Molecular and phenotypic characterization of endophytic Sebacinoid strains

The order Sebacinales belongs to a taxonomically, ecologically, and physiologically diverse group of fungi within the Phylum Basidiomycota. Using several molecular techniques they were detected all over the world. Few isolates, classified into the clade B, are available at the moment: the root–colonizing mutualistic fungus Piriformospora indica, various Sebacina vermifera isolates from autotrophic orchids, as well as Piriformospora glomeralium (ex multinucleate rhizoctonia DAR29830, Warcup). All of them were described as growth promoting and resistance–inducing fungi. In the present work, seven Sebacinales isolates of the clade B were characterized molecularly and phenotypically. In addition, the presence in Germany of fungi closely related to P. indica was proven. Phylogenetic analyses conducted using DNA sequences from the 28S and the translation elongation factor 1–α gene (TEF) showed that analyzed Sebacinales isolates represent at least 3 distinct groups of isolates. Further, three independent environmental samples, collected from two different areas in Germany were examined. The analysis demonstrated that the sample respresented organisms closely related to P. indica. The analyzed introns of their TEF gene had the same sequence. This finding suggests that one genotype is present in different area of Germany which is associated with roots of taxonomically diverse plants, including Anthyllis, Medicago and Lolium. Moreover, the fungal genome sizes of five sebacinoid isolates were estimated using Pulsed Field Gel Electrophoresis (PFGE) and real–time PCR based on the absolute quantification of a single copy gene (TEF). The fungi have at least 4 to 7 chromosomes and a genome size ranging from 21 to 26 Mb. Morphological, physiological and molecular studies of the multinucleate rhizoctonia DAR29830 isolate proved that this strain is the closest related to P. indica, the most investigated member of the Sebacinales. Therefore it is designed in this study as a provisional new species named Piriformospora glomeralium sp. nov. Seven Sebacinales strains were tested for extracellular enzyme production including peroxidase, laccase, protease, pectinase, and cellulase. In general, the enzymatic test demonstrated strong protease activity in cultures of all the analyzed fungi. The orchid mycorrhiza isolates showed stark laccase activity and lack of cellulase production in axenic culture whereas the Piriformospora strains were able to digest this plant cell wall component. Only one isolate proved to secrete pectinase. All Sebacinales isolates except P. indica and P. glomeralium that were co–cultured with a fungal competitor (Rhizoctonia solani) generated significant amounts of laccase. The presence of a plant host, life or dead, had noticeable impact on laccase secretion by P. indica but no influence on P. glomeralium enzyme exudation. In addition, production of laccase, peroxidase, esterase, and lipase by P. indica in response to plant host was spectrophotometrically investigated. Diversity in laccase and lipase activity was observed mostly in presence of decay plant material. Differences in the enzyme profile for the analyzed Sebacinales strains agreed mostly with their phylogenetic position. In addition, genome estimation as well as karyotype analysis clearly confirmed the phylogenetic study and proved that Sebacina vermifera should be considered as complex of isolates. Moreover, laccase secretion by P. indica can be induced only by the presence of the plant symbiotic partner.Die Ordnung der Sebacinales gehört zu einer taxonomisch, ökologisch und physiologisch diversen Gruppe von Pilzen im Phylum der Basidiomyceten. Weltweit können sie durch verschiedene molekulare Techniken detektiert werden. Wenige Isolate, klassifiziert im Untergruppe B, sind zurzeit verfügbar: der Wurzel–kolonisierende, mutualistische Pilz Piriformospora indica, verschiedene Sebacina vermifera Isolate von autotrophen Orchideen, genauso wie Piriformospora glomeralium (ex multinucleate rhizoctonia DAR29830, Warcup). Für alle wurde beschrieben, dass sie das Wachstum fördern und Resistenz vermitteln gegen biotischen und abiotischen Stress. In der vorliegenden Arbeit wurden sieben Sebacinales–Isolate der Untergruppe B molekularbiologisch und phänotypisch charakterisiert. Zusätzlich wurde das Vorkommen eines zu P. indica nahe verwandten Genotyps in Deutschland bewiesen. Phylogenetische Analysen, unter Verwendung von DNA–Sequenzen aus 28S und dem Translationselongationsfaktor 1–α Gen (TEF), zeigten, dass diese Organismen mindestens drei verschiedene Isolat–Gruppen repräsentieren. Zudem wurden drei unabhängige Proben aus zwei verschiedenen Orten Deutschlands untersucht. Die Analyse zeigte, dass sie eng verwandt sind mit P. indica. Die analysierten Introns des TEF–Gens haben die gleiche Sequenz. Dies impliziert, dass ein Genotyp, welcher in verschiedenen Gegenden Deutschland vorkommt, mit den Wurzeln taxonomisch diverser Pflanzen, inklusive Anthyllis, Medicago und Lolium, assoziiert ist. Darüber hinaus wurde die Genomgröße von fünf Sebacinoid–Isolaten durch Pulsed Field Gel Electrophorese (PFGE) und quantitative Real–time PCR bestimmt. Die Pilze besitzen zwischen vier und sieben Chromosomen und die Genomgröße variiert von 21 bis 26 Mb. Morphologische, physiologische und molekulare Untersuchungen des multinuklearen Rhizoctonia Isolates zeigten, dass dieser Stamm am engsten mit P. indica verwandt ist und daher in dieser Abhandlung als Piriformospora glomeralium sp. nov. benannt wird. Sieben Sebacinales–Stämme wurden auf extrazelluläre Ezymproduktion von Peroxidase, Laccase, Protease, Pectinase und Zellulase getestet. Generell zeigten diese Tests eine starke Peroxidase–Aktivität der Kulturen bei allen analysierten Pilzen. Die Orchideen–Mycorrhiza–Isolate zeigten eine starke Laccase–Aktivität und ein Fehlen von Zellulase–Produktion in axenischen Kulturen während die Piriformospora–Stämme diese Zellwandkomponente zersetzen konnten. Nur ein Isolat sekretierte Pectinase. Alle Sebacinales Isolate außer P. indica und P. glomeralium co–kultivert mit einem pilzlichen Konkurrenten (Rhizoctonia solani) bildeten signifikante Mengen an Laccase. Die Gegenwart des Wirts, tot oder lebendig, hatte einen bemerkenswerten Einfluss auf die Laccase–Produktion von P. indica und keinen Einfluss auf die Enzymexudation von P. glomeralium. Zusätzlich wurde die Produktion von Laccase, Peroxidase, Esterase und Lipase bei P. indica in Bezug auf den Wirt photospektrometrisch untersucht. Unterschiede in Laccase– und Lipase–Aktivität wurden vor allem in Gegenwart von totem Pflanzenmaterial beobachtet. Unterschiede in den Enzymprofilen der analysierten Sebacinales–Stämme korrelieren mit ihrer phylogenetischen Position. Außerdem bestätigen die Genomgrößenbestimmung und die Karyotypen–Analyse, dass Sebacina vermifera als ein Komplex von Isolaten angesehen werden sollte. Zudem kann die Laccase–Sekretion bei P. indica nur durch Anwesenheit eines symbiotischen Partners induziert werden

