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BACKGROUND: The use of DNA metabarcoding has become an increasingly popular technique to infer feeding relationships in polyphagous herbivores and predators. Understanding host plant preference of native and invasive herbivore insects can be helpful in establishing effective integrated pest management (IPM) strategies. The invasive Halyomorpha halys and native Pentatoma rufipes are piercing‐sucking stink bug pests that are known to cause economic damage in commercial fruit orchards. RESULTS: In this study, we performed molecular gut content analysis (MGCA) on field‐collected specimens of these two herbivorous pentatomids using next‐generation amplicon sequencing (NGAS) of the internal transcribed spacer 2 (ITS2) barcode region. Additionally, a laboratory experiment was set up where H. halys was switched from a mixed diet to a monotypic diet, allowing us to determine the detectability of the initial diet in a time series of ≤3 days after the diet switch. We detected 68 unique plant species from 54 genera in the diet of two stink bug species, with fewer genera found per sample and a smaller diet breadth for P. rufipes than for H. halys. Both stink bug species generally prefer deciduous trees over gymnosperms and herbaceous plants. Landscape type significantly impacted the observed genera in the diet of both stink bug species, whereas season only had a significant effect on the diet of H. halys. CONCLUSION: This study provides further insights into the dietary composition of two polyphagous pentatomid pests and illustrates that metabarcoding can deliver a relevant species‐level resolution of host plant preference. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Hatcheries nationwide suffer from unexplained acute production failures, termed crashes. The microbiota of oysters relates to larval health with previous studies showing that some bacterial species have positive and others negative effects on oyster health. To investigate microbial correlates of crashes, we collected samples from every batch of oyster larvae (Crassostrea virginica) produced by the Horn Point Laboratory Oyster Hatchery since 2021 and analyzed the microbiota of 15 of those batches over their duration in the hatchery, from age of 3 to 5 days until either harvest or complete die off of the batch. Across events, die-offs generally became evident at or after six days of age. We found that the microbiota of oyster larvae appears to respond to die-off events with crashed batches having fundamentally different microbiota than good batches at age 7 to 9 and 9 to 12 days. Crashed batches were often taken over by microeukaryotes and bacterial taxa from the Protobacteria and Bacteroidetes phyla. However, this presumably opportunistic community differed between batches. Observed Vibrio species level groups did not appear to be oyster pathogens and appeared to respond to, rather than precede, crashes. The microbiota of 3 to 5 day old larvae were statistically related to whether a die-off occurred later in the larval batches' life, only when the taxa were first agglomerated to family level. The detection of two microbial species not previously known to associate with oysters, along with an increased presence of Dinophyceae, predominantly the toxin-producing Gyrodinium jinhaense, in 3 to 5 day old oyster larvae was statistically linked with subsequent batch crashes. This study suggests that the health of larval oysters shapes their microbiome. Conversely, it provides hints that the microbiome of larvae, and perhaps harmful algae, may drive hatchery crashes. [ABSTRACT FROM AUTHOR]

Cyanobacteria are known for forming associations with various animals, including sea turtles, yet our understanding of cyanobacteria associated with sea turtles remains limited. This study aims to address this knowledge gap by investigating the diversity of cyanobacteria in biofilm samples from loggerhead sea turtle carapaces, utilizing a 16S rRNA gene amplicon sequencing approach. The predominant cyanobacterial order identified was Nodosilineales, with the genus Rhodoploca having the highest relative abundance. Our results suggest that cyanobacterial communities become more diverse as sea turtles age, as we observed a positive correlation between community diversity and the length of a sea turtle's carapace. Since larger and older turtles predominantly utilize neritic habitats, the shift to a more diverse cyanobacterial community aligned with a change in loggerhead habitat. Our research provides detailed insights into the cyanobacterial communities associated with loggerhead sea turtles, establishing a foundation for future studies delving into this fascinating ecological relationship and its potential implications for sea turtle conservation. [ABSTRACT FROM AUTHOR]

