Fungistatic Activity Mediated by Volatile Organic Compounds Is Isolate-Dependent in Trichoderma sp. "atroviride B".

Trichoderma spp. produce multiple bioactive volatile organic compounds (VOCs). While the bioactivity of VOCs from different Trichoderma species is well documented, information on intraspecific variation is limited. The fungistatic activity of VOCs emitted by 59 Trichoderma sp. "atroviride B" isolates against the pathogen Rhizoctonia solani was investigated. Eight isolates representing the two extremes of bioactivity against R. solani were also assessed against Alternaria radicina, Fusarium oxysporum f. sp. lycopersici and Sclerotinia sclerotiorum. VOCs profiles of these eight isolates were analyzed using gas chromatography-mass spectrometry (GC-MS) to identify a correlation between specific VOCs and bioactivity, and 11 VOCs were evaluated for bioactivity against the pathogens. Bioactivity against R. solani varied among the fifty-nine isolates, with five being strongly antagonistic. All eight selected isolates inhibited the growth of all four pathogens, with bioactivity being lowest against F. oxysporum f. sp. lycopersici. In total, 32 VOCs were detected, with individual isolates producing between 19 and 28 VOCs. There was a significant direct correlation between VOC number/quantity and bioactivity against R. solani. 6-pentyl-α-pyrone was the most abundant VOC produced, but 15 other VOCs were also correlated with bioactivity. All 11 VOCs tested inhibited R. solani growth, some by >50%. Some of the VOCs also inhibited the growth of the other pathogens by >50%. This study demonstrates significant intraspecific differences in VOC profiles and fungistatic activity supporting the existence of biological diversity within Trichoderma isolates from the same species, a factor in many cases ignored during the development of biological control agents.

Update of the CHIP (CT in Head Injury Patients) decision rule for patients with minor head injury based on a multicenter consecutive case series.

OBJECTIVE: To update the existing CHIP (CT in Head Injury Patients) decision rule for detection of (intra)cranial findings in adult patients following minor head injury (MHI). METHODS: The study is a prospective multicenter cohort study in the Netherlands. Consecutive MHI patients of 16 years and older were included. Primary outcome was any (intra)cranial traumatic finding on computed tomography (CT). Secondary outcomes were any potential neurosurgical lesion and neurosurgical intervention. The CHIP model was validated and subsequently updated and revised. Diagnostic performance was assessed by calculating the c-statistic. RESULTS: Among 4557 included patients 3742 received a CT (82%). In 383 patients (8.4%) a traumatic finding was present on CT. A potential neurosurgical lesion was found in 73 patients (1.6%) with 26 (0.6%) patients that actually had neurosurgery or died as a result of traumatic brain injury. The original CHIP underestimated the risk of traumatic (intra)cranial findings in low-predicted-risk groups, while in high-predicted-risk groups the risk was overestimated. The c-statistic of the original CHIP model was 0.72 (95% CI 0.69-0.74) and it would have missed two potential neurosurgical lesions and one patient that underwent neurosurgery. The updated model performed similar to the original model regarding traumatic (intra)cranial findings (c-statistic 0.77 95% CI 0.74-0.79, after crossvalidation c-statistic 0.73). The updated CHIP had the same CT rate as the original CHIP (75%) and a similar sensitivity (92 versus 93%) and specificity (both 27%) for any traumatic (intra)cranial finding. However, the updated CHIP would not have missed any (potential) neurosurgical lesions and had a higher sensitivity for (potential) neurosurgical lesions or death as a result of traumatic brain injury (100% versus 96%). CONCLUSIONS: Use of the updated CHIP decision rule is a good alternative to current decision rules for patients with MHI. In contrast to the original CHIP the update identified all patients with (potential) neurosurgical lesions without increasing CT rate.

Neurocognitive and behavioural profile in Panayiotopoulos syndrome.

AIM: To determine neurocognitive performance and behavioural problems in children with Panayiotopoulos syndrome. METHOD: All 18 children (10 females, 8 males; mean age 4y 7mo; SD 1y 10mo) diagnosed with Panayiotopoulos syndrome at the Kempenhaeghe Epilepsy Center in the Netherlands between 2010 and 2017 were analysed retrospectively. All underwent a neuropsychological/behavioural assessment, an academic assessment, and a 24-hour electroencephalogram. RESULTS: Mean full-scale IQ (93.5; range 76-123; p=0.04) and performance IQ (93.2; range 76-126; p=0.04) were within the normal range, although significantly lower compared to the normative mean. Verbal IQ (96.3; range 76-118) and processing speed (96.1; range 74-114) were not significantly lower. Simple auditory/visual reaction times, visual attention, visual-motor integration, and verbal memory were significantly lower compared to normative values. On average, patients with Panayiotopoulos syndrome were 8 months behind in arithmetic speed and 11 months behind in reading speed for the number of months in school. Behavioural questionnaires revealed significantly higher scores on reported internalizing behavioural problems. INTERPRETATION: Children with Panayiotopoulos syndrome demonstrated diffuse cognitive dysfunction in full-scale IQ, performance IQ, visual attention, visual-motor integration, and verbal memory. A high incidence of internalizing behavioural problems was reported. This strongly suggests neuropsychological and behavioural comorbidity in children with Panayiotopoulos syndrome. WHAT THIS PAPER ADDS: Children with Panayiotopoulos syndrome are at risk for cognitive deficits in various cognitive domains. Children with Panayiotopoulos syndrome are also prone to internalizing behavioural problems. Mild-to-severe academic underachievement was present in more than half of the children with Panayiotopoulos syndrome.

External validation of computed tomography decision rules for minor head injury: prospective, multicentre cohort study in the Netherlands.

OBJECTIVE: To externally validate four commonly used rules in computed tomography (CT) for minor head injury. DESIGN: Prospective, multicentre cohort study. SETTING: Three university and six non-university hospitals in the Netherlands. PARTICIPANTS: Consecutive adult patients aged 16 years and over who presented with minor head injury at the emergency department with a Glasgow coma scale score of 13-15 between March 2015 and December 2016. MAIN OUTCOME MEASURES: The primary outcome was any intracranial traumatic finding on CT; the secondary outcome was a potential neurosurgical lesion on CT, which was defined as an intracranial traumatic finding on CT that could lead to a neurosurgical intervention or death. The sensitivity, specificity, and clinical usefulness (defined as net proportional benefit, a weighted sum of true positive classifications) of the four CT decision rules. The rules included the CT in head injury patients (CHIP) rule, New Orleans criteria (NOC), Canadian CT head rule (CCHR), and National Institute for Health and Care Excellence (NICE) guideline for head injury. RESULTS: For the primary analysis, only six centres that included patients with and without CT were selected. Of 4557 eligible patients who presented with minor head injury, 3742 (82%) received a CT scan; 384 (8%) had a intracranial traumatic finding on CT, and 74 (2%) had a potential neurosurgical lesion. The sensitivity for any intracranial traumatic finding on CT ranged from 73% (NICE) to 99% (NOC); specificity ranged from 4% (NOC) to 61% (NICE). Sensitivity for a potential neurosurgical lesion ranged between 85% (NICE) and 100% (NOC); specificity from 4% (NOC) to 59% (NICE). Clinical usefulness depended on thresholds for performing CT scanning: the NOC rule was preferable at a low threshold, the NICE rule was preferable at a higher threshold, whereas the CHIP rule was preferable for an intermediate threshold. CONCLUSIONS: Application of the CHIP, NOC, CCHR, or NICE decision rules can lead to a wide variation in CT scanning among patients with minor head injury, resulting in many unnecessary CT scans and some missed intracranial traumatic findings. Until an existing decision rule has been updated, any of the four rules can be used for patients presenting minor head injuries at the emergency department. Use of the CHIP rule is recommended because it leads to a substantial reduction in CT scans while missing few potential neurosurgical lesions.

The NADPH Oxidases Nox1 and Nox2 Differentially Regulate Volatile Organic Compounds, Fungistatic Activity, Plant Growth Promotion and Nutrient Assimilation in Trichoderma atroviride.

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity.

Draft Genome Sequences of Two New Zealand Xanthomonas campestris pv. campestris Isolates, ICMP 4013 and ICMP 21080.

Xanthomonas campestris pv. campestris is a necrotrophic bacterial pathogen of crucifers. We report here the draft genome sequences of isolates ICMP 4013 and ICMP 21080 from New Zealand. These sequences will facilitate the identification of race-specific factors in X. campestris pv. campestris.

Sequence Analysis of SSR-Flanking Regions Identifies Genome Affinities between Pasture Grass Fungal Endophyte Taxa.

Fungal species of the Neotyphodium and Epichloë genera are endophytes of pasture grasses showing complex differences of life-cycle and genetic architecture. Simple sequence repeat (SSR) markers have been developed from endophyte-derived expressed sequence tag (EST) collections. Although SSR array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa, the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects. In contrast, nonrepetitive sequence regions that flank SSRs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes. Consistent patterns of relationships between specific taxa were apparent across multiple target loci, confirming previous studies of genome evolution based on variation of individual genes. Evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content. Results were compatible with the current view that some asexual Neotyphodium species arose following interspecific hybridisation between sexual Epichloë ancestors. Phylogenetic analysis of SSR-flanking regions, in combination with the results of previous studies with other EST-derived SSR markers, further permitted characterisation of Neotyphodium isolates that could not be assigned to known taxa on the basis of morphological characteristics.

Assessment of genetic diversity in cultivars of white clover (Trifolium repens L.) detected by SSR polymorphisms.

White clover (Trifolium repens L.) is an important temperate pasture legume that plays a key role as a companion to grass species, such as perennial ryegrass (Lolium perenne L.). Due to the outbreeding nature of white clover, cultivars are highly heterogeneous. Genetic diversity was assessed using 16 elite cultivars from Europe, North and South America, Australia, and New Zealand. Fifteen simple sequence repeat markers that detect single, codominant polymorphic genetic loci were selected for the study. The genetic relationships among individuals were compared using phenetic clustering, and those among cultivars were compared using nonmetric multidimensional scaling. Intrapopula tion variability exceeded interpopulation variability, with substantial overlap among populations and weak interpopula tion differentiation. No obvious or significant differentiation was observed on the basis of morphology or geographic origin of the cultivars. The number of parental genotypes used to derive each cultivar was not a major determinant of genome-wide genetic diversity. The outcomes of this assessment of genetic variation in elite white clover germplasm pools have important implications for the feasibility of molecular marker-based cultivar discrimination, and will be used to assist the design of linkage disequilibrium mapping strategies for marker-trait association.

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichloë isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichloë species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.