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.

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.

Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata.

BACKGROUND: Boxwood blight disease caused by Calonectria henricotiae and C. pseudonaviculata is of ecological and economic significance in cultivated and native ecosystems worldwide. Prior research has focused on understanding the population genetic and genomic diversity of C. henricotiae and C. pseudonaviculata, but gene family evolution in the context of host adaptation, plant pathogenesis, and trophic lifestyle is poorly understood. This study applied bioinformatic and phylogenetic methods to examine gene family evolution in C. henricotiae, C. pseudonaviculata and 22 related fungi in the Nectriaceae that vary in pathogenic and saprobic (apathogenic) lifestyles. RESULTS: A total of 19,750 gene families were identified in the 24 genomes, of which 422 were rapidly evolving. Among the six Calonectria species, C. henricotiae and C. pseudonaviculata were the only species to experience high levels of rapid contraction of pathogenesis-related gene families (89% and 78%, respectively). In contrast, saprobic species Calonectria multiphialidica and C. naviculata, two of the closest known relatives of C. henricotiae and C. pseudonaviculata, showed rapid expansion of pathogenesis-related gene families. CONCLUSIONS: Our results provide novel insight into gene family evolution within C. henricotiae and C. pseudonaviculata and suggest gene family contraction may have contributed to limited host-range expansion of these pathogens within the plant family Buxaceae.

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.

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.

Competing sexual-asexual generic names in Agaricomycotina (Basidiomycota) with recommendations for use.

With the change to one scientific name for fungal taxa, generic names typified by species with sexual or asexual morph types are being evaluated to determine which names represent the same genus and thus compete for use. In this paper generic names of the Agaricomycotina (Basidiomycota) were evaluated to determine synonymy based on their type. Forty-seven sets of sexually and asexually typified names were determined to be congeneric and recommendations are made for which generic name to use. In most cases the principle of priority is followed. However, 16 generic names are recommended for use that do not have priority and thus need to be protected: Aleurocystis over Matula; Armillaria over Acurtis and Rhizomorpha; Asterophora over Ugola; Botryobasidium over Acladium, Allescheriella, Alysidium, Haplotrichum, Physospora, and Sporocephalium; Coprinellus over Ozonium; Coprinopsis over Rhacophyllus; Dendrocollybia over Sclerostilbum and Tilachlidiopsis; Diacanthodes over Bornetina; Echinoporia over Echinodia; Neolentinus over Digitellus; Postia over Ptychogaster; Riopa over Sporotrichum; Scytinostroma over Artocreas, Michenera, and Stereofomes; Tulasnella over Hormomyces; Typhula over Sclerotium; and Wolfiporia over Gemmularia and Pachyma. Nine species names are proposed for protection: Botryobasidium aureum, B. conspersum, B. croceum, B. simile, Pellicularia lembosporum (syn. B. lembosporum), Phanerochaete chrysosporium, Polyporus metamorphosus (syn. Riopa metamorphosa), Polyporus mylittae (syn. Laccocephalum mylittae), and Polyporus ptychogaster (syn. Postia ptychogaster). Two families are proposed for protection: Psathyrellaceae and Typhulaceae. Three new species names and 30 new combinations are established, and one lectotype is designated.

Peronospora kuewa, sp. nov., a new downy mildew species infecting the endangered Hawaiian plant Plantago princeps var. princeps.

Plantago princeps var. princeps is an endangered native Hawaiian plant, and part of the recovery plan includes repopulation using plants grown in a nursery. However, disease pressure from downy mildew is hindering repopulation efforts. The organism associated with the downy mildew was determined to be a Peronospora species with brown, ellipsoid conidia measuring 21 by 16 µm on average, which was morphologically different from validly described species of Peronospora that infect Plantago species, but it was morphologically similar to the invalidly published species Peronospora lanceolatae (Art. 40.1). Comparison of mitochondrial cytochrome oxidase subunit I (cox1), mitochondrial cytochrome oxidase subunit II (cox2), nuclear internal transcribed spacer (ITS), and nuclear 28S rRNA D1-D2 (28S) loci revealed the unknown Peronospora to be molecularly divergent from Peronospora alta and Peronsopora plantaginis, but very similar to Peronospora from Plantago lanceolata, the type host of P. lanceolatae. Phylogenetic trees inferred with maximum likelihood and Bayesian inference from a concatenated alignmaent and individual gene trees confirmed the divergence of the unknown Peronospora from P. alta and P. plantaginis and its similarity to P. lanceolatae. However, attempts to inoculate Plantago lanceolata with the strain from Plantago princeps var. princeps were unsuccessful, which, in conjunction with divergence in ITS, suggests that the unknown Peronospora is specific to Plantago princeps var. princeps. Herein, the Peronospora strain on Plantago princeps var. princeps is described as the new species Peronospora kuewa based on morphology, molecular phylogenetics, and host specificity. In addition, Peronospora gaponenkoae is described here to honor Nina Ivanova Gaponenko on the basis of her description of P. lanceolatae.

