Optimizing liquid fermentation for Wolfiporia cocos: gene expression and biosynthesis of pachymic acid and mycelial biomass.

Wolfiporia cocos, a versatile fungus acclaimed for its nutritional and therapeutic benefits in Traditional Chinese Medicine, holds immense potential for pharmaceutical and industrial applications. In this study, we aimed to optimize liquid fermentation techniques and culture medium composition to maximize mycelial biomass (MB) yield, pachymic acid (PA) concentration, and overall PA production. Additionally, we investigated the molecular basis of our findings by quantifying the expression levels of genes associated with PA and MB biosynthesis using quantitative real-time polymerase chain reaction. Under the optimized fermentation conditions, significant results were achieved, with maximum MB reaching 6.68 g l-1, PA content peaking at 1.25 mg g-1, and a total PA yield of 4.76 g l-1. Notably, among the four examined genes, squalene monooxygenase, exhibited enhanced expression at 0.06 ratio under the optimized conditions. Furthermore, within the realm of carbohydrate-active enzymes, the glycoside hydrolases 16 family displayed elevated expression levels at 21 ratios, particularly during MB production. This study enhances understanding of genetic mechanism governing MB and PA production in W. cocos, highlighting the roles of squalene monooxygenase and glycoside hydrolases 16 carbohydrate-active enzymes.

Changes in global DNA methylation under climatic stress in two related grasses suggest a possible role of epigenetics in the ecological success of polyploids.

Polyploidization drives the evolution of grasses and can result in epigenetic changes, which may have a role in the creation of new evolutionary lineages and ecological speciation. As such changes may be inherited, they can also influence adaptation to the environment. Populations from different regions and climates may also differ epigenetically; however, this phenomenon is poorly understood. The present study analyzes the effect of climatic stress on global DNA methylation based on a garden collection of two related mountain grasses (the narrow endemic diploid Festuca tatrae and the more widely distributed mixed-ploidy F. amethystina) with different geographic ranges and ecological niches. A lower level of DNA methylation was observed for F. tatrae, while a higher mean level was obtained for the diploid and tetraploid of F. amethystina; with the tetraploids having a higher level of global methylated DNA than the diploids. The weather conditions (especially insolation) measured 24 h prior to sampling appeared to have a closer relationship with global DNA methylation level than those observed seven days before sampling. Our findings suggest that the level of methylation during stress conditions (drought, high temperature and high insolation) may be significantly influenced by the ploidy level and bioclimatic provenance of specimens; however an important role may also be played by the intensity of stress conditions in a given year.

Seed variability of Sisymbriumpolymorphum (Murray) Roth (Brassicaceae) across the Central Palaearctic.

This study presents the results of investigation of the micromorphology and variability of Sisymbriumpolymorphum seeds collected in 49 localities in the core range and isolated populations of Central Europe, Eastern Europe and Central Asia. In addition, we compared the ultrastructure of the seeds of S. polymorphum with that of the closely-related species S.loeselii and S.linifolium. The seeds were measured with a stereoscopic microscope and a biometric programme (ImageJ) and micromorphological studies were performed by scanning electron microscopy (SEM). The seed variability showed intraspecific stability of the ultrastructure and low variability of metric features within the studied range. Studied species showed differences in the ultrastructure, which will be valuable for diagnostic purposes. We present and describe, for the first time, the ultrastructure of S.polymorphum seeds.

Tetraploids expanded beyond the mountain niche of their diploid ancestors in the mixed-ploidy grass Festuca amethystina L.

One promising area in understanding the responses of plants to ongoing global climate change is the adaptative effect of polyploidy. This work examines whether there is a coupling between the distribution of cytotypes and their biogeographical niche, and how different niches will affect their potential range. The study uses a range of techniques including flow cytometry, gradient and niche analysis, as well as distribution modelling. In addition, climatic, edaphic and habitat data was used to analyse environmental patterns and potential ranges of cytotypes in the first wide-range study of Festuca amethystina-a mixed-ploidy mountain grass. The populations were found to be ploidy homogeneous and demonstrate a parapatric pattern of cytotype distribution. Potential contact zones have been identified. The tetraploids have a geographically broader distribution than diploids; they also tend to occur at lower altitudes and grow in more diverse climates, geological units and habitats. Moreover, tetraploids have a more extensive potential range, being six-fold larger than diploids. Montane pine forests were found to be a focal environment suitable for both cytotypes, which has a central place in the environmental space of the whole species. Our findings present polyploidy as a visible driver of geographical, ecological and adaptive variation within the species.

