The Impatiens Downy Mildew Epidemic in the United States Is Caused by New, Introgressed Lineages of Plasmopara destructor with Prominent Genotypic Diversity and High Evolutionary Potential.

Impatiens downy mildew (IDM) caused by Plasmopara destructor is currently the primary constraint on the production and use of impatiens (Impatiens walleriana) as bedding plants worldwide. Downy mildew has been documented since the 1880s from wild-grown Impatiens spp. but epidemic outbreaks of the disease affecting the commercially grown, ornamental I. walleriana were only reported for the first time in 2003 in the United Kingdom and in 2004 in the United States. Here, we assess the genetic diversity, level of differentiation, and population structure from 623 samples associated with current and preepidemic IDM outbreaks, by genotyping the samples with simple sequence repeat markers. P. destructor population structure following the emergence of IDM in the United States is subdivided into four genetic lineages characterized by high genetic diversity, mixed reproduction mode, inbreeding, and an excess of heterozygosity. P. destructor genotypes are significantly differentiated from preepidemic IDM samples from hosts other than I. walleriana but no geographical or temporal subdivision is evident. P. destructor samples from different Impatiens spp. show significant but very low levels of differentiation in the analysis of molecular variance test that did not hold in discriminant analysis of principal components analyses. The same was observed between samples of P. destructor and P. velutina recovered from I. walleriana. The finding of shared genotypes in samples from different countries and lack of differentiation among U.S. and Costa Rican samples indicate the occurrence of international movement of the pathogen. Our study provides the first high-resolution analysis of the diversity of P. destructor populations and the IDM epidemic that may be instrumental for disease management and breeding efforts.

Mitochondrial Loci Enable Specific Quantitative Real-Time PCR Detection of the Pathogen Causing Contemporary Impatiens Downy Mildew Epidemics.

Impatiens downy mildew (IDM) disease is a primary constraint on the production of Impatiens walleriana, a popular and economically important floriculture plant. IDM is caused by the biotrophic. oomycete Plasmopara destructor that emerged as a pathogen of I. walleriana in the 2000s. To enable P. destructor detection and quantification, a hydrolysis-probe-based quantitative PCR diagnostic assay was developed based on unique orientation and order of the mitochondrial cytochrome c oxidase subunit1 (cox1) and ATP synthase subunit alpha (atp1) genes in the genus Plasmopara. Nucleotide sequences and analysis of the cox1/atp1 region distinguished P. destructor and its sister-species P. obducens, consistent with prior phylogenetic analyses using cox2 and rDNA markers. Specificity for P. destructor was incorporated into a hydrolysis probe targeting the cox1 gene and flanking primers that amplified across the cox1/atp1 intergenic region. The limit of detection was 0.5 fg/µl of P. destructor DNA (∼100 plasmid copies/µl), with amplification efficiency = 0.95. The assay was validated against a panel of target and nontarget oomycetes, which showed that the primers were specific for Plasmopara spp., while the probe was specific for P. destructor infecting both I. walleriana and I. balsamina. Testing of Impatiens tissue collected from 23 locations across 13 states indicated all samples with IDM symptoms tested positive for P. destructor. Asymptomatic plants from two locations also tested positive for P. destructor.

Population Genomics Trace Clonal Diversification and Intercontinental Migration of an Emerging Fungal Pathogen of Boxwood.

Boxwood blight was first documented in Europe, prior to its recent colonization of North America, where it continues to have significant negative impacts on the ornamental industry. Due to near genetic uniformity in the two sister species of fungal plant pathogens that cause boxwood blight, understanding historical disease emergence and predicting future outbreaks is limited. The goal of this research was to apply population genomics to understand the role of pathogen diversification and migration in disease emergence. Specifically, we tested whether the primary pathogen species Calonectria pseudonaviculata has remained genetically isolated from its European-limited sister species C. henricotiae, while diversifying into clonal lineages that have migrated among continents. Whole-genome sequencing identified 1,608 single-nucleotide polymorphisms (SNPs) in 67 C. pseudonaviculata isolates from four continents and 1,017 SNPs in 13 C. henricotiae isolates from Europe. Interspecific genetic differentiation and an absence of shared polymorphisms indicated lack of gene flow between the sister species. Tests for intraspecific genetic structure in C. pseudonaviculata identified four genetic clusters, three of which corresponded to monophyletic phylogenetic clades. Comparison of evolutionary divergence scenarios among the four genetic clusters using approximate Bayesian computation indicated that the two C. pseudonaviculata genetic clusters currently found in the United States were derived from different sources, one from the first genetic cluster found in Europe and the second from an unidentified population. Evidence for multiple introductions of this pathogen into the United States and intercontinental migration indicates that future introductions are likely to occur and should be considered in plant disease quarantine regulation.

Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

Evidence for the Role of CYP51A and Xenobiotic Detoxification in Differential Sensitivity to Azole Fungicides in Boxwood Blight Pathogens.

Boxwood blight, a fungal disease of ornamental plants (Buxus spp.), is caused by two sister species, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Compared to Cps, Che is documented to display reduced sensitivity to fungicides, including the azole class of antifungals, which block synthesis of a key fungal membrane component, ergosterol. A previous study reported an ergosterol biosynthesis gene in Cps, CYP51A, to be a pseudogene, and RNA-Seq data confirm that a functional CYP51A is expressed only in Che. The lack of additional ergosterol biosynthesis genes showing significant differential expression suggests that the functional CYP51A in Che could contribute to reduced azole sensitivity when compared to Cps. RNA-Seq and bioinformatic analyses found that following azole treatment, 55 genes in Cps, belonging to diverse pathways, displayed a significant decrease in expression. Putative xenobiotic detoxification genes overexpressed in tetraconazole-treated Che encoded predicted monooxygenase and oxidoreductase enzymes. In summary, expression of a functional CYP51A gene and overexpression of predicted xenobiotic detoxification genes appear likely to contribute to differential fungicide sensitivity in these two sister taxa.

Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen.

Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L50 = 5; N50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

One Clonal Lineage of Calonectria pseudonaviculata Is Primarily Responsible for the Boxwood Blight Epidemic in the United States.

Boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae is destroying cultivated and native boxwood worldwide, with profound negative economic impacts on the horticulture industry. First documented in the United States in 2011, the disease has now occurred in 30 states. Previous research showed that global C. pseudonaviculata populations prior to 2014 had a clonal structure, and only the MAT1-2 idiomorph was observed. In this study, we examined C. pseudonaviculata genetic diversity and population structure in the United States after 2014, following the expansion of the disease across the country over the past 5 years. Two hundred eighteen isolates from 21 states were genotyped by sequencing 11 simple sequence repeat (SSR) loci and by MAT1 idiomorph typing. All isolates presented C. pseudonaviculata-specific alleles, indicating that C. henricotiae is still absent in the U.S. states sampled. The presence of only the MAT1-2 idiomorph and gametic linkage disequilibrium suggests the prevalence of asexual reproduction. The contemporary C. pseudonaviculata population is characterized by a clonal structure and composed of 13 multilocus genotypes (SSR-MLGs) unevenly distributed across the United States. These SSR-MLGs grouped into two clonal lineages (CLs). The predominant lineage CL2 (93% of isolates) is the primary contributor to U.S. disease expansion. The contemporary U.S. C. pseudonaviculata population is not geographically subdivided and not genetically differentiated from the U.S. population prior to 2014, but is significantly differentiated from the main European population, which is largely composed of CL1. Our findings provide insights into the boxwood blight epidemic that are critical for disease management and breeding of resistant boxwood cultivars.

Real-Time PCR Detection of Clarireedia spp., the Causal Agents of Dollar Spot in Turfgrasses.

Dollar spot is one of the most economically important diseases of turfgrasses. Recent taxonomic revisions have placed the dollar spot fungal pathogens in the new genus Clarireedia, with five species described. The main goal of this study was to develop a quantitative real-time PCR (qPCR) molecular detection assay based on the internal transcribed spacer (ITS) of the ribosomal RNA genes to quantify the abundance of Clarireedia spp. from environmental (field) samples. The qPCR assay was able to detect isolates of the four tested Clarireedia spp. but did not cross react with nontarget fungi, including closely related taxa, other turfgrass pathogens, or other fungal species commonly isolated from turfgrass. The assay is capable of detecting as little as 38.0 fg (3.8 × 10-14 g) of Clarireedia genomic DNA in 3 h. The qPCR assay detected Clarireedia spp. in both symptomatic and asymptomatic creeping bentgrass (Agrostis stolonifera) foliar tissue. Clarireedia spp. were rarely detected in the thatch or soil, indicating that these pathogens are not widely distributed in these areas of the environment. The fact that the pathogen was detected in asymptomatic tissue suggests that creeping bentgrass may be able to tolerate a certain quantity of the pathogens in leaves before disease symptoms appear; however, further research is needed to validate this hypothesis.

