The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut.

BACKGROUND: The ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome. RESULTS: The genome of A. anomala was assembled into 108 scaffolds totaling 342,498,352 nt with a GC content of 34.46%. Scaffold N50 was 33.3 Mb and L50 was 5. Nineteen scaffolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identified on both ends of two scaffolds and on one end of another 10 scaffolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identified in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identified throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identified in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identified a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 effectors. CONCLUSIONS: This study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen's life cycle and a solid foundation for studying EFB.

Genetic Diversity Analysis of Anisogramma anomala in the Pacific Northwest and New Jersey.

Anisogramma anomala, a biotrophic ascomycete, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). EFB is endemic in eastern North America, preventing the commercial production of European hazelnut (C. avellana L.). In contrast, the historic absence of A. anomala in the Pacific Northwest (PNW) supported the development of a robust hazelnut industry. Circa 1960, A. anomala was inadvertently introduced into southwestern Washington causing orchard devastation. Distribution of the pathogen in the PNW has been hypothesized to be the result of a single-point introduction. This study aimed to investigate the single-point introduction hypothesis of A. anomala by comparing the genetic diversity of A. anomala samples from the PNW and New Jersey (NJ). Specimens from the main PNW production region [n=60] and an area within the pathogen's native range, NJ [n=151], were genotyped using 15 simple sequence repeat (SSR) markers. The following were used to assess genetic diversity and population structure: allelic summary statistics, discriminant analysis of principal components, network median-joining tree, analysis of multilocus genotypes, and allelic population diversity analysis. Analyses separated the samples into one cluster containing all the PNW isolates, and five clusters of NJ isolates. The PNW samples were nearly genetically uniform, and the NJ isolates were diverse. These findings support the hypothesis that A. anomala in the PNW was derived from a single-point introduction and corroborate previous studies that have shown A. anomala is very diverse in NJ. This indicates that maintaining restrictions on the movement of Corylus into the PNW is important to prevent the introduction of new populations of A. anomala, thus protecting the PNW hazelnut industry.

Population Differentiation Within Anisogramma anomala in North America.

Anisogramma anomala, a biotrophic ascomycete in the order Diaporthales, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). Until recently, little has been documented on its genetic diversity and population structure. In this study, 18 simple sequence repeat markers were used to fingerprint 182 accessions of the fungus originating from across North America. Our results, based on summary statistics of the allelic data, a discriminant analysis of principal components (DAPC) scatterplot, an unweighted pair group method with arithmetic mean (UPGMA) dendrogram, and analysis of multilocus genotypes, show that A. anomala exhibits considerable genetic diversity across multiple populations. Eleven clusters were resolved from the DAPC scatterplot, five of which were validated by statistically supported clusters in the UPGMA dendrogram. The 11 DAPC clusters were statistically significant via an analysis of molecular variance. Dendrogram topology and DAPC scatterplot groups showed some correlation with collection origin; samples collected in proximity tended to cluster together and be genetically similar. However, some locations held populations that were diverse and some populations with a high degree of similarity had disparate origins, suggesting movement by humans. Overall, the results demonstrate the presence of multiple, genetically distinct populations of A. anomala in North America and serve as a reference to assist in understanding and managing EFB.

Development of Genomic Resources for the Powdery Mildew, Erysiphe pulchra.

Powdery mildews (PMs) are important plant pathogens causing widespread damage. Here, we report the first draft genome of Erysiphe pulchra, the causative agent of PM of flowering dogwood, Cornus florida. The assembled genome was 63.5 Mbp and resulted in formation of 19,442 contigs (N50 = 11,686 bp) that contained an estimated 6,860 genes with a genome coverage of 62×. We found 102 candidate secreted effector proteins (CSEPs) in E. pulchra similar to E. necator genes that are potentially involved in disease development. This draft genome is an initial step for understanding the evolutionary history of the PMs and will also provide insight into evolutionary strategies that led to the wide host expansion and environmental adaptations so effectively employed by the PM lineages.

A Real-Time PCR Assay for Early Detection of Eastern Filbert Blight.

Eastern filbert blight (EFB) is a devastating disease of European hazelnut, Corylus avellana, which causes economic losses in Oregon, where 99% of the U.S. crop is produced. The causal fungus, Anisogramma anomala, is native to eastern North America, where it is found associated with the American hazelnut (C. americana). Although C. americana is tolerant, EFB causes cankers, branch dieback, and death of C. avellana. Detection and identification of A. anomala is time consuming using conventional methods because the fungus can only be cultured from sporulating perithecia and the disease symptoms and signs only show 12 to 16 months after infection. In this study, a TaqMan real-time polymerase chain reaction (PCR) assay based on a ribosomal DNA internal transcribed spacer was developed for A. anomala. The assay was validated with multiple isolates of A. anomala, closely related species, common environmental microorganisms, and over 100 C. avellana samples. The real-time PCR assay detected as low as 0.12 pg of A. anomala genomic DNA, and positively diagnosed EFB on 82% of asymptomatic plants as early as 15 weeks from infection. The real-time PCR assay is more sensitive and faster than traditional diagnostic methods. It can facilitate hazelnut breeding and disease management by early and accurate diagnosis of EFB.

Haplotyping of Cornus florida and C. kousa chloroplasts: Insights into species-level differences and patterns of plastic DNA variation in cultivars.

Chloroplast DNA is a part of plant non-nuclear genome, and is of particular interest for lineage studies. Moreover, the non-coding regions of cpDNA display higher mutation rates than the conserved coding cpDNA, which has been employed for phylogenetic and population research. We analyzed the cpDNA of 332 gDNA samples from collections of Cornus florida and C. kousa (commercial cultivars, breeding selections, and wild kousa accessions from Asia), using the chlorotyping system developed on North America-native, wild accessions of C. florida. Our results indicated significant differences in chlorotype frequencies between the two species. Cornus florida samples were represented by all major chlorotypes previously described, whereas all C. kousa samples analyzed had only one of the chlorotype patterns shown by C. florida. The chlorotyping analytic panel was then expanded by sequencing the targeted three non-coding cpDNA regions. Results indicated a major difference in the maternally-inherited cpDNA between the two closely related Big-Bracted Cornus species. Chlorotype diversity and differences in the proportion of informative sites in the cpDNA regions of focus emphasized the importance of proper loci choice for cpDNA-based comparative studies between the closely related dogwood species. Phylogenetic analyses of the retrieved sequences for the other species of Cornus provided information on the relative utility of the cpDNA regions studied and helped delineate the groups (Big-Bracted, Cornelian Cherries, Blue/White-Fruited) within the genus. Genealogical relationships based on the cpDNA sequences and the inferred chlorotype networks indicated the need for continued analyses across further non-coding cpDNA regions to improve the phylogenetic resolution of dogwoods.

Current surgical treatment of thoracic empyema in adults.

