Smut fungus (Langdonia walkerae) incidence is lower in two bunchgrass species (Aristida stricta and A. beyrichiana) after fires early in the year.

PREMISE: In frequently burned southeastern USA pine-grassland communities, wiregrass (Aristida stricta and A. beyrichiana) are dominant bunchgrasses whose flowers are infected during flowering by a smut fungus (Langdonia walkerae). We hypothesized that because prescribed fire timing affects wiregrass flowering patterns, it could affect smut incidence (occurrence of smut on plants) and severity of infection in inflorescences and spikelets. Because soil order could influence plant susceptibility, we hypothesized that these patterns would differ between soil orders. We hypothesized differences between species as representative of geographic variation in this ecosystem. METHODS: We surveyed the incidence and severity of L. walkerae in wiregrass populations (85 populations at 14 sites) that had been prescription burned at different times during the previous year. We used binomial regressions to test whether incidence and severity differed by burn day, soil order, or species, with site as a random effect. RESULTS: Fires that occurred in the winter were associated with significantly lower incidence than fires later in the year (as the months progressed into summer). Plants growing on Spodosol soils were significantly less likely to be infected than those on other soils. More variation in incidence, however, was explained by site, suggesting that site-specific characteristics were important. Smut severity in inflorescences and spikelets was greater overall in populations of A. stricta than in southern populations (A. beyrichiana). CONCLUSIONS: Our findings indicate that fire timing and soil order affect L. walkerae incidence in wiregrass plants, but neither appears to be associated with greater severity. Patterns of smut infection are related to site history and geographic variation.

Genetic Diversity and Fungicide Sensitivity of Cytospora plurivora on Peach.

Cytospora plurivora D.P. Lawr., L.A. Holland & Trouillas has been associated with recent premature peach tree decline in South Carolina, but very little is known about the pathogen or chemical control options. Ninety-three C. plurivora isolates were collected in 2016 and 2017 from 1-year-old peach wood and symptomatic scaffold limbs, respectively, from orchards in six towns in South Carolina. Six unique genotypes were identified based on substantial ITS1-5.8S-ITS2 sequence variability and classified G1 to G6. Three of the genotypes (G2, G3, and G6) were isolated in high frequency in multiple locations of both years. In addition to the genotypic variation, multiple phenotypes were observed between and within genotype groups. Species identity was determined using additional gene loci: ACT, TUB, and EF, and isolates were found to belong to C. plurivora for all genotype groups. All tested genotypes were sensitive to thiophanate-methyl (FRAC 1) but exhibited slightly lower sensitivity to propiconazole and difenoconazole (both FRAC 3). Boscalid, fluopyram (both FRAC 7s), azoxystrobin, and pyraclostrobin (both FRAC 11s) were ineffective in vitro at inhibiting mycelial growth of C. plurivora genotypes. Field inoculation of peach and nectarine trees revealed that all genotypes developed twig cankers with differences in virulence. G1 was most virulent, and G6 was least virulent. This study provides a link between the C. plurivora genetic variability and virulence and provides fungicide sensitivity information that could be used to improve disease management practices.

Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods.

Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.

Flying After Concussion and Symptom Recovery in College Athletes and Military Cadets.

Importance: Concussions are a common occurrence in young athletes. Hypobaric hypoxemia, such as that experienced during airplane travel, can potentially cause alterations to cerebral blood flow and increased neuroinflammatory response. It remains unknown whether flying early after a concussion may influence the clinical course of injury. Objective: To determine whether there is an association between concussion recovery and airplane travel in collegiate athletes and military cadets. Design, Setting, and Participants: This cohort study was conducted by the National Collegiate Athletic Association and US Department of Defense Concussion Assessment, Research, and Education Consortium from August 3, 2014, to September 13, 2018. Participant groups were categorized by those who flew within 72 hours of injury and those who did not fly. All participants included in the final analyses had complete data of interest and only 1 injury during the study. Data analysis was performed from September 2018 to March 2020. Main Outcomes and Measures: Recovery outcome measures were defined as time (in days) from injury to return to activity, school, and baseline symptoms. Symptom and headache severity scores were derived from the Sports Concussion Assessment Tool-Third Edition. Scores for both groups were taken at baseline and a median of 2 days after injury. Results: A total of 92 participants who flew (mean [SD] age, 19.1 [1.2] years; 55 male [59.8%]) and 1383 participants who did not fly (mean [SD] age, 18.9 [1.3] years; 809 male [58.5%]) were included in the analysis of symptom recovery outcomes (analysis 1). Similarly, 100 participants who flew (mean [SD] age, 19.2 [1.2] years; 63 male [63.0%]) and 1577 participants who did not fly (mean [SD] age, 18.9 [1.3] years; 916 male [58.1%]) were included in the analysis of symptom severity outcomes (analysis 2). No significant group differences were found regarding recovery outcome measures. Likewise, there were no group differences in symptom (estimated mean difference, 0.029; 95% CI, -0.083 to 0.144; P = .67) or headache (estimated mean difference, -0.007; 95% CI, -0.094 to 0.081; P = .91) severity scores. Conclusions and Relevance: Airplane travel early after concussion was not associated with recovery or severity of concussion symptoms. These findings may help guide future recommendations on flight travel after concussion in athletes.