ABSTRACT Current rDNA reference sequence databases are tailored towards shorter DNA markers, such as parts of the 16/18S marker or the internally transcribed spacer (ITS) region. However, due to advances in long‐read DNA sequencing technologies, longer stretches of the rDNA operon are increasingly used in environmental sequencing studies to increase the phylogenetic resolution. There is, therefore, a growing need for longer rDNA reference sequences. Here, we present the ribosomal operon database (ROD), which includes eukaryotic full‐length rDNA operons fished from publicly available genome assemblies. Full‐length operons were detected in 34.1% of the 34,701 examined eukaryotic genome assemblies from NCBI. In most cases (53.1%), more than one operon variant was detected, which can be due to intragenomic operon copy variability, allelic variation in non‐haploid genomes, or technical errors from the sequencing and assembly process. The highest copy number found was 5947 in Zea mays. In total, 453,697 unique operons were detected, with 69,480 operon variant clusters remaining after intragenomic clustering at 99% sequence identity. The operon length varied extensively across eukaryotes, ranging from 4136 to 16,463 bp, which will lead to considerable polymerase chain reaction (PCR) bias during amplification of the entire operon. Clustering the full‐length operons revealed that the different parts (i.e., 18S, 28S, and the hypervariable regions V4 and V9 of 18S) provide divergent taxonomic resolution, with 18S, the V4 and V9 regions being the most conserved. The ROD will be updated regularly to provide an increasing number of full‐length rDNA operons to the scientific community. [ABSTRACT FROM AUTHOR]

ABSTRACT It is a global priority to better manage the biosphere, but action must be informed by comprehensive data on the abundance and distribution of species. The acquisition of such information is currently constrained by high costs. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive sequencing protocols are critical as the census of all animal species is likely to require the analysis of a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By enabling the sequencing of highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until recently, constrained adoption. However, the improved performance of its latest flow cells (R10.4.1) erases this barrier. This study demonstrates that a MinION flow cell can characterise an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow. [ABSTRACT FROM AUTHOR]

ABSTRACT Microhaplotypes are small linked genomic regions comprising two or more single‐nucleotide polymorphisms (SNPs) that are being applied in forensics and are emerging in wildlife monitoring studies and genomic epidemiology. Typically, targeted in non‐coding regions, microhaplotypes in exonic regions can be designed with larger amplicons to capture functional non‐synonymous sites and minimise insertion/deletion (indel) polymorphisms. Quality control is an important first step for high‐confidence genotyping to counteract such false‐positive variants. As genetic markers with higher polymorphism compared to biallelic SNPs, it is critical to ensure sequencing errors across the microhaplotype amplicon are filtered out to avoid introducing false‐haplotypes. We developed the MhGeneS pipeline which works in tandem with Seq2Sat to help validate microhaplotype genotyping of the coding region of genes, with broader applicability to any microhaplotype profiling. We genotyped microhaplotype regions of the Zfx (≅ 160 bp) and Zfy (≅ 140 bp) genes, as well as an exon of the prion protein (Prnp) gene (≅ 370 bp) in caribou (Rangifer tarandus) using paired‐end Illumina technology. As important quality metrics affecting microhaplotype calling, we identified the sequencing error rate profile related to the overlap or non‐overlap of paired‐end reads as well as the read depth as significant. In the case of Prnp, we achieved confident microhaplotype calling through MhGeneS by removing small sections of the 5′ and 3′ amplicons and using a minimum read depth of 20. Read depth and sequence trimming may be locus‐specific, and validation of these parameters is recommended before the high‐throughput profiling of samples. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex class I chain‐related genes A and B (MICA and MICB) play a role as ligands in activating the NKG2D receptor expressed in natural killer cells, γδ T‐cells and αβ CD8 T‐cells and have been defined in human diseases and haematopoietic stem cell transplantation (HSCT). MICA and MICB alleles were genotyped at the three‐field level by amplicon‐based next‐generation sequencing (NGS) using a MiSeqDx system and compared with the results from previous studies in healthy South Korean donors. Exons 2–5 of MICA and exons 2–4 of MICB were amplified using a multiplex polymerase chain reaction (PCR). Sequence reads of ≥ 51 depth counts were consistently obtained for each sample exon, and target exons were determined to match reference sequences contained in the IPD‐IMGT/HLA database. MICA and MICB alleles were tested using exon combinations. The program was designed to recognise specific sequences and discriminate between the MICA*008:01:01/*027 alleles. A total of 22 alleles were found in MICA and MICB. We observed 1 HLA‐C ~ HLA‐B ~ MICA ~ MICB ~ HLA‐DRB1 haplotype with significant linkage disequilibrium between alleles at all neighbouring HLA loci. These results are consistent with previous microarray results. Genotyping of MICA and MICB was possible using 11‐loci HLA genes. We updated the distribution of MICA and MICB based on three‐field allele and haplotype frequencies containing linkage disequilibrium in South Koreans using amplicon‐based NGS. These data suggest that high‐resolution MICA and MICB typing data obtained using NGS may aid in performing HSCT and disease association studies. [ABSTRACT FROM AUTHOR]