Endophytic Life Strategies Decoded by Genome and Transcriptome Analyses of the Mutualistic Root Symbiont Piriformospora indica

Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead barley roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyle strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarray analysis, argues for a biphasic root colonization strategy of P. indica. This is supported by a cytological study that shows an early biotrophic growth followed by a cell death-associated phase. About 10% of the fungal genes induced during the biotrophic colonization encoded putative small secreted proteins (SSP), including several lectin-like proteins and members of a P. indica-specific gene family (DELD) with a conserved novel seven-amino acids motif at the C-terminus. Similar to effectors found in other filamentous organisms, the occurrence of the DELDs correlated with the presence of transposable elements in gene-poor repeat-rich regions of the genome. This is the first in depth genomic study describing a mutualistic symbiont with a biphasic lifestyle. Our findings provide a significant advance in understanding development of biotrophic plant symbionts and suggest a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi

Conidial Morphogenesis and Septin-Mediated Plant Infection Require Smo1, a Ras GTPase-Activating Protein in Magnaporthe oryzae

The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria, and asci. All attempts to clone the SMO1 gene by map-based cloning or complementation have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of SMO1 results in abnormal, nonadherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. SMO1 is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskeleton at the appressorium pore. Smol physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31â127 anaesthetic procedures in 30â874 children with a mean age of 6·35 years (SD 4·50) were included. The incidence of perioperative severe critical events was 5·2% (95% CI 5·0â5·5) with an incidence of respiratory critical events of 3·1% (2·9â3·3). Cardiovascular instability occurred in 1·9% (1·7â2·1), with an immediate poor outcome in 5·4% (3·7â7·5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10â000. This was independent of type of anaesthesia. Age (relative risk 0·88, 95% CI 0·86â0·90; p<0·0001), medical history, and physical condition (1·60, 1·40â1·82; p<0·0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0·99, 0·981â0·997; p<0·0048 for respiratory critical events, and 0·98, 0·97â0·99; p=0·0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia. Funding European Society of Anaesthesiology

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6.35 years (SD 4.50) were included. The incidence of perioperative severe critical events was 5.2% (95% CI 5.0-5.5) with an incidence of respiratory critical events of 3.1% (2.9-3.3). Cardiovascular instability occurred in 1.9% (1.7-2.1), with an immediate poor outcome in 5.4% (3.7-7.5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0.88, 95% CI 0.86-0.90; p<0.0001), medical history, and physical condition (1.60, 1.40-1.82; p<0.0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0.99, 0.981-0.997; p<0.0048 for respiratory critical events, and 0.98, 0.97-0.99; p=0.0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia