Decoding the RNA virome of the tree parasite Armillaria provides new insights into the viral community of soil-borne fungi.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

Cryphonectria carpinicola discovered in Japan: first report of the sexual state on Carpinus tree.

Cryphonectria carpinicola is an ascomycetous fungus that has been regularly found in its asexual form on European hornbeam (Carpinus betulus) in Europe over the past two decades. Here we describe the discovery of C. carpinicola in Japan and report for the first time its sexual state on Carpinus species. No symptomatic trees were observed, but stromata were found saprotrophically on broken branches of Carpinus species on the forest floor. The sexual structures of C. carpinicola resembled that of other Cryphonectria species and strongly resembled those of the closely related species C. radicalis. A phylogenetic tree based on the internal transcribed spacer sequences showed monophyly for the Japanese and European isolates of C. carpinicola. Further studies on the distribution and host range of C. carpinicola in Japan and on the life history strategies of this fungus are needed.

A Multiplex PCR Approach to Determine Vegetative Incompatibility Genotypes and Mating Type in Cryphonectria parasitica.

This chapter presents a genotyping assay that uses DNA isolated from axenic cultures of Cryphonectria parasitica, which discriminates the six known diallelic vic loci and the two mating idiomorphs (MAT gene) based on (i) modified primer, labeled with a fluorescent dye, (ii) multiplex polymerase chain reaction (multiplex-PCR), and (iii) capillary electrophoresis system. Alternatively, we show that the same primer set is suitable for conventional PCR of each vic locus and MAT gene using nonmodified primer and agarose gel electrophoresis.

A new tetra-segmented splipalmivirus with divided RdRP domains from Cryphonectria naterciae, a fungus found on chestnut and cork oak trees in Europe.

Positive-sense (+), single-stranded (ss) RNA viruses with divided RNA-dependent RNA polymerase (RdRP) domains have been reported from diverse filamentous ascomycetes since 2020. These viruses are termed splipalmiviruses or polynarnaviruses and have been characterized largely at the sequence level, but ill-defined biologically. Cryphonectria naterciae, from which only one virus has been reported, is an ascomycetous fungus potentially plant-pathogenic to chestnut and oak trees. We molecularly characterized multiple viruses in a single Portuguese isolate (C0614) of C. naterciae, taking a metatranscriptomic and conventional double-stranded RNA approach. Among them are a novel splipalmivirus (Cryphonectria naterciae splipalmivirus 1, CnSpV1) and a novel fusagravirus (Cryphonectria naterciae fusagravirus 1, CnFGV1). This study focused on the former virus. CnSpV1 has a tetra-segmented, (+)ssRNA genome (RNA1 to RNA4). As observed for other splipalmiviruses reported in 2020 and 2021, the RdRP domain is separately encoded by RNA1 (motifs F, A and B) and RNA2 (motifs C and D). A hypothetical protein encoded by the 5'-proximal open reading frame of RNA3 shows similarity to a counterpart conserved in some splipalmiviruses. The other RNA3-encoded protein and RNA4-encoded protein show no similarity with known proteins in a blastp search. The tetra-segment nature was confirmed by the conserved terminal sequences of the four CnSpV1 segments (RNA1 to RNA4) and their 100% coexistence in over 100 single conidial isolates tested. The experimental introduction of CnSpV1 along with CnFGV1 into a virus free strain C0754 of C. naterciae vegetatively incompatible with C0614 resulted in no phenotypic alteration, suggesting asymptomatic infection. The protoplast fusion assay indicates a considerably narrow host range of CnSpV1, restricted to the species C. naterciae and C. carpinicola. This study contributes to better understanding of the molecular and biological properties of this unique group of viruses.