Characterization of fungal spider pathogens lags far behind their insect counterparts. In addition, little to nothing is known concerning the ecological reservoir and/or fungal entomopathogen community surrounding infection sites. Five infected spider cadavers were identified in the neo-tropical climate of north-central Florida, USA, from three of which viable cultures were obtained. Multi-locus molecular phylogenetic and morphological characterization identified one isolate as a new Gibellula species, here named, Gibellula floridensis, and the other isolates highly similar to Parengyodontium album. The fungal entomopathogen community surrounding infected spiders was sampled at different habitats/trophic levels, including soil, leaf litter, leaf, and twig, and analyzed using ITS amplicon sequencing. These data revealed broad but differential distribution of insect-pathogenic fungi between habitats and variation between sites, with members of genera belonging to Metarhizium and Metacordyceps from Clavicipitaceae, Purpureocillium and Polycephalomyces from Ophiocordyceps, and Akanthomyces and Simplicillium from Cordycipitaceae predominating. However, no sequences corresponding to Gibellula or Parengyodontium, even at the genera levels, could be detected. Potential explanations for these findings are discussed. These data highlight novel discovery of fungal spider pathogens and open the broader question regarding the environmental distribution and ecological niches of such host-specific pathogens. [ABSTRACT FROM AUTHOR]

Most eukaryotic genes generate multiple messenger RNA (mRNA) transcript variants by alternative splicing. The incomplete annotation of gene transcripts in genomic databases can result in improper primer design, adversely affecting the reliability of gene expression measurements by reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR). Hence, we present a workflow combining bioinformatics analyses, to select two to three evolutionarily conserved constitutive exons in rats, mice and humans as target sequences for PCR primer design, with experimental RT‐PCR amplification and amplicon sequencing to confirm the expression and identity of gene transcript targets. The application of this workflow to the characterization of neurodevelopmental biomarker genes identified an unannotated exon in the rat Map2 gene, illustrating the importance of target sequence validation for the development of translational mRNA biomarkers for toxicological and biomedical studies. [ABSTRACT FROM AUTHOR]

This paper proposes an original definition of samples as a kind of data within the relational framework of data. The distinction between scientific objects (e.g., samples, data, models) often needs to be clarified in the philosophy of science to understand their role in the scientific inquiry. The relational framework places data at the forefront of knowledge construction. Their epistemic status depends on their evaluation as potential evidence in a research situation and their ability to circulate among researchers. While samples are significant in data-generating science, their role has been underexplored in the philosophy of data literature. I draw on a case study from data-centric microbiology, viz. amplicon sequencing, to introduce specifications of the relational framework. These specifications capture the distinctive epistemic role of samples, allowing the discussion of their significance in the inquiry process. I argue that samples are necessarily transformed to be considered as evidence, portable in the limits of a situation, and they act as world anchors for claims about a phenomenon. I compare these specifications with other data and evidence frameworks and suggest they are compatible. The paper concludes by considering the extension of these criteria in the context of biobanking. The specifications proposed here help analyze other life sciences cases and deepen our understanding of samples and their epistemological role in scientific research. [ABSTRACT FROM AUTHOR]

Synthetic culture media, such as Zarrouk's medium (ZM), are widely used in industrial Arthrospira cultivation but rely heavily on chemical fertilizers, raising concerns over cost and environmental impact. In natural habitats where Arthrospira blooms, the macronutrient concentrations are much lower than those provided by synthetic media. We hypothesized that natural growth may be facilitated by a microbial consortium. To test this, we developed a lab-scale Arthrospira platensis H53 cultivation system using a newly developed organic compost medium (OCM), designed to mimic the natural nutrient composition and microbial interactions. Compared to ZM, A. platensis H53 grown in OCM exhibited elevated growth by day 7. The specific growth rate in OCM was 0.20 day−1, higher than that of 0.17 day−1 in ZM, with optical density values reaching 1.57, compared to 1.13 in ZM. A 1.63-fold increase in biomass was observed in OCM, despite lower initial macronutrient concentrations. Nutrient use efficiency (NUE) in OCM was significantly improved, with nitrate (NO3−) and phosphate (PO43−) utilization up to 5.8-fold higher. Additionally, A. platensis H53 filaments in OCM were more tightly coiled, indicating a physiological change in response to lowered macronutrient concentrations. Microbial composition analysis using 16S rRNA gene amplicon sequencing revealed the presence of growth-promoting bacteria, including Pontibacter spp., Brevundimonas spp., and Aliihoeflea spp., likely contributing to nutrient cycling and enhanced growth. These findings suggest potential symbiotic interactions between cyanobacteria and non-cyanobacteria in the OCM system, promoting increased growth and productivity. This study is the first to propose such symbiosis in an extremely alkalophilic environment, offering another sustainable alternative to traditional chemical-based Arthrospira cultivation methods. [ABSTRACT FROM AUTHOR]

Background: Cereal diseases caused by insect-transmitted viruses are challenging to forecast and control because of their intermittent outbreak patterns, which are usually attributed to increased population densities of vector insects due to cereal crop rotations and indiscriminate use of pesticides, and lack of resistance in commercial varieties. Root microbiomes are known to significantly affect plant health, but there are significant knowledge gaps concerning epidemics of cereal virus diseases at the microbiome-wide scale under a variety of environmental and biological factors. Results: Here, we characterize the diversity and composition of rice (Oryza sativa) root-associated bacterial communities after infection by an insect-transmitted reovirus, rice black-streaked dwarf virus (RBSDV, genus Fijivirus, family Spinareoviridae), by sequencing the bacterial 16S rRNA gene amplified fragments from 1240 samples collected at a consecutive 3-year field experiment. The disease incidences gradually decreased from 2017 to 2019 in both Langfang (LF) and Kaifeng (KF). BRSDV infection significantly impacted the bacterial community in the rice rhizosphere, but this effect was highly susceptible to both the rice-intrinsic and external conditions. A greater correlation between the bacterial community in the rice rhizosphere and those in the root endosphere was found after virus infection, implying a potential relationship between the rice-intrinsic conditions and the rhizosphere bacterial community. The discrepant metabolites in rhizosphere soil were strongly and significantly correlated with the variation of rhizosphere bacterial communities. Glycerophosphates, amino acids, steroid esters, and triterpenoids were the metabolites most closely associated with the bacterial communities, and they mainly linked to the taxa of Proteobacteria, especially Rhodocyclaceae, Burkholderiaceae, and Xanthomonadales. In addition, the greenhouse pot experiments demonstrated that bulk soil microbiota significantly influenced the rhizosphere and endosphere communities and also regulated the RBSDV-mediated variation of rhizosphere bacterial communities. Conclusions: Overall, this study reveals unprecedented spatiotemporal dynamics in rhizosphere bacterial communities triggered by RBSDV infection with potential implications for disease intermittent outbreaks. The finding has promising implications for future studies exploring virus-mediated plant-microbiome interactions. 2AL1xEb7k2nunVBmbuNCsV Video Abstract [ABSTRACT FROM AUTHOR]

The deterioration of fibre-reinforced sprayed concrete was studied in the Oslofjord subsea tunnel (Norway). At sites with intrusion of saline groundwater resulting in biofilm growth, the concrete exhibited significant concrete deterioration and steel fibre corrosion. Using amplicon sequencing and shotgun metagenomics, the microbial taxa and surveyed potential microbial mechanisms of concrete degradation at two sites over five years were identified. The concrete beneath the biofilm was investigated with polarised light microscopy, scanning electron microscopy and X-ray diffraction. The oxic environment in the tunnel favoured aerobic oxidation processes in nitrogen, sulfur and metal biogeochemical cycling as evidenced by large abundances of metagenome-assembled genomes (MAGs) with potential for oxidation of nitrogen, sulfur, manganese and iron, observed mild acidification of the concrete, and the presence of manganese- and iron oxides. These results suggest that autotrophic microbial populations involved in the cycling of several elements contributed to the corrosion of steel fibres and acidification causing concrete deterioration. [ABSTRACT FROM AUTHOR]