Comparative analysis of extracellular proteomes reveals putative effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata.

Calonectria henricotiae (Che) and C. pseudonaviculata (Cps) are destructive fungal pathogens causing boxwood blight, a persistent threat to horticultural production, landscape industries, established gardens, and native ecosystems. Although extracellular proteins including effectors produced by fungal pathogens are known to play a fundamental role in pathogenesis, the composition of Che and Cps extracellular proteins has not been examined. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics prediction tools, 630 extracellular proteins and 251 cell membrane proteins of Che and Cps were identified in the classical secretion pathway in the present study. In the non-classical secretion pathway, 79 extracellular proteins were identified. The cohort of proteins belonged to 364 OrthoMCL clusters, with the majority (62%) present in both species, and a subset unique to Che (19%) and Cps (20%). These extracellular proteins were predicted to play important roles in cell structure, regulation, metabolism, and pathogenesis. A total of 124 proteins were identified as putative effectors. Many of them are orthologs of proteins with documented roles in suppressing host defense and facilitating infection processes in other pathosystems, such as SnodProt1-like proteins in the OrthoMCL cluster OG5_152723 and PhiA-like cell wall proteins in the cluster OG5_155754. This exploratory study provides a repository of secreted proteins and putative effectors that can provide insights into the virulence mechanisms of the boxwood blight pathogens.

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.

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.

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.

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.

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.

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.

Widespread Occurrence of a CYP51A Pseudogene in Calonectria pseudonaviculata.

Calonectria pseudonaviculata and C. henricotiae are two closely related fungal species responsible for boxwood blight disease of ornamental shrubs (Buxus spp.) in the U.S. and Europe. A previous study has shown isolates of the latter species, which is restricted to Europe, to be less sensitive to tetraconazole, an azole fungicide. In this study, we have analyzed the CYP51 paralogs for polymorphism in 26 genomes, representing geographically disparate populations of C. pseudonaviculata (n = 19) and C. henricotiae (n = 7), from the U.S., Europe, Asia, and New Zealand. The presence of a CYP51A pseudogene and lack of a functional CYP51A paralog in all C. pseudonaviculata genomes examined is a novel discovery for fungi and could have implications for the evolution of resistance to antifungal chemicals.

Prokaryotic taxa play keystone roles in the soil microbiome associated with woody perennial plants in the genus Buxus.

The microbiome associated with ornamental plants has largely been neglected, despite its potential for impacting plant health. This work characterized the composition, diversity, and microbial co-associations in the soil microbiome associated with species and cultivars of plant in the genus Buxus (common name boxwood), a group of woody perennial shrubs commonly used in residential landscapes and found in native ecosystems. Soil was collected from 82 individual curated boxwood accessions at the U.S. National Arboretum National Boxwood Collection. Amplicon libraries targeting archaea, bacteria, and fungi were generated and sequenced using the Illumina MiSeq platform. Identification of individual sequence variants resulted in 275 archaeal, 15,580 bacterial, and 7,525 fungal taxa. Neither spatial distance among samples nor association with different types of boxwood were significant predictors of soil microbiome composition. However, archaeal and bacterial diversity was significantly different in soil from distinct types of boxwood. Co-association networks indicated that archaea and bacteria show greater evidence of being keystone taxa than fungi. Overall, this work demonstrates the potential for targeting specific keystone taxa to shift the soil microbiome associated with these boxwood accessions and that planting different species or cultivars in the landscape may shift the diversity of prokaryotic microorganisms.

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.

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.

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.

Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

BACKGROUND: Plasmopara obducens is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of Impatiens that causes high crop loss. Currently, there are no available methods for the microscopic detection of P. obducens from leaves of impatiens, which may be contributing to the spread of the disease. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. To study this important pathogen, we developed and standardized a FISH technique for the visualization of P. obducens from Impatiens walleriana tissues using a species-specific 24-mer oligonucleotide probe designed to target a region of the rRNA internal transcribed spacer 2 (ITS2). RESULTS: Since P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the P. obducens rRNA-ITS target sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect P. obducens sporangiophores, sporangia and oospores, and hyphae from naturally infected I. walleriana leaves and stems. Cross-reactivity was not observed from plant tissue, and the assay did not react when applied to E. coli with self-ligated plasmids and non-target oomycete species. CONCLUSIONS: This FISH protocol may provide a valuable tool for the study of this disease and could potentially be used to improve early monitoring of P. obducens, substantially reducing the persistence and spread of this destructive plant pathogen.