Infección por SARS-CoV-2 COVID-19: seguimiento de casos sospechosos y contactos en la población pediátrica de un hospital de tercer nivel / SARS-CoV-2 COVID 19 infection: follow-up of suspected cases and contacts in the pediatric population of a tertiary care hospital / Infecção por SARS-CoV-2 COVID 19: monitoramento de casos suspeitos e contatos na população pediátrica de um hospital terciário

La Organización Mundial de la Salud (OMS) declaró la pandemia por SARS-CoV-2 COVID-19 el 11 de marzo de 2020. En Uruguay el 13 de marzo de 2020 se notificaron los primeros casos declarándose el estado de emergencia sanitaria. Desde el inicio de la epidemia hasta el 27 de abril de 2021 se reportaron 22.718 casos en menores de 15 años. Dada esta situación epidemiológica desde el Departamento de Pediatría se implementó el diagnóstico y seguimiento clínico telefónico de los niños y adolescentes (NNA) que consultaron en la Emergencia Pediátrica y constituyeran casos y/o contactos de enfermedad por coronavirus 2019 (COVID-19). Se analizan las características epidemiológicas y clínicas de NNA en seguimiento por sospecha de infección por SARS-CoV-2 en un hospital de tercer nivel entre el 1 de marzo de 2020 y el 28 de febrero de 2021. Se realizó un estudio observacional descriptivo retrospectivo en ese período. Se incluyeron pacientes de 0 a 14 años que consultaron en el Departamento de Emergencia Pediátrica con síntomas sugestivos de COVID-19 o por situación de contacto con un caso confirmado. Fuentes de datos: estadísticas de ingresos, historias clínicas y laboratorio. Variables: fecha de consulta, sexo, edad, procedencia, clínica, contacto COVID-19 confirmado, comorbilidad, resultado de test PCR para SARS-CoV-2, seguimiento y evolución. Análisis estadístico distribución de frecuencias, medidas de resumen y pruebas de significancia, considerando estadísticamente significativo un valor de p ≤0.05. Consideraciones éticas: no se realizó una intervención específica, se garantizó la privacidad y el anonimato de las personas involucradas. Se realizaron 657 consultas. 455 (69,3%) telefónicas, y 202 (30,7%) presenciales. 287 (43,7%) asintomáticos y 370 (56,3%) sintomáticos: rinorrea (27%), fiebre (23%), tos(22%), odinofagia (14%), gastrointestinales (4,8%), dificultad respiratoria (3,6%), entre otros. 394 pacientes (60%) fueron contactos, 254 (64,5%) intrafamiliar, 89 (22,6%) educativo entre otros. 467 (71,1%) tuvieron al menos un seguimiento, 451 (96,6%) telefónico y 16 (3,4%) presencial. 78 (11,9%) fueron positivos, 504 (76,7%) negativos y 75 (11,4%) no realizó test. 646 (98,3%) ambulatorios, uno ingresó a CTI. Ninguno falleció. A través de telemedicina se logró implementar un seguimiento adecuado de casos y contactos COVID-19 e identificar situaciones que requirieran atención presencial. La mayoría cursaron su enfermedad en forma ambulatoria. En los menores de 12 años hubo más infecciones asintomáticas y la fuente de contagio fue un conviviente con mayor frecuencia que en los mayores de 12 años, siendo estas diferencias estadísticamente significativas (p=0,006 y p=0,005). La presentación clínica en esta población fue similar a la reportada. El seguimiento telefónico y la orientación oportunas contribuyeron a la adopción de medidas para disminuir los contagios y la circulación viral y permitieron acompañar al paciente y su familia física y emocionalmente.

