Synergistic removal of pharmaceuticals and antibiotic resistance from ultrafiltered WWTP effluent: Free-floating ARGs exceptionally susceptible to degradation.

To protect the environment and human health, antibiotic resistance genes (ARGs) and persistent pharmaceuticals need to be removed from WWTP effluent prior to its reuse. However, an efficient process for removing free-floating extracellular DNA (exDNA) in combination with a wide range of pharmaceuticals is yet to be reported for real process conditions. As a possible solution, we treated real ultrafiltered WWTP effluent with UV/H2O2 and combined GAC and zeolite sorption. In terms of exDNA, sequencing and high-throughput quantitative PCR (HT-qPCR) showed that exDNA is a potent carrier of numerous ARGs in ultrafiltered WWTP effluent (123 ARGs), including multi-drug efflux pump mexF that became the dominant exARG in GAC effluent over time. Due to the exposure to degradation agents, exDNA was reduced more efficiently than intracellular DNA, and overall levels of ARGs were substantially lowered. Moreover, GAC sorption was particularly effective in the removal of almost all the 85 detected pharmaceutical residues, with fresh GAC demonstrating an efficiency of up to 100%. However, zeolite (Si/Al 0.8) addition was needed to enhance the removal of persistent pollutants such as gabapentin and diclofenac to 57% and up to 100%, respectively. Our combined approach eminently decreases the hazardous effects of pharmaceuticals and antibiotic resistance in the ultrafiltered WWTP effluent, producing effluent suitable for multiple reuse options according to the latest legislation. In addition, we provided similarly promising but less extensive data for surface water and treated greywater.

Extravasation (paravasation) of chemotherapy drugs - updated recommendations (2020) for standard care in the Czech Republic from the cooperation of the Supportive Care Group of the Czech Society for Oncology, Czech Society for Hematology, Oncology Section of the Czech Nurses Association and the Society for Ports and Permanent Catheters.

BACKGROUNDS: Extravasation (paravasation) of chemotherapy drugs is a very significant complication. Preventive and therapeutic interventions reduce the risk of the complication or the extent of its consequences. A working group of authors from expert groups prepared recommendations for standard care. PURPOSE: A basic summary of recommended interventions for daily practice, defined on the basis of knowledge from long-term, proven, evidence-based practice or on the consensus opinions of the expert groups representatives. RESULTS: Preventive measures are essential and include early consideration of long-term venous access devices indications, choice of injection site, venous line control before each chemotherapy drug application, and patient education. The intervention in case of extravasation mainly involves the application of antidotes (DMSO, hyaluronidase, dexrazoxane) and the application of dry cold or heat according to the type of cytostatic drug. Subcutaneous corticosteroids, moist heat or cooling and compression are not recommended. CONCLUSION: The recommended procedures contribute to reducing the risk and consequences of extravasation. The range of recommended interventions can be expanded individually depending on individual clinical site policy and needs.

The importance of arbuscular mycorrhiza for Cyclamen purpurascens subsp. immaculatum endemic in Slovakia.

At present, there is no relevant information on arbuscular mycorrhiza and the effect of the symbiosis on the growth of wild populations of cyclamens. To fill this gap, two populations of Cyclamen purpurascens subsp. immaculatum, endemic in Nízke Tatry (NT) mountains and Velká Fatra (VF) mountains, Slovakia, were studied in situ as well as in a greenhouse pot experiment. For both populations, mycorrhizal root colonization of native plants was assessed, and mycorrhizal inoculation potential (MIP) of the soils at the two sites was determined in 3 consecutive years. In the greenhouse experiment, the growth response of cyclamens to cross-inoculation with arbuscular mycorrhizal fungi (AMF) was tested: plants from both sites were grown in their native soils and inoculated with a Septoglomus constrictum isolate originating either from the same or from the other plant locality. Although the MIP of soil at the NT site was significantly higher than at the VF site, the level of AMF root colonization of C. purpurascens subsp. immaculatum plants in the field did not significantly differ between the two localities. In the greenhouse experiment, inoculation with AMF generally accelerated cyclamen growth and significantly increased all growth parameters (shoot dry weight, leaf number and area, number of flowers, tuber, and root dry weight) and P uptake. The two populations of C. purpurascens subsp. immaculatum grown in their native soils, however, differed in their response to inoculation. The mycorrhizal growth response of NT plants was one-order higher compared to VF plants, and all their measured growth parameters were stimulated regardless of the fungal isolates' origin. In the VF plants, only the non-native (NT originating) isolate showed a significant positive effect on several growth traits. It can be concluded that mycorrhiza significantly increased fitness of C. purpurascens subsp. immaculatum, despite the differences between plant populations, implying that AMF symbionts should be taken into account in conservation programs of this endemic plant.