First report: Co-infection of Sarcococca hookeriana (sweetbox) by Coccinonectria pachysandricola and Calonectria pseudonaviculata causes a foliar disease of sweetbox in Pennsylvania.

Sweetbox (Sarcococca hookeriana) are high value ornamental shrubs susceptible to disease caused by Calonectria pseudonaviculata (Cps) and Coccinonectria pachysandricola (Cpa) (Malapi-Wight et al. 2016; Salgado-Salazar et al. 2019). In July 2018, 18-month old sweetbox with leaf spots and defoliation were observed in a residential landscape in Lancaster County, Pennsylvania. Small tan leaf spots grew to cover half of the leaf, developing a concentric banding with dark brown rings and a yellow halo (Sup. Doc. 1: Sup. Fig. 1). The symptoms agreed with those of Cpa disease of sweetbox reported from Washington D.C. (Salgado-Salazar et al. 2019). Diseased plants were located ~1.5 m from Buxus sempervirens with boxwood blight. Morphological and genetic characterization of isolated fungi and pathogenicity tests followed Salgado-Salazar et al. (2019) (Sup. Doc. 2). White to salmon pink spore masses developed on the abaxial leaf surface after humid chamber incubation. Two distinct fungal cultures were recovered (JAC 18-61, JAC 18-79) on potato dextrose agar (Fisher Scientific, Pittsburg, PA). JAC 18-61 presented cultural and morphological characteristics as described for Cps (Crous et al. 2002). JAC 18-79 produced flat, filamentous, light salmon colonies with tan centers and white filiform borders containing pale pink sporodochia, verticillate and simple conidiophores (x̄: 61.8 ± 20.12 µm, N = 20) with lateral, cylindrical phialides (x̄ = 18.1 ± 5.83 x 2.4 ± 0.7 µm, N = 20), and ellipsoid, hyaline conidia without septa (x̄ = 15.2 ± 1.9 x 3.3 ± 0.7 µm, N = 20). Sexual structures and chlamydospores were not observed. The characteristics of JAC 18-79 agree with those reported for Cpa (Salgado-Salazar et al. 2019). Bidirectional sequencing of the ITS, beta-TUB, and RPB1 and RPB2 regions was performed as described (Salgado-Salazar et al. 2019). BLASTn comparisons against NCBI GenBank revealed JAC 18-61 sequences (MT318150 and MT328399) shared 100% identity with Cps sequences (JX535321 and JX535307 from isolate CB002). Sequences from JAC 18-79 (MT318151, MT341237 to MT341239) were 100% identical to Cpa sequences (MH892596, MH936775, MH936703 from isolate JAC 16-20 and JF832909, isolate CBS 128674). The genome of JAC 18-79 was sequenced and yielded an assembly of 26.3 Mb (204 contigs > 1000 bases, N50 = 264.3 kb, 92x coverage, JABAHV0000000000) that contained the MAT1-2 mating-type idiomorph and shared 98.9% similarity with Cpa BPI910731. Isolate JAC 18-61 (Cps) caused lesions on wounded and unwounded sweetbox and boxwood leaves (Sup. Table 1). In general, JAC 18-79 (Cpa) infected only wounded leaves of both hosts; however, in one trial, one unwounded sweetbox and two unwounded boxwood plants developed lesions, possibly due to the presence of natural wounds. Control plants did not develop symptoms. These results diverge to some degree from previous reports of Cpa infecting unwounded sweetbox and not infecting wounded boxwood (Salgado-Salazar et al. 2019). These results indicate that virulence variation among Cpa isolates might occur. Plating of symptomatic tissue and examination of spores fulfilled Koch's postulates for both pathogens. To our knowledge, this is the first report of Cpa blight on sweetbox in Pennsylvania, and the second U.S. report of the disease. This is also the first report of co-infection of Cpa and Cps on diseased sweetbox foliage. Given the capacity of Cpa to infect both sweetbox and boxwood, inspection for Cpa on both hosts is advisable.

Global distribution of mating types shows limited opportunities for mating across populations of fungi causing boxwood blight disease.