Background Aims Methods Results Conclusions Sewage sludge biochar has been widely used in agriculture for various purposes; however, knowledge about its residual effect on the soil microbial community is limited.This study aimed to evaluate changes in the microbial community 5 years after the application of sewage sludge biochar.Biochar was produced from sewage sludge obtained at a wastewater treatment pyrolyzed at 300°C (Biochar300) and 500°C (Biochar500). Both biochar treatments were applied in corn fields for two growing seasons (2014/2015; 2015/2016). Biochar was not applied from the third (2016/2017) to the seventh (2020/2021) growing season to assess the residual effect. Soil samples were collected after harvesting in 2020/2021 growing season, therefore in the fifth year subject to the residual effects of biochar. The microbial community was evaluated by accessing bacterial and fungal groups using DNA techniques. Amplicon sequencing analysis was performed with the samples by Illumina MiSeq 250 bp.At genus level, the Lachnospira, Faecalibacterium, Bacteroides, Epulopiscium, and Parabacteroides were affected by Biochar500. Furthermore, at upper levels of taxonomy, this biochar treatment increased the relative abundance of bacterial classes Clostridiaceae and Bacteroidia and the fungal families Chaetomiaceae and Aspergillaceae. Concerning the relationship between microbial community and soil chemical properties, soil phosphorus (P) and calcium (Ca) were significantly affected by biochar treatments within the bacterial community; the fungal ones affected significantly soil P and potential acidity (H + Al).Even under the residual effect of 5 years, the application of biochar altered the microbial community on corn plantation, especially concerning on the richness and the abundance of some groups. Biochar500 was the most responsive among the treatments. [ABSTRACT FROM AUTHOR]

Background: Clustering of sequences into operational taxonomic units (OTUs) and denoising methods are a mainstream stopgap to taxonomically classifying large numbers of 16S rRNA gene sequences. Environment-specific reference databases generally yield optimal taxonomic assignment. Results: We developed SpeciateIT, a novel taxonomic classification tool which rapidly and accurately classifies individual amplicon sequences (https://github.com/Ravel-Laboratory/speciateIT). We also present vSpeciateDB, a custom reference database for the taxonomic classification of 16S rRNA gene amplicon sequences from vaginal microbiota. We show that SpeciateIT requires minimal computational resources relative to other algorithms and, when combined with vSpeciateDB, affords accurate species level classification in an environment-specific manner. Conclusions: Herein, two resources with new and practical importance are described. The novel classification algorithm, SpeciateIT, is based on 7th order Markov chain models and allows for fast and accurate per-sequence taxonomic assignments (as little as 10 min for 107 sequences). vSpeciateDB, a meticulously tailored reference database, stands as a vital and pragmatic contribution. Its significance lies in the superiority of this environment-specific database to provide more species-resolution over its universal counterparts. [ABSTRACT FROM AUTHOR]

The root-knot nematode (RKN) causes significant yield loss in tomatoes. Understanding the interaction of biocontrol agents (BCAs)-nematicides-soil microbiomes and RKNs is essential for enhancing the efficacy of biocontrol agents and nematicides to curb RKN damage to crops. The present study aimed to evaluate the in vitro effectiveness of BACa and nematicide against RKN and to apply the amplicon sequencing to assess the interaction of Bacillus velezensis (VB7) and Trichoderma koningiopsis (TK) against RKNs. Metagenomic analysis revealed the relative abundance of three phyla such as Proteobacteria (42.16%), Firmicutes (19.57%), and Actinobacteria (17.69%) in tomato rhizospheres. Those tomato rhizospheres treated with the combined application of B. velezensis VB7 + T. koningiopsis TK and RKN had a greater frequency of diversity and richness than the control. RKN-infested tomato rhizosphere drenched with bacterial and fungal antagonists had the maximum diversity index of bacterial communities. A strong correlation with a maximum number of interconnection edges in the phyla Proteobacteria, Firmicutes, and Actinobacteria was evident in soils treated with both B. velezensis VB7 and T. koningiopsis TK challenged against RKN in infected soil. The present study determined a much greater diversity of bacterial taxa observed in tomato rhizosphere soils treated with B. velezensis VB7 and T. koningiopsis TK than in untreated soil. It is suggested that the increased diversity and abundance of bacterial communities might be responsible for increased nematicidal properties in tomato plants. Hence, the combined applications of B. velezensis VB7 and T. koningiopsis TK can enhance the nematicidal action to curb RKN infecting tomatoes. [ABSTRACT FROM AUTHOR]