Surprising spectra of root-associated fungi in submerged aquatic plants.

Similarly to plants from terrestrial ecosystems, aquatic species harbour wide spectra of root-associated fungi (RAF). However, comparably less is known about fungal diversity in submerged roots. We assessed the incidence and diversity of RAF in submerged aquatic plants using microscopy, culture-dependent and culture-independent techniques. We studied RAF of five submerged isoetid species collected in four oligotrophic freshwater lakes in Norway. Levels of dark septate endophytes (DSE) colonization differed among the lakes and were positively related to the organic matter content and negatively related to pH. In total, we identified 41 fungal OTUs using culture-dependent and culture-independent techniques, belonging to Mucoromycotina, Chytridiomycota, Glomeromycota, Ascomycota as well as Basidiomycota. Sequences corresponding to aquatic hyphomycetes (e.g. Nectria lugdunensis, Tetracladium furcatum and Varicosporium elodeae) were obtained. Eight arbuscular mycorrhizal taxa belonging to the orders Archaeosporales, Diversisporales and Glomerales were also detected. However, the vast majority of the fungal species detected (e.g. Ceratobasidium sp., Cryptosporiopsis rhizophila, Leptodontidium orchidicola, and Tuber sp.) have previously been known only from roots of terrestrial plants. The abundance and phylogenetic distribution of mycorrhizal as well as nonmycorrhizal fungi in the roots of submerged plants have reshaped our views on the fungal diversity in aquatic environment.

Long-term tracing of Rhizophagus irregularis isolate BEG140 inoculated on Phalaris arundinacea in a coal mine spoil bank, using mitochondrial large subunit rDNA markers.

During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.

Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes.

Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as 'endophytes' have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems.

Ericaceous dwarf shrubs affect ectomycorrhizal fungal community of the invasive Pinus strobus and native Pinus sylvestris in a pot experiment.

This study aimed to elucidate the relationship between ericaceous understorey shrubs and the diversity and abundance of ectomycorrhizal fungi (EcMF) associated with the invasive Pinus strobus and native Pinus sylvestris. Seedlings of both pines were grown in mesocosms and subjected to three treatments simulating different forest microhabitats: (a) grown in isolation and grown with (b) Vaccinium myrtillus or (c) Vaccinium vitis-idaea. Ericaceous plants did not act as a species pool of pine mycobionts and inhibited the ability of the potentially shared species Meliniomyces bicolor to form ectomycorrhizae. Similarly, Ericaceae significantly reduced the formation of Thelephora terrestris ectomycorrhizae in P. sylvestris. EcMF species composition in the mesocosms was strongly affected by both the host species and the presence of an ericaceous neighbour. When grown in isolation, P. strobus root tips were predominantly colonised by Wilcoxina mikolae, whereas those of P. sylvestris were more commonly colonised by Suillus and Rhizopogon spp. Interestingly, these differences were less evident (Suillus + Rhizopogon spp.) or absent (W. mikolae) when the pines were grown with Ericaceae. P. strobus exclusively associated with Rhizopogon salebrosus s.l., suggesting the presence of host specificity at the intrageneric level. Ericaceous plants had a positive effect on colonisation of P. strobus root tips by R. salebrosus s.l. This study demonstrates that the interaction of selective factors such as host species and presence of ericaceous plants may affect the realised niche of the ectomycorrhizal fungi.