Introduction: the World Health Organization (WHO) declared the SARS-CoV2 COVID 19 pandemic on March 11, 2020. In Uruguay, on March 13, 2020, the first cases were reported, and a state of health emergency was set up. From the beginning of the epidemic until April 27, 2021, 22,718 cases were reported in children under 15 years of age. Given this epidemiological situation, the Department of Pediatrics implemented the telephone consultations and clinical follow-up of children and adolescents (CHA) who consulted in the Pediatric Emergency and became cases and/or contacts of coronavirus disease 2019 (COVID 19). Objective: to analyze the epidemiological and clinical characteristics of children under follow-up for suspected SARS-CoV-2 infection in a tertiary care hospital between March 1, 2020 and February 28, 2021. Material and methods: a retrospective descriptive observational study was carried out in the period mentioned above. Patients from 0 to 14 years old who consulted in the Pediatric Emergency Department with symptoms of COVID 19 or because of a contact with a confirmed case were included. Data sources: admission statistics, medical records and laboratory. Variables: consultation date, sex, age, origin, clinic, confirmed COVID 19 contact, comorbidity, PCR test result for SARS-CoV-2, follow-up and evolution. Statistical analysis frequency distribution, summary measures and significance tests, a value of p≤0.05 was regarded as statistically significant. Ethical considerations: no specific intervention was performed, the privacy and anonymity of the people involved were guaranteed. 657 consultations were made. 455 (69.3%) by telephone, and 202 (30.7%) in person. 287 (43.7%) asymptomatic and 370 (56.3%) symptomatic: runny nose (27%), fever (23%), cough (22%), sore throat (14%), gastrointestinal (4.8%), respiratory distress (3.6%) among others. 394 patients (60%) were contacts, 254 (64.5%) intrafamily, 89 (22.6%) at school, among others. 467 (71.1%) had at least 1 follow-up, 451 (96.6%) by telephone and 16 (3.4%) in person. 78 (11.9%) were positive, 504 (76.7%) negative and 75 (11.4%) did not perform the test. 646 (98.3%) outpatients, 1 admitted to ICU. None died. Conclusions: through telemedicine, we could implement an adequate follow-up of COVID 19 cases and contacts and identify situations that required in-person care. In most cases, the disease was resolved in an outpatient manner. Patients under 12 were more asymptomatic and the source of infection was a cohabitant more frequently than in those over 12 years of age, these differences being statistically significant (p=0.006 and p=0.005). The clinical presentation in this population was similar to reported data. The timely telephone follow-up and orientation contributed to the adoption of measures to reduce infections and viral circulation and enabled patients and families to feel physically and emotionally accompanied.

Introdução: a Organização Mundial da Saúde (OMS) declarou a pandemia de SARS-CoV2 COVID 19 eno dia 11 de março de 2020. No Uruguai, em 13 de março de 2020, foram notificados os primeiros casos e foi declarado o estado de emergência sanitária. Desde o início da epidemia até 27 de abril de 2021, foram notificados 22.718 casos em menores de 15 anos. Perante esta situação epidemiológica, o Departamento de Pediatria implementou o diagnóstico telefónico e o acompanhamento clínico das crianças e adolescentes (CA) que consultaram na Emergência Pediátrica e constituíram casos e/ou contatos de doença por coronavírus 2019 (COVID 19). Objetivo: analisar as características epidemiológicas e clínicas de crianças em acompanhamento por suspeita de infecção por SARS-CoV-2 em um hospital terciário entre 1º de março de 2020 e 28 de fevereiro de 2021. Materiais e métodos: foi realizado um estudo observacional descritivo retrospectivo no período mencionado acima. Foram incluídos pacientes de 0 a 14 anos que consultaram no pronto-socorro pediátrico com sintomas sugestivos de COVID 19 ou devido ao contato com um caso confirmado. Fontes de dados: estatísticas de admissão, prontuários e laboratório. Variáveis: data da consulta, sexo, idade, procedência, clínica, contato confirmado de COVID 19, comorbidade, resultado do teste PCR para SARS-CoV-2, acompanhamento e evolução. Distribuição de frequência da análise estatística, medidas sumárias e testes de significância, considerando-se um valor de p≤0,05 como estatisticamente significativo. Considerações éticas: não foi realizada nenhuma intervenção específica, foi garantida a privacidade e o anonimato das pessoas envolvidas. Foram feitas 657 consultas. 455 (69,3%) por telefone e 202 (30,7%) pessoalmente. 287 (43.7%) assintomáticos y 370 (56.3%) sintomáticos: rinorreia (27%), febre(23%), tosse(22%), odinofagia (14%), gastrointestinal (4.8%), dificuldade respiratória (3.6%) entre outros. 394 pacientes (60%) foram contatos, 254 (64,5%) intrafamiliares, 89 (22,6%) na escola, entre outros. 467 (71,1%) tiveram pelo menos 1 acompanhamento, 451 (96,6%) por telefone e 16 (3,4%) pessoalmente. 78 (11,9%) foram positivos, 504 (76,7%) negativos e 75 (11,4%) não realizaram o teste. 646 (98,3%) pacientes ambulatoriais, 1 internado na UTI. Nenhum deles morreu. Conclusões: por meio da telemedicina, foi possível implementar um acompanhamento adequado dos casos e contatos da COVID 19 e identificar situações que exigiram atendimento presencial. A maioria atendeu sua doença de forma ambulatorial. Nos menores de 12 anos houve mais infecções assintomáticas e a fonte de infecção foi um coabitante mais do que nos maiores de 12 anos, sendo essas diferenças estatisticamente significativas (p=0,006 p=0,005). Essa população foi semelhante aos dados registrados. O acompanhamento e orientação oportuna por telefone contribuíram para a adoção de medidas de redução de infecções e circulação viral e permitiram que o paciente e sua família se sentissem acompanhados física e emocionalmente.