Boxwood blight is a disease threat to natural and managed landscapes worldwide. To determine mating potential of the fungi responsible for the disease, Calonectria pseudonaviculata and C. henricotiae, we characterized their mating-type (MAT) loci. Genomes of C. henricotiae, C. pseudonaviculata and two other Calonectria species (C. leucothoes, C. naviculata) were sequenced and used to design PCR tests for mating-type from 268 isolates collected from four continents. All four Calonectria species have a MAT locus that is structurally consistent with the organization found in heterothallic ascomycetes, with just one idiomorph per individual isolate. Mating type was subdivided by species: all C. henricotiae isolates possessed the MAT1-1 idiomorph, whereas all C. pseudonaviculata isolates possessed the MAT1-2 idiomorph. To determine the potential for divergence at the MAT1 locus to present a barrier to interspecific hybridization, evolutionary analysis was conducted. Phylogenomic estimates showed that C. henricotiae and C. pseudonaviculata diverged approximately 2.1 Mya. However, syntenic comparisons, phylogenetic analyses, and estimates of nucleotide divergence across the MAT1 locus and proximal genes identified minimal divergence in this region of the genome. These results show that in North America and parts of Europe, where only C. pseudonaviculata resides, mating is constrained by the absence of MAT1-1. In regions of Europe where C. henricotiae and C. pseudonaviculata currently share the same host and geographic range, it remains to be determined whether or not these two recently diverged species are able to overcome species barriers to mate.

Genome resources for the stem and bark canker pathogens Corinectria fuckeliana, Neonectria hederae and N. punicea.

Corinectria fuckeliana, Neonectria hederae, and N. punicea are fungi in the family Nectriaceae that cause canker diseases of numerous hardwood trees, conifers, and woody perennials, often leading to plant mortality. Here, we report draft genome sequences for these three phytopathogenic fungal species. The genome sizes are consistent with those reported for other members of the Nectriaceae (28 to 43 Mb). These are the first genome resources available for C. fuckeliana, N. hederae, and N. punicea. These genome sequences may provide insights into the mechanisms of virulence and pathogenicity employed by these three destructive plant pathogens, and are resources suitable for the development of molecular markers that could be used for species identification, diagnostic tools and barcodes, and population studies.

Coccinonectria pachysandricola, Causal Agent of a New Foliar Blight Disease of Sarcococca hookeriana.

Woody plants of the Buxaceae, including species of Buxus, Pachysandra, and Sarcococca, are widely grown evergreen shrubs and groundcovers. Severe leaf spot symptoms were observed on S. hookeriana at the U.S. National Arboretum in Washington, DC, in 2016. Affected plants were growing adjacent to P. terminalis exhibiting Volutella blight symptoms. Fungi isolated from both hosts were identical based on morphology and multilocus phylogenetic analysis and were identified as Coccinonectria pachysandricola (Nectriaceae, Hypocreales), causal agent of Volutella blight of Pachysandra species. Pathogenicity tests established that Co. pachysandricola isolated from both hosts caused disease symptoms on P. terminalis and S. hookeriana, but not on B. sempervirens. Artificial inoculations with Pseudonectria foliicola, causal agent of Volutella blight of B. sempervirens, did not result in disease on P. terminalis or S. hookeriana. Wounding enhanced infection by Co. pachysandricola and Ps. foliicola on all hosts tested but was not required for disease development. Genome assemblies were generated for the Buxaceae pathogens that cause Volutella diseases: Co. pachysandricola, Ps. buxi, and Ps. foliicola; these ranged in size from 25.7 to 28.5 Mb. To our knowledge, this foliar blight of S. hookeriana represents a new disease for this host and is capable of causing considerable damage to infected plants.

Boxwood blight: an ongoing threat to ornamental and native boxwood.

Boxwood blight is an emerging disease of ornamental and native boxwood plants in the family Buxaceae. First documented in the 1990s at a single location in England, the disease is now reported throughout Europe, Asia, New Zealand, and North America. To address the growing concern over boxwood blight, ongoing research focuses on multiple biological and genetic aspects of the causal pathogens and susceptible host plants. Characterization of genetic variation among the Calonectria fungi that cause boxwood blight shows that two unique sister species with different geographic distributions incite the disease. Studies of the pathogen life cycle show the formation of long-lived survival structures and that host infection is dependent on inoculum density, temperature, and humidity. Host range investigations detail high levels of susceptibility among boxwood as well as the potential for asymptomatic boxwood infection and for other plants in the family Buxaceae to serve as additional hosts. Multiple DNA-based diagnostic assays are available, ranging from probe-based quantitative PCR assays to the use of comparative genomics to develop robust diagnostic markers or provide whole genome-scale identifications. Though many questions remain, the research that continues to address boxwood blight demonstrates the importance of applying a multidisciplinary approach to understand and control emerging plant diseases.

