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Charcoal kilns, old structures used for charcoal production in the forest, preserve a charcoal-enriched topsoil representing a suitable proxy for studying the long-term effect of biochar addition to soil. Two kiln platforms located at Gelbison and Vesole mountain sites in Southern Italy were selected due to their comparable climates but distinct parent rocks. We conducted standard soil chemical analyses and used next-generation sequencing to explore bacterial and fungal microbiome. Anthracology identified charcoal species, while scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS) characterized charcoal particles. Reflectance Fourier transform infrared spectroscopy (DRIFTS) assessed biochar surface oxidation. Additionally, a bioassay with soybean, maize, and Tomato investigated the impact of kiln soil on plant performance. Our results showed that kiln soils did not exhibit higher pH, cation exchange capacity, or greater richness in cations. EDS and FTIR analyses showed that charcoal buried in forest soil for decades undergoes significant oxidation, with increased O/C ratio and the presence of oxygenated functional groups. Charcoal surfaces were selectively enriched with Ca2+ on limestone substrate sites but with Al and Si over sedimentary (flysch) substrate. While differences in the kiln soil and its surroundings were noticeable, they were not drastic in terms of microbial diversity and composition. Surprisingly, the bioassay indicated that the kiln microbiota had a more positive impact on plant growth compared to external forest soil. In conclusion, this study highlights the unique nature of kiln microsites and begins to unveil the enduring effects of charcoal accumulation on soil chemistry and microbiota in forest soil. Highlights: Kiln soils did not exhibit higher pH, cation exchange capacity, or greater richness in cations. Charcoal in forest kilns underwent significant oxidation with increased O/C ratio and presence of oxygenated functional groups. Charcoal surface showed different enrichment of Ca, Al, and Si influenced by the parent rock. Kiln microbiota exhibited a positive influence on plant growth, hinting at ecological benefits. [ABSTRACT FROM AUTHOR]

To effectively protect the marine environment, it is crucial to establish effective environ mental monitoring platforms. Traditional marine environmental monitoring methods heavily rely on morphological identification and field expertise, with the sampling process being disruptive and potentially destructive to vulnerable marine environments. In light of emerging biomonitoring needs and biodiversity declines, we reviewed the urgently needed, ongoing advances in developing effective, noninvasive, and innovative monitoring methods and systems to examine the complex marine environment for better strategic conservation and protection, using the coral ecosystem as one of the representative forefront examples in marine protection. This review summarizes current trends and efforts in transitioning into more standardizable and automatable utilizations of environmental DNA metabarcoding-based monitoring strategies and high-resolution underwater optical imaging monitoring systems as two of the promising pillars for the next generation of noninvasive biomonitoring and associated applications. The assistance of artificial intelligence for environmental DNA metabarcoding and high-resolution underwater optical imaging into an empowered, all-rounded monitoring platform for enhanced monitoring capacity is discussed as a highly potent direction for future research exploration. This review will be a cornerstone reference for the future development of artificial intelligence-assisted, noninvasive, and innovative marine environmental monitoring systems. [ABSTRACT FROM AUTHOR]

The propagation of antibiotic resistance in environments, particularly aquatic environments that serve as primary pathways for antibiotic resistance genes (ARGs), poses significant health risks. The impact of nutrients, as key determinants of bacterial growth and metabolism, on the propagation of ARGs, particularly extracellular ARGs (eARGs), remains poorly understood. In this study, we collected microorganisms from the Yangtze River and established a series of microcosms to investigate how variations in nutrient levels and delivery frequency affect the relative abundance of intracellular ARGs (iARGs) and eARGs in bacterial communities. Our results show that the relative abundance of 7 out of 11 representative eARGs in water exceeds that of iARGs, while 8 iARGs dominate in biofilms. Notably, iARGs and eARGs consistently exhibited opposite responses to nutrient variation. When nutrient levels increased, iARGs in the water also increased, with the polluted group (COD = 333.3 mg/L, COD:N:P = 100:3:0.6, m/m) and the eutrophic group (COD = 100 mg/L, COD:N:P = 100:25:5, m/m) showing 1.2 and 3.2 times higher levels than the normal group (COD = 100 mg/L, COD:N:P = 100:10:2, m/m), respectively. In contrast, eARGs decreased by 6.7% and 8.4% in these groups. On the other hand, in biofilms, higher nutrient levels led to an increase in eARGs by 1.5 and 1.7 times, while iARGs decreased by 17.5% and 50.1% in the polluted and eutrophic groups compared to the normal group. Moreover, while increasing the frequency of nutrient delivery (from 1 time/10 d to 20 times/10 d) generally did not favor iARGs in either water or biofilm, it selectively enhanced eARGs in both. To further understand these dynamics, we developed an ARGs-nutrient model by integrating the Lotka–Volterra and Monod equations. The results highlight the complex interplay of bacterial growth, nutrient availability, and mechanisms such as horizontal gene transfer and secretion influencing ARGs' propagation, driving the opposite trend between these two forms of ARGs. This contrasting response between iARGs and eARGs contributes to a dynamic balance that stabilizes bacterial resistance levels amid nutrient fluctuations. This study offers helpful implications regarding the persistence of bacterial resistance in the environment. [ABSTRACT FROM AUTHOR]