Terminal restriction fragment length measurement errors are affected mainly by fragment length, G+C nucleotide content and secondary structure melting point.

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi-natural grasslands.

Glomus intraradices, an arbuscular mycorrhizal fungus (AMF), is frequently found in a surprisingly wide range of ecosystems all over the world. It is used as model organism for AMF and its genome is being sequenced. Despite the ecological importance of AMF, little has been known about their population structure, because no adequate molecular markers have been available. In the present study we analyse for the first time the intraspecific genetic structure of an AMF directly from colonized roots in the field. A recently developed PCR-RFLP approach for the mitochondrial rRNA large subunit gene (mtLSU) of these obligate symbionts was used and complemented by sequencing and primers specific for a particularly frequent mtLSU haplotype. We analysed root samples from two agricultural field experiments in Switzerland and two semi-natural grasslands in France and Switzerland. RFLP type composition of G. intraradices (phylogroup GLOM A-1) differed strongly between agricultural and semi-natural sites and the G. intraradices populations of the two agricultural sites were significantly differentiated. RFLP type richness was higher in the agricultural sites compared with the grasslands. Detailed sequence analyses which resolved multiple sequence haplotypes within some RFLP types even revealed that there was no overlap of haplotypes among any of the study sites except between the two grasslands. Our results demonstrate a surprisingly high differentiation among semi-natural and agricultural field sites for G. intraradices. These findings will have major implications on our views of processes of adaptation and specialization in these plant/fungus associations.

The cultivation bias: different communities of arbuscular mycorrhizal fungi detected in roots from the field, from bait plants transplanted to the field, and from a greenhouse trap experiment.

The community composition of arbuscular mycorrhizal fungi (AMF) was investigated in roots of four different plant species (Inula salicina, Medicago sativa, Origanum vulgare, and Bromus erectus) sampled in (1) a plant species-rich calcareous grassland, (2) a bait plant bioassay conducted directly in that grassland, and (3) a greenhouse trap experiment using soil and a transplanted whole plant from that grassland as inoculum. Roots were analyzed by AMF-specific nested polymerase chain reaction, restriction fragment length polymorphism screening, and sequence analyses of rDNA small subunit and internal transcribed spacer regions. The AMF sequences were analyzed phylogenetically and used to define monophyletic phylotypes. Overall, 16 phylotypes from several lineages of AMF were detected. The community composition was strongly influenced by the experimental approach, with additional influence of cultivation duration, substrate, and host plant species in some experiments. Some fungal phylotypes, e.g., GLOM-A3 (Glomus mosseae) and several members of Glomus group B, appeared predominantly in the greenhouse experiment or in bait plants. Thus, these phylotypes can be considered r strategists, rapidly colonizing uncolonized ruderal habitats in early successional stages of the fungal community. In the greenhouse experiment, for instance, G. mosseae was abundant after 3 months, but could not be detected anymore after 10 months. In contrast, other phylotypes as GLOM-A17 (G. badium) and GLOM-A16 were detected almost exclusively in roots sampled from plants naturally growing in the grassland or from bait plants exposed in the field, indicating that they preferentially occur in late successional stages of fungal communities and thus represent the K strategy. The only phylotype found with high frequency in all three experimental approaches was GLOM A-1 (G. intraradices), which is known to be a generalist. These results indicate that, in greenhouse trap experiments, it is difficult to establish a root-colonizing AMF community reflecting the diversity of these fungi in the field roots because fungal succession in such artificial systems may bias the results. However, the field bait plant approach might be a convenient way to study the influence of different environmental factors on AMF community composition directly under the field conditions. For a better understanding of the dynamics of AMF communities, it will be necessary to classify AMF phylotypes and species according to their life history strategies.

Cooccurring Gentiana verna and Gentiana acaulis and their neighboring plants in two Swiss upper montane meadows harbor distinct arbuscular mycorrhizal fungal communities.