SimpleSynteny: a web-based tool for visualization of microsynteny across multiple species.

Defining syntenic relationships among orthologous gene clusters is a frequent undertaking of biologists studying organismal evolution through comparative genomic approaches. With the increasing availability of genome data made possible through next-generation sequencing technology, there is a growing need for user-friendly tools capable of assessing synteny. Here we present SimpleSynteny, a new web-based platform capable of directly interrogating collinearity of local genomic neighbors across multiple species in a targeted manner. SimpleSynteny provides a pipeline for evaluating the synteny of a preselected set of gene targets across multiple organismal genomes. An emphasis has been placed on ease-of-use, and users are only required to submit FASTA files for their genomes and genes of interest. SimpleSynteny then guides the user through an iterative process of exploring and customizing genomes individually before combining them into a final high-resolution figure. Because the process is iterative, it allows the user to customize the organization of multiple contigs and incorporate knowledge from additional sources, rather than forcing complete dependence on the computational predictions. Additional tools are provided to help the user identify which contigs in a genome assembly contain gene targets and to optimize analyses of circular genomes. SimpleSynteny is freely available at: http://www.SimpleSynteny.com.

Genetic Variation of the Pathogen Causing Impatiens Downy Mildew Predating and Including Twenty-first Century Epidemics on Impatiens walleriana.

Impatiens downy mildew (IDM) of cultivated Impatiens walleriana has had a significant economic impact on the ornamental horticulture industry in the United States and globally. Although recent IDM outbreaks started in 2003, downy mildews on noncultivated Impatiens species have been documented since the 1880s. To understand the relationship between the pathogen causing recent epidemics and the pathogen historically present in the United States, this work characterized genetic variation among a collection of 1,000 samples on 18 plant hosts. Samples included collections during recent IDM epidemics and historical herbarium specimens. Ten major genotypes were identified from cloned rDNA amplicon sequencing and endpoint SNP genotyping. Three genotypes accounted for >95% of the samples, with only one of these three genotypes found on samples predating recent IDM outbreaks. Based on phylogenetic analysis integrating data from three markers and the presence of individual genotypes on multiple Impatiens species, there was some evidence of pathogen-specific infection of I. noli-tangere, but the distinction between genotypes infecting I. walleriana and I. balsamina was not upheld. Overall, this work provides evidence that the majority of rDNA genotypes recovered from recent IDM epidemics are different from historical U.S. genotypes, and that these genotypes can infect Impatiens spp. other than I. walleriana.

Newly Emerged Populations of Plasmopara halstedii Infecting Rudbeckia Exhibit Unique Genotypic Profiles and Are Distinct from Sunflower-Infecting Strains.

The oomycete Plasmopara halstedii emerged at the onset of the 21st century as a destructive new pathogen causing downy mildew disease of ornamental Rudbeckia fulgida (rudbeckia) in the United States. The pathogen is also a significant global problem of sunflower (Helianthus annuus) and is widely regarded as the cause of downy mildew affecting 35 Asteraceae genera. To determine whether rudbeckia and sunflower downy mildew are caused by the same genotypes, population genetic and phylogenetic analyses were performed. A draft genome assembly of a P. halstedii isolate from sunflower was generated and used to design 15 polymorphic simple sequence repeat (SSR) markers. SSRs and two sequenced phylogenetic markers measured differentiation between 232 P. halstedii samples collected from 1883 to 2014. Samples clustered into two main groups, corresponding to host origin. Sunflower-derived samples separated into eight admixed subclusters, and rudbeckia-derived samples further separated into three subclusters. Pre-epidemic rudbeckia samples clustered separately from modern strains. Despite the observed genetic distinction based on host origin, P. halstedii from rudbeckia could infect sunflower, and exhibited the virulence phenotype of race 734. These data indicate that the newly emergent pathogen populations infecting commercial rudbeckia are a different species from sunflower-infecting strains, notwithstanding cross-infectivity, and genetically distinct from pre-epidemic populations infecting native rudbeckia hosts.