The enhancement of valley water retentiveness in climate change conditions.

River system retentiveness must be enhanced to increase multidimensional environmental sustainability and thus ameliorate the effects of climate change and the occurrence of extreme hydrological events. The aim of the article is to demonstrate how ecohydrological Nature-Based Solutions can be combined with conventional infrastructure to improve WBSRCE benefits (Water, Biodiversity, ecosystem Services, Resilience, Culture, Education) by taking a holistic approach to multifunctional reservoir design. The paper proposes a new form of lateral reservoir, which is built without disturbing the meandering river and is supplied with good quality water through a monitoring and regulatory system; its design is based on thorough analysis of hydrological pulses and suspended matter and nutrients fluxes. The regulatory system also includes an innovative Sequential Sedimentation and Biofiltration System. Lateral reservoirs increase water retentiveness in the river valley by lifting ground water level and restoring surrounding wetlands, thus enhancing biodiversity, ecosystem services for society and the resilience of the river system to climate change. An integrative understanding of the interplay between hydrology and biocenosis can be used to enhance river system sustainability potential (WBSR) and harmonise societal needs with biosphere sustainability through culture and education (CE).

A catchment-scale assessment of the sanitary condition of treated wastewater and river water based on fecal indicators and carbapenem-resistant Acinetobacter spp.

Urbanization and population growth have created considerable sanitation challenges in cities and communities in many parts of Europe and the world. As such, it is imperative to identify the most environmentally-harmful microbiological and chemical sources of pollution, these being wastewater treatment plants (WWTPs) which release wastewater of low quality. In the present manuscript, an extensive study was performed of the sanitary conditions of river water and treated wastewater from seventeen WWTPs of various sizes along the Pilica River catchment in central Poland, with the aim of identifying "hot spots" in terms of most serious sources of sanitary hazards. The bacteriological risk for the river, including fecal indicator bacteria (FIB) such as coliforms, E.coli, enterococci, C. perfringens, and carbapenem-resistant Acinetobacter spp. (CRA) were assessed using classical microbiological methods, and the physicochemical parameters were also tested. The WWTPs, particularly the small ones (<2000 people equivalent, PE) demonstrated significant variation regarding the physicochemical parameters. Carbapenem-resistant Acinetobacter spp. bacteria growing at 42 °C were found in the effluent wastewaters of all tested municipal WWTPs, and in most of the Pilica River water samples, presenting a potential hazard to public health. A positive correlation was identified between E. coli and CRA abundance in treated wastewater; however, no such relationship was found in river water. It was found that seven small treatment plants discharged wastewater with very different microbiological parameters. Moreover, three small treatment plants serving only 0.56% of the population in the studied area continuously released extremely high microbiological contamination, constituting as much as 54-82% of fecal indicator bacteria loads in the area studied. Our findings show that this type of comprehensive analysis may enable assessment of the use of the entire catchment area, thus identifying the most serious threats to surface water quality and guiding the actions needed to improve the worst operating WWTPs.

The impact of WWTP size and sampling season on the prevalence of antibiotic resistance genes in wastewater and the river system.

There is a growing concern about the fate of antibiotic resistance genes (ARGs) during wastewater treatment and their potential impacts on the receiving water bodies. We hypothesised that the quantity of ARGs in effluents may be related to the size of wastewater treatment plants (WWTPs) and sampling season. To date, only several attempts have been made to investigate the impact of the above factors at the catchment scale. Therefore, the goal of the present study was to explore possible differences in the quantity of ARGs in treated wastewater from small, medium-sized and large WWTPs in the catchment of the Pilica River (9258 km2). The impact of treated wastewater on the concentration of ARGs was also determined along the river continuum from upland to lowland segments to the point of confluence with the Vistula (342 km). Treated effluent was sampled in 17 WWTPs, and river water was sampled in 7 sampling sites in four seasons. The concentrations of blaTEM, tet(A), ermF, sul1 and aac(6')-Ib-cr genes, the integrase gene intI1 and the 16S rRNA gene were analysed by quantitative PCR. The physical and chemical parameters and nutrient concentrations (23 various parameters) in the analysed samples were determined. The highest absolute concentrations of the studied genes were noted in effluent samples from small WWTPs (p < 0.01). The concentration of ARGs (gene copies/mL) peaked in winter and spring samples (p < 0.04). The results of statistical analyses indicate that in small WWTPs, the absolute concentration of ARGs can be predicted based on the biochemical oxygen demand, in routine water analyses. However, none of the studied parameters supported predictions of ARG abundance in medium-sized and large WWTPs or in river water.

