The Relationship between Fungal Diversity and Invasibility of a Foliar Niche—The Case of Ash Dieback

European ash (Fraxinus excelsior) is threatened by the invasive ascomycete Hymenoscyphus fraxineus originating from Asia. Ash leaf tissues serve as a route for shoot infection but also as a sporulation substrate for this pathogen. Knowledge of the leaf niche partitioning by indigenous fungi and H. fraxineus is needed to understand the fungal community receptiveness to the invasion. We subjected DNA extracted from unwashed and washed leaflets of healthy and diseased European ash to PacBio sequencing of the fungal ITS1-5.8S-ITS2 rDNA region. Leaflets from co-inhabiting rowan trees (Sorbus aucuparia) served as a reference. The overlap in leaflet mycobiomes between ash and rowan was remarkably high, but unlike in rowan, in ash leaflets the sequence read proportion, and the qPCR-based DNA amount estimates of H. fraxineus increased vigorously towards autumn, concomitant with a significant decline in overall fungal richness. The niche of ash and rowan leaves was dominated by epiphytic propagules (Vishniacozyma yeasts, the dimorphic fungus Aureobasidion pullulans and the dematiaceous hyphomycete Cladosporium ramotenellum and H. fraxineus), and endophytic thalli of biotrophs (Phyllactinia and Taphrina species), the indigenous necrotroph Venturia fraxini and H. fraxineus. Mycobiome comparison between healthy and symptomatic European ash leaflets revealed no significant differences in relative abundance of H. fraxineus, but A. pullulans was more prevalent in symptomatic trees. The impacts of host specificity, spatiotemporal niche partitioning, species carbon utilization profiles and life cycle traits are discussed to understand the ecological success of H. fraxineus in Europe. Further, the inherent limitations of different experimental approaches in the profiling of foliicolous fungi are addressed.

ThefruRBAOperon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood

Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogenStreptococcus pyogenes(the group AStreptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where thefrulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed thatfruRBAformed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entirefruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, andN-acetyl-d-galactosamine. The inactivation ofsloR, afruAhomolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruRand ΔfruBmutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruAmutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment.

ISOLATION AND CHARACTERIZATION OF AN ACRYLAMIDE-DEGRADING Burkholderia sp. STRAIN DR.Y27

ABSTRACT Several local bacteria have been isolated from glyphosate-contaminated soils at various locations throughout Malaysia. Quantitative monitoring of acrylamide degradation was performed using High Performance Liquid Chromatography (HPLC) whilst bacterial growth was carried out by plate counting. The isolate was tentatively identified as Burkholderia sp. strain DR.Y27 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny. Highest growth was obtained at acrylamide concentrations of between 100 to 2000 mg L-1. Complete degradation of 850 mg L-1 of acrylamide occurs after ten days of incubation with concomitant cell growth. The isolate grew optimally in between pH 6.0 and 8.0. The effect of incubation temperature on the growth of this isolate shows an optimum growth at 30°C. Glucose, lactose, maltose, fructose, mannitol, citric acid and sucrose at an initial concentration of 1.0% (w/v) supported growth with glucose being the best carbon source. Aliphatic amides such as 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, propionamide and urea supported growth with increasing assimilative capability from 2-chloroacetamide to urea. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide. Keywords: isolation, characterization, acrylamide-degrading, Bacterium

Effect of Long-Term Starvation on the Survival, Recovery, and Carbon Utilization Profiles of a Bovine Escherichia coli O157:H7 Isolate from New Zealand

ABSTRACTThe ability to maintain a dual lifestyle of colonizing the ruminant gut and surviving in nonhost environments once shed is key to the success ofEscherichia coliO157:H7 as a zoonotic pathogen. Both physical and biological conditions encountered by the bacteria are likely to change during the transition between host and nonhost environments. In this study, carbon starvation at suboptimal temperatures in nonhost environments was simulated by starving a New Zealand bovineE. coliO157:H7 isolate in phosphate-buffered saline at 4 and 15°C for 84 days. Recovery of starved cells on media with different nutrient availabilities was monitored under aerobic and anaerobic conditions. We found that the New Zealand bovineE. coliO157:H7 isolate was able to maintain membrane integrity and viability over 84 days and that the level of recovery depended on the nutrient level of the recovery medium as well as the starvation temperature. In addition, a significant difference in carbon utilization was observed between starved and nonstarved cells.

Carbon utilization profiles of Fusarium virguliforme isolates

Fusarium virguliforme is the cause of sudden death syndrome in soybean. Physiological variability among isolates of the fungus is unknown. One way to measure physiologic variability is to analyze growth on different carbon sources. The carbon source utilization profiles of 18 F. virguliforme isolates were examined using the Biolog FF 96-well microplate, which contains 95 different carbon sources. The utilization of dextrin, d-mannitol, maltotriose, d-lactic acid methyl ester, N-acetyl-d-galactosamine, salicin, d-trehalose, and l-alanine differed significantly among isolates (P = 0.05). Carbon sources were grouped into 3 clusters based on their ability to promote growth of F. virguliforme, after calculating Euclidean distances among them. About 12% of the carbon sources promoted a high amount of mycelial growth, 39% promoted a medium amount of growth, and 49% promoted a low amount of mycelial growth; the latter was not significantly different from the water blank control. A hierarchical tree diagram was produced for the 18 isolates based on their carbon source utilization profiles using Ward’s hierarchical analysis method. Two main clusters of isolates were formed. One cluster represented greater average mycelial growth on all of the carbon sources than the other cluster. In this study, variability in carbon source utilization among F. virguliforme isolates was evident, but the results were not associated with geographic origin of the isolates, year collected, or published data on aggressiveness. Additional research is needed to determine if these carbon utilization profiles are associated with other biological characteristics, like spore germination, propagule formation, and saprophytic competitiveness.

Global Carbon Utilization Profiles of Wild-Type, Mutant, and Transformant Strains of Hypocrea jecorina

ABSTRACT The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, d-sorbitol, d-ribose, d-galactose, l-arabinose, N-acetyl-d-glucosamine, maltotriose, and β-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on γ-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (±10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function.