Introduction: Microalgae form an essentia! group of benthic organisms that respond swiftly to environmental changes. They are widely used as bioindicators of anthropogenic stressors in freshwater ecosystems. We aimed to assess the responses of microalgae communities to multiple environmental stressors in the Kinzig River catchment, home to a long-term ecological monitoring site, in Germany. Methods: We used a photosynthetic biomass proxy alongside community composition of diatoms assessed by digital light microscopy, and of microalgae by 18S-V9 amplicon sequencing, to characterise microalgae at 19 sampling sites scattered across the catchment. Results: Our results revealed significant effects of physical and chemical factors on microalgae biomass and community compositions. We found that conductivity, water temperature and pH were the most important factors affecting microalgae community composition, as observed in both microscopy and amplicon analysis. In addition to these three variables, the effect of total phosphate on all microalgae, together with water discharge on the diatom (Bacillariophyta) communities, as assessed by amplicon analysis, may reveal taxon-specific variations in the ecological responses of different microalgal groups. Discussion: Our results highlighted the complex relationship between various environmental variables and microalgae biomass and community composition. Further investigations, involving the collection of time series data, are required to fully understand the underlying biotic and abiotic parameters that influence these microalgae communities. [ABSTRACT FROM AUTHOR]

It is increasingly recognized that different genetic variants of hosts can uniquely shape their microbiomes. Invasive species often evolve in their introduced ranges, but little is known about the potential for their microbial associations to change during invasion as a result. We asked whether host genotype (G), microbial environment (E), or their interaction (G × E) affected the composition and diversity of host-associated microbiomes in Centaurea solstitialis (yellow starthistle), a Eurasian plant that is known to have evolved novel genotypes and phenotypes and to have altered microbial interactions, in its severe invasion of CA, USA. We conducted an experiment in which native and invading plant genotypes were inoculated with native and invaded range soil microbial communities. We used amplicon sequencing to characterize rhizosphere bacteria in both the experiment and the field soils from which they were derived. We found that native and invading plant genotypes accumulated different microbial associations at the family level in each soil community, often counter to differences in family abundance between soil communities. Root associations with potentially beneficial Streptomycetaceae were particularly interesting, as these were more abundant in the invaded range field soil and accumulated on invading genotypes. We also found that bacterial diversity is higher in invaded soils, but that invading genotypes accumulated a lower diversity of bacteria and unique microbial composition in experimental inoculations, relative to native genotypes. Thus variation in microbial associations of invaders was driven by the interaction of plant G and microbial E, and rhizosphere microbial communities appear to change in composition in response to host evolution during invasion. [ABSTRACT FROM AUTHOR]

Commercial livestock producers need to prioritize genetic progress for health and efficiency traits to address productivity, welfare, and environmental concerns but face challenges due to limited pedigree information in extensive multi‐sire breeding scenarios. Utilizing pooled DNA for genotyping and integrating seminal microbiome information into genomic models could enhance predictions of male fertility traits, thus addressing complexities in reproductive performance and inbreeding effects. Using the Angus Australia database comprising genotypes and pedigree data for 78,555 animals, we simulated percentage of normal sperm (PNS) and prolificacy of sires, resulting in 713 sires and 27,557 progeny in the final dataset. Publicly available microbiome data from 45 bulls was used to simulate data for the 713 sires. By incorporating both genomic and microbiome information our models were able to explain a larger proportion of phenotypic variation in both PNS (0.94) and prolificacy (0.56) compared to models using a single data source (e.g., 0.36 and 0.41, respectively, using only genomic information). Additionally, models containing both genomic and microbiome data revealed larger phenotypic differences between animals in the top and bottom quartile of predictions, indicating potential for improved productivity and sustainability in livestock farming systems. Inbreeding depression was observed to affect fertility traits, which makes the incorporation of microbiome information on the prediction of fertility traits even more actionable. Crucially, our inferences demonstrate the potential of the semen microbiome to contribute to the improvement of fertility traits in cattle and pave the way for the development of targeted microbiome interventions to improve reproductive performance in livestock. [ABSTRACT FROM AUTHOR]

The Altai Mountains (ALE) and the Greater Khingan Mountains (GKM) in northern China are forest regions dominated by coniferous trees. These geographically isolated regions provide an ideal setting for studying microbial biogeographic patterns. In this study, we employed high-throughput techniques to obtain DNA sequences of soil myxomycetes, bacteria, and fungi and explored the mechanisms underlying the assembly of both local and cross-regional microbial communities in relation to environmental factors. Our investigation revealed that the environmental heterogeneity in ALE and GKM significantly affected the succession and assembly of soil bacterial communities at cross-regional scales. Specifically, the optimal environmental factors affecting bacterial Bray-Curtis similarity were elevation and temperature seasonality. The spatial factors and climate change impact on bacterial communities under the geographical barriers surpassed that of local soil microenvironments. The assembly pattern of bacterial communities transitions from local drift to cross-regional heterogeneous selection. Environmental factors had a relatively weak influence on myxomycetes and fungi. Both soil myxomycetes and fungi faced considerable dispersal limitation at local and cross-regional scales, ultimately leading to weak geographical distribution patterns. [ABSTRACT FROM AUTHOR]