The global decline of amphibian populations, with 40.7% of species classified as threatened, calls for innovative and ethical approaches in conservation genetics. Molecular biology advancements have introduced environmental DNA (eDNA) analysis, primarily focusing on aquatic environments. However, the present study explores a novel non-invasive protocol using water samples to extract DNA from terrestrial and semi-terrestrial amphibians, specifically the endangered Italian endemic salamander, Salamandrina perspicillata (Savi, 1821). Unlike traditional invasive methods involving tissue sampling, this protocol immerses animals briefly, eliminating the need for digit or tail amputations or manipulation for buccal swabs. The study validated the protocol through DNA extraction, amplification, and sequencing, yielding results comparable to traditional methods. The non-invasive nature of the protocol aligns with the 3Rs principles (Replace, Reduce, Refine) and offers a streamlined, stress-minimizing alternative for studying protected and endangered species. Future experiments should also explore further refinements, including reduced soaking times and additional applications, such as skin microbiota analysis. This protocol represents a significant step towards ethical and effective research practices in amphibian conservation genetics, encouraging a paradigm shift in wildlife research ethics. Continued innovation in non-invasive methodologies is essential for comprehensive understanding and robust conservation strategies amid the ongoing biodiversity crisis. [ABSTRACT FROM AUTHOR]

Background: Hereditary Spastic Paraplegias (HSP) are genetic neurodegenerative disorders affecting the corticospinal tract. No established neuroimaging biomarker is associated with this condition. Methods: A total of 46 patients affected by HSP, genetically and clinically evaluated and tested with SPRS scores, and 46 healthy controls (HC) matched by age and gender underwent a single-voxel Magnetic Resonance Spectroscopy sampling (MRS) of bilateral pre-central and pre-frontal regions. MRS data were analyzed cross-sectionally (at T0 and T1) and longitudinally (T0 vs. T1). Results: Statistically significant data showed that T0 mI/Cr in the pre-central areas of HSP patients was higher than in HC. In the left (L) pre-central area, NAA/Cr was significantly lower in HSP than in HC. In the right (R) pre-frontal area, NAA/Cr was significantly lower in HSP patients than in HC. HSP SPG4 subjects had significantly lower Cho/Cr concentrations in the L pre-central area compared to HC. Among the HSP subjects, non-SPG4 patients had significantly higher mI/Cr in the L pre-central area compared to SPG4 patients. In the R pre-frontal area, NAA/Cr was reduced, and ml/Cr was higher in non-SPG4 patients compared to SPG4 patients. Comparing "pure" and "complex" forms, NAA/Cr was higher in pHSP than in cHSP in the R pre-central and R pre-frontal areas. The longitudinal analysis, which involved fewer patients (n = 30), showed an increase in mI/Cr concentration in the L pre-frontal area among HSP subjects with respect to baseline. The patients had significantly higher SPRS scores at follow-up, with a significant positive correlation between SPRS scores and mI/Cr in the L precentral area, while in bilateral pre-frontal areas, lower SPRS scores corresponded to higher NAA/Cr concentrations. To explore the discriminating power of MRS in correctly identifying HSP and controls, an inference tree methodology classified HSP subjects and controls with an overall accuracy of 73.9%, a sensitivity of 87.0%, and a specificity of 60.9%. Conclusion: This pilot study indicates that brain MRS is a valuable approach that could potentially serve as an objective biomarker in HSP. [ABSTRACT FROM AUTHOR]