The Rise of the Concussion Clinic for Diagnosis of Pediatric Mild Traumatic Brain Injury.

It was not too long ago that being struck violently in the head or "getting your bell rung" was often brushed off as nothing to worry about. These days the pendulum has swung and there is growing appreciation that this mild traumatic brain injury (mTBI), often called concussion, must be taken seriously and is now on the forefront of public health concerns. As a growing body of research continues to expand our understanding of concussion, the paradigm of diagnosis and treatment is transforming rapidly. The recent rise of specialty concussion clinics across the country has grown out of a need for safe, effective, and efficient evaluation of these injuries by healthcare professionals qualified to implement diagnostic and management strategies that align with the latest evidence-based practice guidelines. Due to the complex nature of each injury, a comprehensive multidisciplinary team can provide a valuable individualized approach to concussion care.

When in doubt, sit it out! Pediatric concussion-an update.

Concussion is a major public health issue that has received much publicity in recent years. Reported concussions are increasing across all ages, including the highest rates in pediatric age ranges. Due to the age-related physiological responses and differential recovery of symptoms after concussion, special consideration must be given to youth concussions. Concussion can affect each child differently and likely affects children differently than adults. Until the past decade, there has been a scarcity of scientifically rigorous studies examining concussions in children. This increasing amount of research and consensus has begun to influence the definition, evaluation, management, return to school and play recommendations, prevention, and education relating to pediatric concussion. The purpose of this article is a review of relevant research published in the last few years (2015-2017) with highlights of these updates summarized for clinical use.

Biology, ecology and ultrastructure of Ascobotryozyma and Botryozyma, unique commensal nematode-associated yeasts.

Ascobotryozyma (anamorph Botryozyma) is an ascomycetous yeast genus characterized by its unusual morphology and ability to attach to free-living nematodes in the genus Panagrellus. Other than species descriptions for four taxa little is known about these yeasts. Investigations were carried out to determine the ecological relationship and specificity with nematodes in addition to examining basic cell structure, thallus development and sexual reproduction. Based on previous examinations of the yeast and nematode it was hypothesized the fungus did not parasitize the nematode but instead remained attached to nematodes for dispersal. Electron micrographs supported our hypotheses and revealed a specialized cell wall and mucilage that aid in attachment to the nematode cuticle. Tests to determine the ability of Ascobotryozyma and Botryozyma to attach to different nematode taxa revealed that attachment varies across taxonomic groups. Examinations of thallus cells, thallus growth, cell fusion and ascosporogenesis are presented. This study provides insight into an otherwise cryptic yeast genus.

Improved methods for axenic culture of Labyrinthula terrestris, causal agent of rapid blight of turfgrasses.

The genus Labyrinthula is a group of unicellular microorganisms with spindle-shaped cells that move in an ectoplasmic network. Most Labyrinthula species are saprotrophic and found in coastal marine or estuarine habitats; however, exceptions exist, such as Labyrinthula terrestris , a terrestrial plant pathogen that causes rapid blight on cool-season turfgrasses. Labyrinthula spp. can be grown in culture, which facilitates studies on their biology and pathology. However, axenic culture of L. terrestris has always been challenging. We modified the most commonly used Labyrinthula growth medium, serum seawater agar (SSA), and designed 2 media for improved pure culture, modified SSA (MSSA) and grass extract SSA (GESSA). A comparative assessment of these 2 media and basic SSA was made to measure the growth responses of 18 L. terrestris isolates. Results indicate that the average colony area was greatest on GESSA followed by MSSA, while cultures lived longest on MSSA followed by GESSA. We also suggest an improved long-term culture technique to maintain viable L. terrestris isolates for at least 2 years.

Botrytis caroliniana, a new species isolated from blackberry in South Carolina.

Blackberry fruits symptomatic for gray mold were collected from three commercial blackberry fields in northwestern South Carolina. Single-spore isolates were generated and two distinct phenotypes were discovered in each location; one sporulated on PDA and one did not. One isolate of each phenotype and location (six isolates total) were selected for in depth molecular and morphological characterization. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) coding sequence alignment revealed Botrytis cinerea as the sporulating phenotype and a new yet undescribed species as the non-sporulating phenotype. The new Botrytis sp., described herein as Botrytis caroliniana, was most closely related genetically to B. fabiopsis and B. galanthina, the causal agents of gray mold disease of broad bean and snowdrop, respectively. It produces smaller conidia than either B. fabiopsis or B. galanthina, and sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) also indicated that the Botrytis isolates represent a separate and distinct species. The new species is pathogenic on blackberry fruits and broad bean leaves, which distinguishes it further from B. galanthina. The new species formed white to pale gray colonies with short, tufted aerial mycelium and produced black sclerotia on PDA at 20 C. To our knowledge this is only the third Botrytis species discovered to cause disease on blackberry in the United States.

Development of molecular markers and preliminary investigation of the population structure and mating system in one lineage of black morel (Morchella elata) in the Pacific Northwestern USA.

Phylogenetic analysis of LSU/ITS sequence data revealed two distinct lineages among 44 morphologically similar fruiting bodies of natural black morels (Morchella elata group) sampled at three non-burn locations in the St Joe and Kanisku National Forests in northern Idaho. Most of the sampled isolates (n = 34) represented a dominant LSU/ITS haplotype present at all three sites and identical to the Mel-12 phylogenetic lineage (GU551425) identified in a previous study. Variation at 1-3 nucleotide sites was detected among a small number of isolates (n = 6) within this well supported clade (94%). Four isolates sampled from a single location were in a well supported clade (97%) distinct from the dominant haplotypes and may represent a previously un-sampled, cryptic phylogenetic species. Species-specific SNP and SCAR markers were developed for Mel-12 lineage isolates by cloning and sequencing AFLP amplicons, and segregation of AFLP markers were studied from single ascospore isolates from individual fruiting bodies. Based on the segregation of AFLP markers within single fruiting bodies, split decomposition analyses of two SCAR markers, and population genetic analyses of SNP, SCAR, and AFLP markers, it appears that members of the Morchella sp. Mel-12 phylogenetic lineage are heterothallic and outcross in nature similar to yellow morels. This is the first set of locus-specific molecular markers that has been developed for any Morchella species, to our knowledge. These markers will prove to be valuable tools to study mating system, gene flow and genetic structure of black morels at various spatial scales with field-collected fruiting bodies and eliminate the need to culture samples in vitro.

The histopathology of Phaeocryptopus gaeumannii on Douglas-fir needles.

Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincides with budbreak and shoot elongation. Colonization within needles is exclusively intercellular and increases continuously during Jul-May. No intracellular hyphae or haustoria were observed, but hyphae closely appressed to mesophyll and palisade cell walls are abundant by 3-5 mo after initial infection. Pseudothecial primordia begin to form in epistomatal chambers Oct-Apr, 4-9 mo after initial infection. Pseudothecial primordia developing in the epistomatal chamber are connected to the endophytic thallus by specialized cells in the substomatal chamber that have thickened apical walls and resemble phialides but are not involved in asexual reproduction. The apical wall thickenings instead appear to function as reinforcement against the turgor pressure of the guard cells, allowing cytoplasmic continuity to be maintained between the developing pseudothecium and vegetative hyphae within the needle. Concurrent with the formation of pseudothecial primorida, epiphytic hyphae emerge from the periphery of developing pseudothecia, grow across the needle surface, form numerous anastomoses and reenter the needle by producing appressoria above unoccupied stomata. Epiphytic hyphae and their associated appressoria gradually become more abundant during Oct-Jan.

A/C(i) curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance.