The transferability of microsatellite loci from a homoploid to a polyploid hybrid complex: an example from fine-leaved Festuca species (Poaceae).

BACKGROUND: Microsatellite loci, or single sequence repeats (SSR), are widely used as powerful markers in population genetics. They represent an attractive tool for studying plants such as grasses, whose evolution is driven by hybridisation and polyploidization. However, the development of microsatellite markers has been challenging and time-consuming, especially for non-model organisms lacking available genome-wide sequence data. One straightforward and low-cost approach is to transfer the SSR loci developed for one species, or complex, to another closely-related one. This work evaluates the transferability of microsatellite loci from homoploid to allopolyploid complexes of fine-leaved Festuca species and to assess their use in two new species. The studied complex (F. amethystina-F. tatrae) is a useful model for research on the local adaptability of grasses with different ploidy levels. Since both species can be considered as rare or threatened (F. tatrae-as a mountain and narrow endemic species and F. amethystina-a mountain species with relict lowland populations), any tool enabling studies on genetic diversity and population genetics, such as SSR markers, could also be very useful in a conservation context. METHODS: The ploidy level within populations was estimated using flow cytometry. One diploid and one tetraploid population of F. amethystina and a diploid population of F. tatrae were chosen to test the transferability of SSR loci. Because our work describes the transfer of SSR nuclear markers designed originally for F. gautieri, a phylogenetic tree was prepared based on the ITS marker to assess the genetic distance between the studied complexes. The PCR products were separated on a high-resolution agarose gel, intended for SSR marker analysis. Appropriate solutions for the allotetraploid population and whole mixed-ploidy complex were implemented. RESULTS: Flow cytometry confirmed earlier data regarding DNA content in the investigated species and cytotypes. The phylogenetic ITS tree indicated a small genetic distance between F. gautieri complexes and the studied species. Ten microsatellite markers were successfully transferred. All markers were polymorphic. In total, 163 different alleles were scored from the 10 SSR loci. PCoA of accessions revealed well-separated groups corresponding to studied populations. Over 60% of the total variance is explained by differentiation within populations and one third among them. CONCLUSIONS: The transferred markers are valid tools for the study of population genetics and inheritance relationships within cytotypes and species and between them. The presented markers can be used to study inbreeding depression in the Festuca species, and variations in the degrees of genetic diversity between different cytotypes in mountain and lowland areas. Our findings can also be applied to study conservation strategies for ensuring biodiversity at the genetic level in polyploid complexes.

Morphometric traits in the fine-leaved fescues depend on ploidy level: the case of Festuca amethystina L.

BACKGROUND: Polyploid specimens are usually characterized by greater exuberance: they reach larger sizes and/or have a larger number of some organs. Festuca amethystina L. belongs to the section Aulaxyper. Based on morphological features, four subspecies of F. amethystina have been already identified. On the other hand, it has two cytotypes: diploid and tetraploid. The main aim of our study was to distinguish morphological differences between the cytotypes of F. amethystina, assuming that its phenotype differs significantly. METHODS: The nuclear DNA content was measured by flow cytometry in dry leaves from specimens originating from 13 populations of F. amethystina. Several macrometric and micrometric traits of stems, spikelets and leaf blades were taken into account in the comparative analysis of two cytotypes. RESULTS: In the case of cytotypes, specimens of tetraploids were larger than diploids. The conducted morphometric analysis of leaf cross-sections showed significant differences between the cytotypes. DISCUSSION: The research has confirmed for the first time that in the case of F. amethystina the principle of greater exuberance of polyploids is true. Differences between the cytotypes are statistically significant, however, they are not enough to make easy the distinction of cytotypes on the basis of the measurements themselves. Our findings favor the rule known in Festuca taxonomy as a whole, i.e. that the ploidy level can be one of the main classification criteria.

The use of a hybrid Sequential Biofiltration System for the improvement of nutrient removal and PCB control in municipal wastewater.