A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae Is Able to Cross the Species Barrier and Is Deleterious to a New Host.

Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.

Cryphonectria carpinicola sp. nov. Associated with hornbeam decline in Europe.

Since the early 2000s, reports on declining hornbeam trees (Carpinus betulus) are spreading in Europe. Two fungi are involved in the decline phenomenon: One is Anthostoma decipiens, but the other etiological agent has not been identified yet. We examined the morphology, phylogenetic position, and pathogenicity of yellow fungal isolates obtained from hornbeam trees from Austria, Georgia and Switzerland, and compared data with disease reports from northern Italy documented since the early 2000s. Results demonstrate distinctive morphology and monophyletic status of Cryphonectria carpinicola sp. nov. as etiological agent of the European hornbeam decline. Interestingly, the genus Cryphonectria splits into two major clades. One includes Cry. carpinicola together with Cry. radicalis, Cry. decipiens and Cry. naterciae from Europe, while the other comprises species known from Asia-suggesting that the genus Cryphonectria has developed at two evolutionary centres, one in Europe and Asia Minor, the other in East Asia. Pathogenicity studies confirm that Car. betulus is a major host species of Cry. carpinicola. This clearly distinguished Cry. carpinicola from other Cryphonectria species, which mainly occur on Castanea and Quercus.

Long-read sequencing reveals the evolutionary drivers of intra-host diversity across natural RNA mycovirus infections.

Intra-host dynamics are a core component of virus evolution but most intra-host data come from a narrow range of hosts or experimental infections. Gaining broader information on the intra-host diversity and dynamics of naturally occurring virus infections is essential to our understanding of evolution across the virosphere. Here we used PacBio long-read HiFi sequencing to characterize the intra-host populations of natural infections of the RNA mycovirus Cryphonectria hypovirus 1 (CHV1). CHV1 is a biocontrol agent for the chestnut blight fungus (Cryphonectria parasitica), which co-invaded Europe alongside the fungus. We characterized the mutational and haplotypic intra-host virus diversity of thirty-eight natural CHV1 infections spread across four locations in Croatia and Switzerland. Intra-host CHV1 diversity values were shaped by purifying selection and accumulation of mutations over time as well as epistatic interactions within the host genome at defense loci. Geographical landscape features impacted CHV1 inter-host relationships through restricting dispersal and causing founder effects. Interestingly, a small number of intra-host viral haplotypes showed high sequence similarity across large geographical distances unlikely to be linked by dispersal.