A review of the recent literature on treatment modalities of adult thoracic empyema was conducted in order to expose the controversies and verify where consensus exists. Critical reading filtered through clinical experience was the method followed. The roles of surgical drainage, lavage techniques, debridement via VATS, decortication, thoracoplasty and open window thoracostomy were considered using the Oxford Center of Evidence Based Medicine criteria. The roles of the different therapeutical modalities were interpreted in the light of the triphasic nature of empyema thoracis. The randomised controlled trials came up with conflicting results. With two exceptions all of the papers reviewed provide level (2b) or below evidences. The lack of a single ideal treatment modality or policy reflects the complexity of the diagnosis and staging of this heterogeneous disease. Basic elements of intervention--drainage, different evacuation techniques, decortication, thoracoplasty and open window thoracostomy--are well-established technical modalities; however, neither a universally acceptable primary modality nor the gold standard of their sequence is available. Drainage remains to be the initial treatment modality in Phase I disease. Debridement via VATS is a safe, reliable and efficient method in the fibrinopurulent phase. Organised pleural callus requires formal decortication. Open window thoracostomy is a simple and safe procedure for high-risk patients and results in quick detoxication. Thoracoplasty kept its final role in pleural space management. Acute postoperative bronchial stump insufficiency requires immediate surgery. Evacuation of toxic material is mandatory. No single-stage procedure offers a solution. An optimised agressivity treatment modality should be tailored to the condition of the patient and to the potential of the persisting cavity. Decision-making involves a triad consisting of the aetiology of empyema (i.e. primary vs secondary), general condition of the patient and stage of disease, while considering the triphasic nature of development of thoracic empyema. The current attitudes show that the present concepts are based mainly on expert opinion. Flexibility and patience on behalf of the surgeon and nursing staff, the patient and the hospital management, as well as a good understanding of the complexity of this condition are the cornerstones of the treatment. No exclusive sequence of procedures leading to a uniformly predictable successful outcome is available. Individualised approaches can be recommended based on institutional practice and local protocols. Thoracic empyema in general seems to remain resilient to fit completely into the categories of evidence-based medical approach.

A new device for the identification of lymph nodes at lung cancer surgery.

In order to provide a precise lymph node mapping during lung cancer surgery a sterilizable plastic tray moulded in the shape of the mediastinum and lungs is presented by the author. The device makes lymph node mapping simpler, safer, quicker and methodically more structured. A positive impact is expected as a result of usage of the device from making pathologist's work easier and facilitating the flux of information on the surgeon-pathologist-oncologist-pneumonologist chain to be more disinformation-free.

Lung biopsy using harmonic scalpel: a randomised single institute study.

OBJECTIVE: Applicability of harmonic scalpel in lung biopsy was investigated in a randomised single institute study. METHODS: Safety of the method, morbidity, drainage duration and in-hospital stays were compared in two randomised groups of patients in which either ultrasonic harmonic scalpel (n:20) or endostapler (n:20) were used for pulmonary biopsies during VATS. RESULTS: An advantage of 16min in average operation time was found in favour of the harmonic scalpel (30.75 vs 46.9min) which was significant. There were no differences in average drainage duration (40.2 vs 30.6h) and pleural fluid volume (258 vs 232ml). Minor complication rates (3 vs 3) were identical and in-hospital stays (7.6 vs 7.2 days) were also similar. CONCLUSIONS: Overall, the vibration transmission method was shown not to be inferior to the standard endostapling technique. A safe new method offers an alternative technique for peripherial lung biopsy.

Cornus×elwinortonii and Cornus×rutgersensis (Cornaceae), new names for two artificially produced hybrids of big-bracted dogwoods.

Big-bracted dogwoods (Cornus sp.) are well-known plants in North America and eastern Asia where they occur as wild, generally spring-flowering understory trees. They are also popular ornamental landscape plants, and many economically important cultivars are propagated and sold across North America, Europe, and Asia. Starting in the late 1960s, Elwin Orton of Rutgers University in New Jersey (USA) utilized three geographically disjunct species of dogwoods, Cornusflorida (eastern North America), Cornusnuttallii (western North America), and Cornuskousa (East Asia), in an extensive interspecific hybridization program. He was successful in developing the first-ever interspecific F1 hybrids of these species, several of which have become staple items in the ornamental nursery trade due to their enhanced ornamental qualities and resistance to diseases. The original F1 plants are still alive at Rutgers University. While they have been available for decades in horticultural commerce, the interspecific hybrid crosses were never formally described and their scientific hybrid names were never published. For the Cornuskousa × Cornusflorida hybrids, the name Cornus 'rutgersensis' has been used on occasion in the horticultural trade, but without proper citation and description. Here, it is formally named Cornus×rutgersensis Mattera, T. Molnar, & Struwe, hybr. nov. For the Cornuskousa × Cornusnuttallii hybrids, no previous name has been used, and it is hereby named Cornus×elwinortonii Mattera, T. Molnar, & Struwe, hybr. nov. The need for providing scientific names for commonly used horticultural hybrids is discussed. Holotype material for both hybrid names was collected from the original F1 hybrids for full documentation, typification, and description. The comparative intermediate development of leaves, inflorescence structures, and fruit types of the hybrids and their parents is discussed and illustrated. Etymology, phenology, and cultivation aspects of these hybrids and their cultivars including backcrosses to Cornuskousa are also presented.

