Non-target analysis of Danish wastewater treatment plant effluent: Statistical analysis of chemical fingerprinting as a step toward a future monitoring tool.

In an attempt to discover and characterize the plethora of xenobiotic substances, this study investigates chemical compounds released into the environment with wastewater effluents. A novel non-targeted screening methodology based on ultra-high resolution Orbitrap mass spectrometry and nanoflow ultra-high performance liquid chromatography together with a newly optimized data-processing pipeline were applied to effluent samples from two state-of-the-art and one small wastewater treatment facility. In total, 785 molecular structures were obtained, of which 38 were identified as single compounds, while 480 structures were identified at a putative level. Most of these substances were therapeutics and drugs, present as parent compounds and metabolites. Using R packages Phyloseq and MetacodeR, originally developed for bioinformatics, significant differences in xenobiotic presence in the wastewater effluents between the three sites were demonstrated.

Sequential analysis of δ15 N in guard hair suggests late gestation is the most critical period for muskox calf recruitment.

RATIONALE: Analysis of stable isotopes in tissue and excreta may provide information about animal diets and their nutritional state. As body condition may have a major influence on reproduction, linking stable isotope values to animal demographic rates may help unravel the drivers behind animal population dynamics. METHODS: We performed sequential analysis of δ15 N values in guard hair from 21 muskoxen (Ovibos moschatus) from Zackenberg in high arctic Greenland. We were able to reconstruct the dietary history for the population over a 5-year period with contrasting environmental conditions. We examined the linkage between guard hair δ15 N values in 12 three-month periods and muskox calf recruitment to detect critical periods for muskox reproduction. Finally, we conducted similar analyses of the correlation between environmental conditions (snow depth and air temperature) and calf recruitment. RESULTS: δ15 N values exhibited a clear seasonal pattern with high levels in summer and low levels in winter. However, large inter-annual variation was found in winter values, suggesting varying levels of catabolism depending on snow conditions. In particular δ15 N values during January-March were linked to muskox recruitment rates, with higher values coinciding with lower calf recruitment. δ15 N values were a better predictor of muskox recruitment rates than environmental conditions. CONCLUSIONS: Although environmental conditions may ultimately determine the dietary δ15 N signal in muskox guard hairs, muskox calf recruitment was more strongly correlated with δ15 N values than ambient snow and temperature. The period January-March, corresponding to late gestation, appears particularly critical for muskox reproduction.

Pruning Wound Protection Products Induce Alterations in the Wood Mycobiome Profile of Grapevines.

Fungal pathogens involved in grapevine trunk diseases (GTDs) may infect grapevines throughout their lifetime, from nursery to vineyard, via open wounds in stems, canes or roots. In vineyards, pruning wound protection products (PWPPs) offer the best means to reduce the chance of infection by GTD fungi. However, PWPPs may affect non-target microorganisms that comprise the natural endophytic mycobiome residing in treated canes, disrupting microbial homeostasis and indirectly influencing grapevine health. Using DNA metabarcoding, we characterized the endophytic mycobiome of one-year-old canes of cultivars Cabernet Sauvignon and Syrah in two vineyards in Portugal and Italy and assessed the impact of established and novel PWPPs on the fungal communities of treated canes. Our results reveal a large fungal diversity (176 taxa), and we report multiple genera never detected before in grapevine wood (e.g., Symmetrospora and Akenomyces). We found differences in mycobiome beta diversity when comparing vineyards (p = 0.01) but not cultivars (p > 0.05). When examining PWPP-treated canes, we detected cultivar- and vineyard-dependent alterations in both alpha and beta diversity. In addition, numerous fungal taxa were over- or under-represented when compared to control canes. Among them, Epicoccum sp., a beneficial genus with biological control potential, was negatively affected by selected PWPPs. This study demonstrates that PWPPs induce alterations in the fungal communities of grapevines, requiring an urgent evaluation of their direct and indirect effects on plants health with consideration of factors such as climatic conditions and yearly variations, in order to better advise viticulturists and policy makers.

Muskoxen homogenise soil microbial communities and affect the abundance of methanogens and methanotrophs.

Grazing herbivores may affect soil microbial communities indirectly by impacting soil structure and vegetation composition. In high arctic environments, this impact is poorly elucidated, while having potentially wide-reaching effects on the ecosystem. This study examines how a key arctic herbivore, the muskox Ovibos moschatus, affects the soil microbial community in a high arctic fen. Environmental DNA was extracted from soil samples taken from grazed control plots and from muskox exclosures established 5 years prior. We sequenced amplicons of the 16S rRNA gene to provide insight into the microbial communities. We found that in the grazed control plots, microbial communities exhibited high evenness and displayed highly similar overall diversity. In plots where muskoxen had been excluded, microbial diversity was significantly reduced, and had more uneven intra-sample populations and overall lower ecological richness and evenness. We observed that the composition of microbial communities in grazed soils were significantly affected by the presence of muskoxen, as seen by elevated relative abundances of Bacteroides and Firmicutes, two major phyla found in muskox faeces. Furthermore, an increase in relative abundance of bacteria involved in degradation of recalcitrant carbohydrates and cycling of nitrogen was observed in grazed soil. Ungrazed soils displayed increased abundances of bacteria potentially involved in anaerobic oxidation of methane, whereas some methanogens were more abundant in grazed soils. This corroborates a previous finding that methane emissions are higher in arctic fens under muskox grazing. Our results show that the presence of large herbivores stimulates soil microbial diversity and has a homogenizing influence on the inter-species dynamics in soil microbial communities. The findings of this study, thus, improve our understanding of the effect of herbivore grazing on arctic ecosystems and the derived methane cycling.