The community composition of arbuscular mycorrhizal fungi (AMF) was analyzed in roots of Gentiana verna, Gentiana acaulis, and accompanying plant species from two species-rich Swiss alpine meadows located in the same area. The aim of the study was to elucidate the impact of host preference or host specificity on the AMF community in the roots. The roots were analyzed by nested PCR, restriction fragment length polymorphism screening, and sequencing of ribosomal DNA small-subunit and internal transcribed spacer regions. The AMF sequences were analyzed phylogenetically and used to define monophyletic sequence types. The AMF community composition was strongly influenced by the host plant species, but compositions did not significantly differ between the two sites. Detailed analyses of the two cooccurring gentian species G. verna and G. acaulis, as well as of neighboring Trifolium spp., revealed that their AMF communities differed significantly. All three host plant taxa harbored AMF communities comprising multiple phylotypes from different fungal lineages. A frequent fungal phylotype from Glomus group B was almost exclusively found in Trifolium spp., suggesting some degree of host preference for this fungus in this habitat. In conclusion, the results indicate that within a relatively small area with similar soil and climatic conditions, the host plant species can have a major influence on the AMF communities within the roots. No evidence was found for a narrowing of the mycosymbiont spectrum in the two green gentians, in contrast to previous findings with their achlorophyllous relatives.

Acaulospora alpina, a new arbuscular mycorrhizal fungal species characteristic for high mountainous and alpine regions of the Swiss Alps.

Acaulospora alpina sp. nov. forms small (65-85 microm diam), dark yellow to orange-brown spores laterally on the neck of hyaline to subhyaline sporiferous saccules. The spores have a three-layered outer spore wall, a bi-layered middle wall and a three-layered inner wall. The surface of the second layer of the outer spore wall is ornamented, having regular, circular pits (1.5-2 microm diam) that are as deep as wide and truncated conical. A "beaded" wall layer as found in most other Acaulospora spp. is lacking. The spore morphology of A. alpina resembles that of A. paulinae but can be differentiated easily by the unique ornamentation with the characteristic pits and by the spore color. A key is presented summarizing the morphological differences among Acaulospora species with an ornamented outer spore wall. Partial DNA sequences of the ITS1, 5.8S subunit and ITS2 regions of ribosomal DNA show that A. alpina and A. paulinae are not closely related. Acaulospora lacunosa, which has similar color but has generally bigger spores, also has distinct rDNA sequences. Acaulospora alpina is a characteristic member of the arbuscular mycorrhizal fungal communities in soils with pH 3.5-6.5 in grasslands of the Swiss Alps at altitudes between 1800 and 2700 m above sea level. It is less frequent at 1300-1800 m above sea level, and it so far has not been found in the Alps below 1300 m or in the lowlands of Switzerland.

Communities of arbuscular mycorrhizal fungi in arable soils are not necessarily low in diversity.

Communities of arbuscular mycorrhizal fungi (AMF) in five agricultural field sites of different management intensities were studied. Variable regions of the ribosomal RNA genes were used to detect and identify AMF directly within colonized roots. Roots from a continuous maize monoculture showed low AMF diversity, in agreement with previous reports on molecular diversity of AMF in agricultural soils. In contrast, a substantially higher diversity of AMF was found throughout the long term 'DOK' field experiment, where organic and conventional agricultural practices have been compared side by side since 1978. In this experiment, a 7-year crop rotation is performed under lower levels of inorganic fertilizer input and chemical pest control. These results are in good agreement with analyses of the spore community previously conducted in these field sites. In a third site, an organically managed leek field with soil of very high phosphate content reflecting the highly intensive conventional field history and intensive tillage, we detected a low-diversity community comparable to the maize monoculture. In addition to fungi from Glomus group A, which have previously been reported to dominate arable soils, we regularly found members of the genera Scutellospora, Paraglomus and Acaulospora. The genus Acaulospora was shown to occur more frequently early in the growing season, suggesting that the life history strategy of AMF may influence the active community at a given time. These data show that the diversity of AMF is not always low in arable soils. Furthermore, low-input agriculture involving crop rotation may provide better conditions to preserve AMF diversity, by preventing the selection for the few AMF taxa tolerating high nutrient levels.

Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N.

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3'-phosphoadenosine 5'-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.