide adaptive evolution within major clades of soft pines (Pinus subgenus Strobus).

Estimates from molecular data for the fraction of new nonsynonymous mutations that are adaptive vary strongly across plant species. Much of this variation is due to differences in life history strategies as they influence the effective population size (Ne ). Ample variation for these estimates, however, remains even when comparisons are made across species with similar values of Ne . An open question thus remains as to why the large disparity for estimates of adaptive evolution exists among plant species. Here, we have estimated the distribution of deleterious fitness effects (DFE) and the fraction of adaptive nonsynonymous substitutions (α) for 11 species of soft pines (subgenus Strobus) using DNA sequence data from 167 orthologous nuclear gene fragments. Most newly arising nonsynonymous mutations were inferred to be so strongly deleterious that they would rarely become fixed. Little evidence for long-term adaptive evolution was detected, as all 11 estimates for α were not significantly different from zero. Nucleotide diversity at synonymous sites, moreover, was strongly correlated with attributes of the DFE across species, thus illustrating a strong consistency with the expectations from the Nearly Neutral Theory of molecular evolution. Application of these patterns to genome-wide expectations for these species, however, was difficult as the loci chosen for the analysis were a biased set of conserved loci, which greatly influenced the estimates of the DFE and α. This implies that genome-wide parameter estimates will need truly genome-wide data, so that many of the existing patterns documented previously for forest trees (e.g. little evidence for signature of selection) may need revision.

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

The genome of tolypocladium inflatum: evolution, organization, and expression of the cyclosporin biosynthetic gene cluster.

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.