Identification and Characterization of 33 Bacillus cereus sensu lato Isolates from Agricultural Fields from Eleven Widely Distributed Countries by Whole Genome Sequencing.

The phylogeny, identification, and characterization of 33 B. cereus sensu lato isolates originating from 17 agricultural soils from 11 countries were analyzed on the basis of whole genome sequencing. Phylogenetic analyses revealed all isolates are divided into six groups, which follows the generally accepted phylogenetic division of B. cereus sensu lato isolates. Four different identification methods resulted in a variation in the identity of the isolates, as none of the isolates were identified as the same species by all four methods-only the recent identification method proposed directly reflected the phylogeny of the isolates. This points to the importance of describing the basis and method used for the identification. The presence and percent identity of the protein product of 19 genes potentially involved in pathogenicity divided the 33 isolates into groups corresponding to phylogenetic division of the isolates. This suggests that different pathotypes exist and that it is possible to differentiate between them by comparing the percent identity of proteins potentially involved in pathogenicity. This also reveals that a basic link between phylogeny and pathogenicity is likely to exist. The geographical distribution of the isolates is not random: they are distributed in relation to their division into the six phylogenetic groups, which again relates to different ecotypes with different temperature growth ranges. This means that we find it easier to analyze and understand the results obtained from the 33 B. cereus sensu lato isolates in a phylogenetic, patho-type and ecotype-oriented context, than in a context based on uncertain identification at the species level.

Fungicides and the Grapevine Wood Mycobiome: A Case Study on Tracheomycotic Ascomycete Phaeomoniella chlamydospora Reveals Potential for Two Novel Control Strategies.

Phaeomoniella chlamydospora is a tracheomycotic fungus that colonizes the xylem of grapevines (Vitis vinifera L.), causing wood discoloration, brown wood streaking, gummosis, and wood necrosis, which negatively affect the overall health, productivity, and life span of vines. Current control strategies to prevent or cope with P. chlamydospora infections are frequently ineffective. Moreover, it is unclear how fungicides commonly applied in vineyards against downy and powdery mildew agents affect the wood mycobiome, including wood pathogens such as P. chlamydospora. In this study, we used next-generation sequencing to assess the effects of foliar spray of grapevines with inorganic (copper oxychloride and sulfur), synthetic (penconazole and fosetyl-aluminum), and natural (Blad) fungicides currently used against the downy and powdery mildews. The subjects of our investigation were (i) the resident wood mycobiome, (ii) the early colonization by a consortium of fungal wood endophytes (ACEA1), (iii) the wood colonization success of P. chlamydospora, and (iv) the in planta interaction between P. chlamydospora and ACEA1, under greenhouse conditions, in rooted grapevine cuttings of cv. Cabernet Sauvignon. The data obtained suggest that the resident mycobiome is affected by different fungicide treatments. In addition, the early colonization success of the endophytes composing ACEA1 varied in response to fungicides, with relative abundances of some taxa being overrepresented or underrepresented when compared with the control. The wood colonization by P. chlamydospora comported significant changes in the mycobiome composition, and in addition, it was greatly affected by the foliar spray with Blad, which decreased the relative abundance of this pathogen 12-fold (4.9%) when compared with the control (60.7%) and other treatments. The presence of the pathogen also decreased considerably when co-inoculated into the plant with ACEA1, reaching relative abundances between 13.9% and 2.0%, depending on the fungicide treatment applied. This study shows that fungicides sprayed to prevent infections of powdery and downy mildews have an effect on non-target fungi that colonize the endosphere of grapevines. We suggest two potential control strategies to fight P. chlamydospora, namely, the foliar spray with Blad and the use of ACEA1. Further studies to confirm these results are required.

Characterization of the Wood Mycobiome of Vitis vinifera in a Vineyard Affected by Esca. Spatial Distribution of Fungal Communities and Their Putative Relation With Leaf Symptoms.

Esca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina® next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (Debaryomyces, Trematosphaeria, Biatriospora, Lopadostoma, and Malassezia) and numerous hitherto unreported species. Esca-associated fungi Phaeomoniella chlamydospora and Fomitiporia sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., Eutypa spp., Inonotus hispidus). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting 'tiger stripes' symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of esca.