Background: Forest fire is very common in all ecosystems of the world that affects both vegetation and soil and is also helpful in maintaining the diversity and stability of ecosystems. The effect of forest fires and prescribed fires on forest soil is very complex. It affects the organic matter, macro and micronutrients, and physical properties of soil, such as texture, color, pH, and bulk density, as well as soil biota. Fire is a natural factor in the forest that temporarily reduces the vegetation on the soil surface. After a fire, the soil can experience long-term, medium-term, and short-term changes, which are different depending on the type of soil characteristics, weather conditions, duration, intensity, and frequency of the fire. The Zagros forest ecosystem is generally in the coppice form, and these forests are not safe from fire, which constantly affects them. Considering that the long-term stability of these forests depends on maintaining the soil quality, it is essential to investigate the effects of fire on soil properties. This study aimed to investigate the effect of fire on Zagros forests in Kermanshah province, Gahvareh city in the Lerini region. Methods: The studied area is a part of Zagros forests in Kermanshah province, Gahvareh city, the Lerini region located at 34° 10' 35″ E and 46° 34' 28″ N, with an average height of 1468 m above sea level. In this research, the points were determined by measuring with a meter on transects, and they were sampled regularly in specific time intervals (October, November, February, April, and June). The distance between the samples in each row was 20 m and the distance between the rows was 50 m, and three rows were determined in each of the burnt and control areas. Some soil characteristics, such as pH, electrical conductivity (EC), organic carbon, absorbable phosphorus, soil respiration, microbial biomass carbon, substrate-induced respiration, and metabolic quotient, were measured in soil samples. The effect of the treatments on all measured variables was investigated by the two-way ANOVA. Means were compared by Duncan's Multiple Range Test (DMRT). Data were analyzed using the Windows version of SAS (version 9.4, SAS Institute Inc., Cary, NC). Results: The soil pH decreased with the passage of time and a decrease in air temperature under the crown (unburnt and burned) and outside the unburnt crown. An upward trend in the pH of the soil in these three places occurred after the cold season. The soil pH ranged from 7.31 to 7.66 in all treatments from October to June. The results of the two-way ANOVA showed that the fire and sampling location did not affect the soil EC in different months of sampling. The amount of absorbable phosphorus increased with the occurrence of fire. The highest and lowest amounts of absorbable phosphorus occurred under and outside the burnt and the control crowns, respectively. The range of organic carbon changes indicates that soil organic carbon has increased over time in all treatments. The highest and the lowest increases were observed under the burnt (1.51%) and the control (unburned) crowns, respectively. Examining the changes indicates that the basic soil respiration was high at the beginning of the fire but decreased gradually, and then the initial value also increased in the last months of sampling. In this research, the amount of basic respiration in the soil of burned areas is not higher than that of unburned areas despite the occurrence of fire and the negative effect on soil organic carbon. According to the results, the amount of substrateinduced respiration decreases under and outside the crown (burnt and control) with the passage of time and the decrease in temperature. According to the ANOVA results, the effects of fire and the sampling location were significant on the microbial metabolic quotient of the soil, although no significant interaction effects on the microbial metabolic quotient were observed in February and June. The highest and the lowest microbial metabolic quotients were measured under (0.94 mg C/mg MBC.day) and outside (0.26 mg C/mg MBC.day) the unburned crown, respectively. With the passage of time and a decrease in temperature, the amount of this parameter decreased in the treatment under the crown of the unburnt area and increased in the three treatments under and outside the burned crown and outside the unburnt crown. At the end of the period, the highest and the lowest microbial metabolic quotients were measured under (0.72 mg C/mg MBC. day) and outside (0.59 mg C/mg MBC. day) the crown of the burned area, respectively. Conclusion: The results showed that the fire was superficial in both places under and outside the crown and did not significantly affect the soil's chemical and biological characteristics. Therefore, no significant changes occurred in indicators such as soil organic carbon and EC, and the fire only increased phosphorus in the burned areas compared to the control. Due to the occurrence of surface fire, combustion did not significantly increase biological indicators. The results of this research demonstrate that the fire changes the soil's chemical and biological characteristics when it occurs with a higher intensity and at a higher temperature. [ABSTRACT FROM AUTHOR]

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity. [ABSTRACT FROM AUTHOR]

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics. [ABSTRACT FROM AUTHOR]