Pericardioperitoneal shunt: further development of the procedure using VATS technique.

We report a modification of the previously described VATS (video-assisted thoracic surgical) method of pericardioperitoneal shunt. Our method was used in 5 patients with pericardial tamponade requiring permanent drainage.

Changing dogmas: history of development in treatment modalities of traumatic pneumothorax, hemothorax, and posttraumatic empyema thoracis.

Development of treatment modalities for chest wounds and traumatic empyema thoracis is reviewed in the light of war experience. Mortality from thoracic injury was more than 50% before World War I and was about 25% during World War I. It came down to 10% in World War II and was about 5% during the Korean War. It improved further during the Vietnam War, until it ranged at 2% to 4%, where no further improvement could be imagined. Thoracic surgery was born in the field hospitals of World War I. Established drainage methods and standardized anesthesia made thoracotomy a standard procedure in World War II. As experience increased in chest trauma, surgical aggression diminished. Drainage ruled primary chest trauma treatment algorithms during the Vietnam War and coexisted with the full arsenal of cardiothoracic surgery when it was needed. Optimization of thoracic surgical aggression includes a case-tailored approach when major chest surgery with or without interventions on the central cardiovascular system is needed. This is where we are now, provided a proper logistic, Medevac system exists. If we let the past fade away, the danger of committing the mistakes of our predecessors increases without having their excuses. Our present is only the past of the future.

Cutting lung parenchyma using the harmonic scalpel--an animal experiment.

OBJECTIVE: Applicability of harmonic scalpel in lung surgery was investigated using an animal model. METHODS: Air tightness, control of bleeding and features of tissue regeneration were compared in a 4-week time frame of investigation in animals in which either surgical stapler or harmonic scalpel were used for pulmonary resection. RESULTS: No significant differences between the two methods were found on a clinical and histopathological basis. CONCLUSIONS: Complete lack of granuloma formation at the resection line and in its vicinity consequently restitutio ad integrum demonstrate the advantage of the harmonic scalpel over the stapler in the circumstances investigated. Overall the vibration transmission method was shown not to be inferior to the standard methods in peripheral lung tissue resection.

European guidelines on structure and qualification of general thoracic surgery.

OBJECTIVE: To update the recommendations for the structural characteristics of general thoracic surgery (GTS) in Europe in order to provide a document that can be used as a guide for harmonizing the general thoracic surgical practice in Europe. METHODS: A task force was created to set the structural, procedural and qualification characteristics of a European GTS unit. These criteria were endorsed by the Executive Committee of the European Society of Thoracic Surgeons and by the Thoracic Domain of the European Association for Cardio-Thoracic Surgery and were validated by the European Board of Thoracic Surgery at European Union of Medical Specialists. RESULTS: Criteria regarding definition and scope of GTS, structure and qualification of GTS unit, training and education and recommendations for subjects of particular interest (lung transplant, oesophageal surgery, minimally invasive thoracic surgery, quality surveillance) were developed. CONCLUSIONS: This document will hopefully represent the first step of a process of revision of the modern thoracic surgeons' curricula, which need to be qualitatively rethought in the setting of the qualification process. The structural criteria highlighted in the present document are meant to help and tackle the challenge of cultural and language barriers as well as of widely varying national training programmes.

Genome-wide microsatellite identification in the fungus Anisogramma anomala using Illumina sequencing and genome assembly.

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the "Seq-to-SSR" approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel "Seq-Assembly-SSR" approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the "Seq-Assembly-SSR" approach has proven to be a successful one for microsatellite discovery.