This article aims to evaluate the efficiency of an innovative hybrid Sequential Biofiltration System (SBS) for removing phosphorus and nitrogen and polychlorinated biphenyls (PCBs) from original municipal wastewater produced by a Wastewater Treatment Plant under authentic operating conditions. The hybrid SBS was constructed with two barriers, a geochemical (filtration beds with limestone, coal and sawdust) and a biological barrier (wetlands with Glyceria, Acorus, Typha, Phragmites), operating in parallel. Significant differences were found between inflow and outflow from the SBS with regard to wastewater contaminant concentrations, the efficiency of removal being 16% (max. 93%) for Total Phosphorus (TP), 25% (max. 93%) for Soluble Reactive Phosphorus (SRP), 15% (max. 97%) for Total Nitrogen (TN), 17% (max. 98%) for NO3-N, and 21% for PCB equivalency (PCB EQ). In the case of PCB EQ concentration, the highest efficiency of 43% was obtained using beds with macrophytes. The SBS removed a significant load of TP (0.415 kg), TN (3.136 kg), and PCB EQ (0.223 g) per square meter per year. The use of low-cost hybrid SBSs as a post-treatment step for wastewater treatment was found to be an effective ecohydrological biotechnology that may be used for reducing point source pollution and improving water quality.

Forest roadsides harbour less competitive habitats for a relict mountain plant (Pulsatilla vernalis) in lowlands.

The long-term survival of relict populations depends on the accessibility of appropriate sites (microrefugia). In recent times, due to the mass extinction of rare species that has resulted from the loss of natural habitats, the question is - Are there any human-made sites that can act as refugial habitats? We examined forest roadside populations of the mountain plant Pulsatilla vernalis in the last large lowland refugium in Central Europe. We compared the habitat conditions and community structure of roadsides with P. vernalis against the forest interior. Light availability and bryophyte composition were the main factors that distinguished roadsides. Pulsatilla occurred on sites that had more light than the forest interior, but were also more or less shaded by trees, so more light came as one-side illumination from the road. Roadsides had also a lower coverage of bryophytes that formed large, dense carpets. At the same time, they were characterised by a greater richness of vascular plants and 'small' bryophytes, which corresponds to a higher frequency of disturbances. In a warming and more fertile Anthropocene world, competition plays the main role in the transformation of forest communities, which is why relict populations have found refugia in extensively disturbed human-made habitats.

The Role of Hydrology in the Polychlorinated Dibenzo--dioxin and Dibenzofuran Distributions in a Lowland River.

Persistent organic pollutants such as polychlorinated dibenzo--dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are environmental contaminants that have widespread distribution and pose a serious threat to aquatic ecosystems. We conducted a study to quantify the distribution, patterns, and transport of PCDDs and PCDFs along the Pilica River in central Poland under different hydrological conditions to estimate the loads of these compounds and understand their fate in aquatic systems. Water samples were collected at five sampling points along the river that represent a range of hydrological conditions including flooding and stable and low water flows. Reduced river water flow was associated with lower average total and toxic equivalent (TEQ) concentrations of PCDDs plus PCDFs: 33.6 pg L and 4.21 pg TEQ L for flooding; 28.3 pg L and 3.6 pg TEQ L for stable flow; 18.4 pg L and 1.0 pg TEQ L for low-water flow. Similar results were observed for daily loadings of total and TEQ concentrations: the highest values were observed during flooding (331.1-839.4 mg d and 27.8-110.7 mg TEQ d), medium under stable hydrological conditions (55.8-121.0 mg d and 7.7-15.3 mg TEQ d), and the lowest values during low water flow (30.9 and 40.3 mg d and 1.4-2.4 mg TEQ d). The results demonstrate that diffuse sources of pollution play a key role during periods of high water flow (i.e., flooding season), whereas point sources of pollution, including municipal and industrial wastewater treatment plant discharges, mainly determine the PCDD and PCDF concentrations seen during low water periods.

Hierarchy of factors exerting an impact on nutrient load of the Baltic Sea and sustainable management of its drainage basin.

The aim of the paper was to evaluate 23 catchment factors that determine total phosphorus and total nitrogen load to the Baltic Sea. Standard correlation analysis and clustering were used. Both phosphorus and nitrogen loads were found to be positively related to the number of pigs and the human population associated with wastewater treatment plants (WWTPs) per km(2), while the number of cattle and agricultural area were found to influence nitrogen rather than phosphorus load, and the area of forests is negatively related to loads of both nutrients. Clustering indicates an overall north-south pattern in the spatial co-occurrence of socio-ecological factors, with some exceptions discussed in the paper. Positive steps in the Baltic Sea region have already been taken, but much remains to be done. The development of coherent response policies to reduce eutrophication in the Baltic Sea should be based on a comprehensive knowledge base, an appropriate information strategy and learning alliance platform in each drainage river catchments.