Background: In Europe, Pinus cembra forests cover subalpine and alpine areas and they are of high conservational and ecological relevance. These forests experience strong seasonality with alternating snow-free and snow covered periods. Although P. cembra is known for mycorrhization and mycorrhizae usually involve fungi, plants and bacteria, the community compositions of fungi and bacteria and their associations in (sub-)alpine P. cembra forests remain vastly understudied. Here, we studied the fungal and bacterial community compositions in three independent (sub-)alpine P. cembra forests and inferred their microbial associations using marker gene sequencing and network analysis. We asked about the effect of snow cover on microbial compositions and associations. In addition, we propose inferring microbial associations across a range of filtering criteria, based on which we infer well justified, concrete microbial associations with high potential for ecological relevance that are typical for P. cembra forests and depending on snow cover. Results: The overall fungal and bacterial community structure was comparable with regards to both forest locations and snow cover. However, occurrence, abundance, and diversity patterns of several microbial taxa typical for P. cembra forests differed among snow-free and snow covered soils, e.g. Russula, Tetracladium and Phenoliphera. Moreover, network properties and microbial associations were influenced by snow cover. Here, we present concrete microbial associations on genus and species level that were repeatedly found across microbial networks, thereby confirming their ecological relevance. Most importantly, ectomycorrhizal fungi, such as Basidioascus, Pseudotomentella and Rhizopogon, as well as saprobic Mortierella changed their bacterial association partners depending on snow cover. Conclusion: This is the first study researching fungal-bacterial associations across several (sub-)alpine P. cembra forests. The poorly investigated influence of snow cover on soil fungi and bacteria, especially those mycorrhizing P. cembra roots, but also saprobic soil organisms, underlines the relevance of forest seasonality. Our findings highlight that the seasonal impact of snow cover has significant consequences for the ecology of the ecosystem, particularly in relation to mycorrhization and nutrient cycling. It is imperative to consider such effects for a comprehensive understanding of the functioning resilience and responsiveness of an ecosystem. [ABSTRACT FROM AUTHOR]

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0–3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR's capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA. [ABSTRACT FROM AUTHOR]

Studying past ecosystems from ancient environmental DNA preserved in lake sediments (sedaDNA) is a rapidly expanding field. This research has mainly involved Holocene sediments from lakes in cool climates, with little known about the suitability of sedaDNA to reconstruct substantially older ecosystems in the warm tropics. Here, we report the successful recovery of chloroplast trnL (UAA) sequences (trnL-P6 loop) from the sedimentary record of Lake Towuti (Sulawesi, Indonesia) to elucidate changes in regional tropical vegetation assemblages during the lake's Late Quaternary paleodepositional history. After the stringent removal of contaminants and sequence artifacts, taxonomic assignment of the remaining genuine trnL-P6 reads showed that native nitrogen-fixing legumes, C 3 grasses, and shallow wetland vegetation (Alocasia) were most strongly associated with >1-million-year-old (>1 Ma) peats and silts (114-98.8 m composite depth; mcd), which were deposited in a landscape of active river channels, shallow lakes, and peat-swamps. A statistically significant shift toward partly submerged shoreline vegetation that was likely rooted in anoxic muddy soils (i.e., peatland forest trees and wetland C 3 grasses (Oryzaceae) and nutrient-demanding aquatic herbs (presumably Oenanthe javanica)) occurred at 76 mcd (~0.8 Ma), ~0.2 Ma after the transition into a permanent lake. This wetland vegetation was most strongly associated with diatom ooze (46-37 mcd), thought to be deposited during maximum nutrient availability and primary productivity. Herbs (Brassicaceae), trees/shrubs (Fabaceae and Theaceae), and C 3 grasses correlated with inorganic parameters, indicating increased drainage of ultramafic sediments and laterite soils from the lakes' catchment, particularly at times of inferred drying. Downcore variability in trnL-P6 from tropical forest trees (Toona), shady ground cover herbs (Zingiberaceae), and tree orchids (Luisia) most strongly correlated with sediments of a predominantly felsic signature considered to be originating from the catchment of the Loeha River draining into Lake Towuti during wetter climate conditions. However, the co-correlation with dry climate-adapted trees (i.e., Castanopsis or Lithocarpus) plus C 4 grasses suggests that increased precipitation seasonality also contributed to the increased drainage of felsic Loeha River sediments. This multiproxy approach shows that despite elevated in situ temperatures, tropical lake sediments potentially comprise long-term archives of ancient environmental DNA for reconstructing ecosystems, which warrants further exploration., (© 2024 The Authors. Geobiology published by John Wiley & Sons Ltd.)