Sarcococca Blight: Use of Whole-Genome Sequencing for Fungal Plant Disease Diagnosis.

Early and accurate diagnosis of new plant pathogens is vital for the rapid implementation of effective mitigation strategies and appropriate regulatory responses. Most commonly, pathogen identification relies on morphology and DNA marker analysis. However, for new diseases, these approaches may not be sufficient for precise diagnosis. In this study, we used whole-genome sequencing (WGS) to identify the causal agent of a new disease affecting Sarcococca hookeriana (sarcococca). Blight symptoms were observed on sarcococca and adjacent Buxus sempervirens (boxwood) plants in Maryland during 2014. Symptoms on sarcococca were novel, and included twig dieback and dark lesions on leaves and stems. A Calonectria sp. was isolated from both hosts and used to fulfill Koch's postulates but morphology and marker sequence data precluded species-level identification. A 51.4-Mb WGS was generated for the two isolates and identified both as Calonectria pseudonaviculata. A single-nucleotide polymorphism at a noncoding site differentiated between the two host isolates. These results indicate that the same C. pseudonaviculata genotype has the ability to induce disease on both plant species. This study marks the first application of WGS for fungal plant pathogen diagnosis and demonstrates the power of this approach to rapidly identify causal agents of new diseases.

A Multiplex Real-Time PCR Assay for the Detection of Puccinia horiana and P. chrysanthemi on Chrysanthemum.

Puccinia horiana, the cause of chrysanthemum white rust, is a regulated fungal plant pathogen in the United States, while P. chrysanthemi, the cause of chrysanthemum brown rust, is a widespread but less destructive pathogen. Accurate identification is essential to enforce quarantine measures, but the two species cannot be differentiated visually in the absence of mature spores or symptoms. A multiplex real-time PCR assay was developed to detect and discriminate between P. chrysanthemi and P. horiana. Species-specific hydrolysis probes labeled with different fluorescent dyes were designed based on the rDNA internal transcribed spacer region. Seven fresh samples and 270 herbarium specimens of chrysanthemum rust were tested with the assay with results confirmed using spore morphology. P. horiana and P. chrysanthemi were accurately detected from all fresh samples, and as little as 1 pg of template DNA was reproducibly detected. Of the herbarium specimens, 99% were positive for at least one species using the multiplex assay with 7% positive for both species. This multiplex assay can discriminate between P. chrysanthemi and P. horiana and provides an additional tool for identification of P. horiana to ensure appropriate application of quarantine measures.

Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi.

BACKGROUND: Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. RESULTS: Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. CONCLUSIONS: These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

Anthracnose disease of centipedegrass turf caused by Colletotrichum eremochloae, a new fungal species closely related to Colletotrichum sublineola.

Colletotrichum is a cosmopolitan, anamorphic fungal genus responsible for anthracnose disease in hundreds of plant species worldwide, including members of the Poaceae. Anthracnose disease of the widely planted, non-native, warm-season lawn grass, Eremochloae ophiuroides (centipedegrass), is commonly encountered in the southern United States, but the causal agent has never been identified. We use DNA sequence data from modern cultures and archival fungarium specimens in this study to determine the identity of the fungus responsible for centipedegrass anthracnose disease and provide experimental confirmation of pathogenicity. C. eremochloae sp. nov., a pathogen of centipedegrass, is proposed based on phylogenetic evidence from four sequence markers (Apn2, Apn2/ Mat1, Sod2, ITS). C. eremochloae isolates from centipedegrass shared common morphology and phenotype with C. sublineola, a destructive pathogen of cultivated sorghum and Johnsongrass weeds (Sorghum halepense, S. vulgaris). Molecular phylogenetic analysis identified C. eremochloae and C. sublineola as closely related sister taxa, but genealogical concordance supported their distinction as unique phylogenetic species. Fixed nucleotide differences between C. eremochloae and C. sublineola were observed from collections of these fungi spanning 105 y, including the 1904 lectotype specimen of C. sublineola. C. eremochloae was identified from a fungarium specimen of centipedegrass intercepted at a USA port from a 1923 Chinese shipment; the multilocus sequence from this specimen was identical to modern samples of the fungus. Thus, it appears that the fungus might have migrated to the USA around the same time that centipedegrass first was introduced to the USA in 1916 from China, where the grass is indigenous. The new species C. eremochloae is described and illustrated, along with a description and discussion of C. sublineola based on the lectotype and newly designated epitype.