In-Tree Behavior of Diverse Viruses Harbored in the Chestnut Blight Fungus, Cryphonectria parasitica.

The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem.IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

Determining the Suitability of MinION's Direct RNA and DNA Amplicon Sequencing for Viral Subtype Identification.

The MinION sequencer is increasingly being used for the detection and outbreak surveillance of pathogens due to its rapid throughput. For RNA viruses, MinION's new direct RNA sequencing is the next significant development. Direct RNA sequencing studies are currently limited and comparisons of its diagnostic performance relative to different DNA sequencing approaches are lacking as a result. We sought to address this gap and sequenced six subtypes from the mycovirus CHV-1 using MinION's direct RNA sequencing and DNA sequencing based on a targeted viral amplicon. Reads from both techniques could correctly identify viral presence and species using BLAST, though direct RNA reads were more frequently misassigned to closely related CHV species. De novo consensus sequences were error prone but suitable for viral species identification. However, subtype identification was less accurate from both reads and consensus sequences. This is due to the high sequencing error rate and the limited sequence divergence between some CHV-1 subtypes. Importantly, neither RNA nor amplicon sequencing reads could be used to obtain reliable intra-host variants. Overall, both sequencing techniques were suitable for virus detection, though limitations are present due to the error rate of MinION reads.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Genetic and Phenotypic Characterization of Cryphonectria hypovirus 1 from Eurasian Georgia.

Cryphonectria hypovirus 1 (CHV-1) infects the chestnut blight fungus Cryphonectria parasitica and acts as a biological control agent against this harmful tree disease. In this study, we screened the recently characterized C. parasitica population in Eurasian Georgia for the presence of CHV-1. We found 62 CHV-1 infected C. parasitica isolates (9.3%) among a total of 664 isolates sampled in 14 locations across Georgia. The prevalence of CHV-1 at the different locations ranged from 0% in the eastern part of the country to 29% in the western part. Sequencing of two specific regions of the viral genome one each in ORFA and ORFB revealed a unique CHV-1 subtype in Georgia. This subtype has a recombinant pattern combining the ORFA region from the subtype F2 and the ORFB region from subtype D. All 62 viral strains belonged to this Georgian CHV-1 subtype (subtype G). The CHV-1 subtype G strongly reduced the parasitic growth of C. parasitica isolates from Georgia, with a more severe effect on the European genepool compared to the Georgian genepool. The CHV-1 subtype detected in Georgia provides a valuable candidate for biological control applications in the Caucasus region.

Are species-pairs diverging lineages? A nine-locus analysis uncovers speciation among species-pairs of the Lobaria meridionalis-group (Ascomycota).

In spite of considerable effort to verify the theory of species-pairs, uncertainty still exists about the relationship between sexually or vegetatively reproducing populations of morphologically indistinguishable, sympatric lichen species. The current paper studies putative species-pairs within the Asian Lobaria meridionalis-group, using a nine-locus and time calibrated species-tree approach. Analyses demonstrate that pairs of sexually or vegetatively reproducing lineages split into highly supported monophyletic clades-confirming molecularly the species-pair concept for the L. meridionalis-group. In the broader context of evolution and speciation dynamics in lichenized fungi, this paper attempts to synthesize molecular findings from the last two decades to promote a more modern perception of the species-pair concept. Taxonomically, eight species were found to currently conform to the L. meridionalis-group, which differentiated during the Pliocene and Pleistocene. The coincidence of paleoclimatic events with estimated dates of divergence support a bioclimatic hypothesis for the evolution of species in the L. meridionalis-group, which also explains their current eco-geographic distribution patterns. Greater recognition for species with a long and independent evolutionary history, which merit high conservation priority, will be especially critical for preserving geographically restricted endemics from Southeast Asia, where habitat loss is driving rapid declines.