The analysis and interpretation of A/C(i) curves (net CO(2) assimilation rate, A, versus calculated substomatal CO(2) concentration, C(i)) is dependent upon a number of underlying assumptions. The influence of the C(i) value at which the A/C(i) curve switches between the Rubisco- and electron transport-limited portions of the curve was examined on A/C(i) curve parameter estimates, as well as the effect of mesophyll CO(2) conductance (g(m)) values on estimates of the maximum rate of Rubisco-mediated carboxylation (V(cmax)). Based on an analysis using 19 woody species from the Pacific Northwest, significant variation occurred in the C(i) value where the Rubisco- and electron transport-limited portions of the curve intersect (C(i_t)), ranging from 20 Pa to 152 Pa and averaging c. 71 Pa and 37 Pa for conifer and broadleaf species, respectively. Significant effects on estimated A/C(i) parameters (e.g. V(cmax)) may arise when preliminary estimates of C(i_t), necessary for the multiple regression analyses, are set either too high or too low. However, when the appropriate threshold is used, a significant relationship between A/C(i) and chlorophyll fluorescence estimates of carboxylation is achieved. The use of the V(cmax) parameter to describe accurately the Rubisco activity from the A/C(i) curve analysis is also dependent upon the assumption that C(i) is approximately equal to chloroplast CO(2) concentrations (C(c)). If leaf mesophyll conductance is low, C(c) will be much lower than C(i) and will result in an underestimation of V(cmax) from A/C(i) curves. A large range of mesophyll conductance (g(m)) values was observed across the 19 species (0.005+/-0.002 to 0.189+/-0.011 mol m(-2) s(-1) for Tsuga heterophylla and Quercus garryana, respectively) and, on average, g(m) was 1.9 times lower for the conifer species (0.058+/-0.017 mol m(-2) s(-1) for conifers versus 0.112+/-0.020 mol m(-2) s(-1) for broadleaves). When this mesophyll limitation was accounted for in V(cmax) estimates, considerable variation still existed between species, but the difference in V(cmax) between conifer and broadleaf species was reduced from c. 11 micromol m(-2) s(-1) to 4 micromol m(-2) s(-1). For example, A/C(i) curve estimates of V(cmax) were 31.2+/-6.2 and 42.2+/-4.4 micromol m(-2) s(-1), and A/C(c) curve estimates were 41.2+/-7.1 micromol m(-2) s(-1) and 45.0+/-4.8 micromol m(-2) s(-1), for the conifer and broadleaf species, respectively.

Botryozyma mucatilis sp. nov., an anamorphic ascomycetous yeast associated with nematodes in poplar slime flux.

A new species of Botryozyma, Botryozyma mucatilis, was isolated from the surface of free-living nematodes, Panagrellus dubius, inhabiting slime flux from hybrid poplars, Populus deltoidesxtrichocarpa, in Oregon, USA. This species was discovered in relatively close proximity to the teleomorphic species Ascobotryozyma americana and Ascobotryozyma cognata, both collected from P. dubius nematodes inhabiting beetle galleries in Populus spp. and Populus and Salix spp., respectively. B. mucatilis is recognized as a distinct species based on molecular and morphological data. Sequence divergence in both the D1/D2 domain of the nuclear large-subunit rDNA and internal transcribed spacer region rDNA, low DNA reassociation values, notably different amplified fragment-length polymorphic fingerprints, and significantly longer cells all support the designation of a novel species.

Ascobotryozyma cognata sp. nov., a new ascomycetous yeast associated with nematodes from wood-boring beetle galleries.

A new species of Ascobotryozyma, A. cognata sp. nov. (anamorph Botryozyma cognata), was isolated from beetle galleries in Idaho, USA. A. cognata was found on the surface of free-living nematodes, Panagrellus dubius, collected from galleries created by the long-horned beetle Saperda calcarata in Populus (aspen), and the weevil Cryptorhynchus lapathi in Salix (willow). A. cognata isolates were collected from similar habits and in relatively close proximity to those of A. americana, the only species described from North America. The recognition of A. cognata as a distinct species was supported by morphological and molecular data. Thallus cells of A. cognata were significantly shorter than those of A. americana. Low DNA reassociation values, notably different randomly amplified polymorphic DNA (RAPD), inter-sequence simple repeat (ISSR), and amplified fragment-length polymorphic (AFLP) fingerprints, and sequence divergence in both the D1/D2 domain of the nuc-LSU rDNA and an additional unidentified region were all consistent with the recognition of a new species.