Folie à deux / Case report: Folie à deux

JMCD, Sexo masculino, 19 años, soltero, sin hijos. Vive en Casas Compartidas de la Fundación Don Bosco. Examen Mental: Refiere que conversa con Dios en su corazón, y ahí siente el "fuego", pero no lo ve ni lo escucha. Dice tener la misión de predicar (sin mayor elaboración), y que no le gusta ninguna Iglesia. Discurso sin alteración del curso formal. Afecto de "templanza" forzada y superioridad. Al ingreso se agita y presenta postura antieconómica (también en el hogar: crucificado). Pone los ojos en blanco y parpadea rápidamente. Sin consciencia de enfermedad. Juicio de realidad alterado. Diagnósticos: Síndrome delirante lúcido. Folie à deux. Discusión: El trastorno de ideas delirantes inducidas, también conocido como Trastorno psicótico compartido o folie à deux es una condición poco común, caracterizada por síntomas psicóticos en 2 o más individuos que mantienen una relación cercana.

JMCD, male, 19 years old, single, childless. Live in shared houses of the Don Bosco Foundation. Mental examination: Refers conversing with God in his heart, and there he feels the "fire", but neither sees nor hears God. He claims to have the mission of preaching (without further processing), and he does not like any church. Speech without altering the formal course. Forced "temperance" affection and superiority. Agitation at Income and presents uneconomical posture (also at home: crucified). He rolls his eyes and blinks rapidly. Without awareness of disease. Reality trial actually altered. Diagnosis: Lucid delusional syndrome. Folie à deux. Discussion: The disorder of induced delusional thoughts, also known as shared psychotic disorder or folie à deux is a rare condition characterized by psychotic symptoms in 2 or more individuals who maintain a close relationship.

Phylogenetic and phenotypic characterisation of Sirococcus castaneae comb. nov. (synonym Diplodina castaneae), a fungal endophyte of European chestnut.

In this paper we resolve the taxonomic status of the fungus Diplodina castaneae (Ascomycetes, Diaporthales, Gnomoniaceae) which occurs on the European chestnut (Castanea sativa) as endophyte and as the causal agent of Javart disease. Specimens from Switzerland, Spain, and Azerbaijan were sequenced at five nuclear loci (ß-tubulin, EF-1α, ITS, LSU, and RPB2). Phylogenies were inferred to place D. castaneae in the Gnomoniaceae family. Moreover, growth rates and morphological characteristics on different agar media were assessed and compared to those of Gnomoniopsis castaneae, which can easily be confused with D. castaneae. Based on morphological and phylogenetic characteristics, we propose to reallocate D. castaneae to the genus Sirococcus, as S. castaneae comb. nov.

Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation.

Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).

Cyanobacterial gardens: the liverwort Frullania asagrayana acts as a reservoir of lichen photobionts.

Cyanobacteria are important mediators of unrelated lichen species, which form epiphytic communities that share the same cyanobiont. No study to date, however, has considered the role of cyanobacteria as mediator between lichens and bryophytes. In the present study, DNA barcoding (16S rDNA, rbcLX) was used to identify filamentous cyanobacteria living in close association with members of an epiphytic liverwort-lichen community on balsam fir in Newfoundland. This study is the first to confirm the presence of Rhizonema strains in boreal forests where they are associated with the liverwort Frullania asagrayana and several lichen species. The majority of cyanobacterial haplotypes can associate with the liverwort, however, some lichen species appear to be more selective for single or closely related haplotypes. Some Rhizonema strains were found exclusively in association with boreal lichens, while others seem to be globally distributed and involved in different lichen symbioses of unrelated fungal lineages and of varying ecological traits. Complex biological interactions in a cyanobacteria-mediated guild are proposed here, which explains composition and dynamics in bryophyte and lichen-dominated epiphytic communities.