Coal mining has led to escalating ecological and environmental issues in significant coal and grain production areas, posing a severe danger to food security. This study examines the disturbance patterns of soil factors and microbial communities in coal and grain production areas, and attempts to understand the impact of subsidence and water accumulation stress on soil characteristics and microbial communities in coal mining subsidence areas with high subsidence levels. Five specific regions of Zhao Gu Yi Mine, situated in Henan Province and under the ownership of Jiaozuo Coal Group, were chosen. Aside from the control group (CK), the study blocks situated in the coal mining subsidence zones consisted of perennial subsidence ponding (PSP), seasonal subsidence ponding (SSP), the neutral zone (NZ), and the horizontal deformation zone (HDZ). The soil nutrient indices and the stoichiometric properties of soil C, N, and P were assessed on the surface of each block. The organization of the soil microbial community was identified using high-throughput sequencing. The findings indicate that: 1. Substantial disparities exist in soil properties and microbial community structure between the subsidence and non-subsidence zones. The levels of soil organic mater (SOM), total nitrogen (TN), total phosphorus (TP), available nitrogen (AN), and available phosphorus (AP) all decrease to different extents in the subsidence area. Additionally, the coal mining subsidence waterlogged area exhibits higher levels compared to the coal mining subsidence non-waterlogged area. Conversely, the soil water content (SWC), C/N ratio, C/P ratio, and N/P ratio all increase to varying degrees. 2. Regarding the composition of the community, the presence of Proteobacteria is considerably greater in the non-water-logged area of coal mining subsidence (NZ, HDZ) compared to the water-logged area and control group (p < 0.05). The prevalence of Firmicutes in the subsidence water area was substantially greater compared to both the subsidence non-waterlogged area and the control group (p < 0.05). The prevalence of Gemmatimonadota is markedly greater in the waterlogged area of mining subsidence compared to the non-waterlogged area and CK (p < 0.05). The Ascomycota population reached its highest value in the neutral zone (NZ), which was significantly greater than the values observed in the seasonal subsidence ponding (SSP) and perennial subsidence ponding (PSP) regions (p < 0.05). On the other hand, the Rozellomycota population had its highest value in the SSP region, which was significantly greater than the values observed in the other regions (p < 0.05). 3. The abundance and variety of soil bacteria and fungi, as well as their important populations, are associated with different levels of soil characteristics. The primary elements that influence the alteration of microbial communities are soil nutrients and soil water content. The presence of coal mine subsidence and water accumulation has a notable impact on the properties of the soil in the surrounding area. This study offers a scientific foundation for reclaiming land affected by subsidence caused by coal mining in regions where coal and grain production are the dominant industries. [ABSTRACT FROM AUTHOR]

The objective of the present study was to find Single Nucleotide Polymorphisms (SNPs) in ESR1 and ESR2 genes and examine their association with egg production in chickens up to 64 weeks of age (EN64). Blood samples were taken from 48 Anand Synthetic White Leghorn (ASWLH) and 48 Anand Bantamised White Leghorn (ABWLH) chickens. Amplicon sequencing was carried out using the IlluminaMiseq platform, and a custom panel was developed to cover the exon regions of these genes. Total 91 SNPs, 59 previously reported and 32 novel SNPs were identified from 96 samples. In conclusion, the genetic variants g.52944897 T>C and rs3181386 inside the ESR2 gene exhibit potential as selection markers linked to EN64 in chickens. [ABSTRACT FROM AUTHOR]

The rhizosphere is the hotspot for microbial enzyme activities and contributes to carbon cycling. Precipitation is an important component of global climate change that can profoundly alter belowground microbial communities. However, the impact of precipitation on conifer rhizospheric microbial populations has not been investigated in detail. In the present study, using high-throughput amplicon sequencing, we investigated the impact of precipitation on the rhizospheric soil microbial communities in two Norway Spruce clonal seed orchards, Lipová Lhota (L-site) and Prenet (P-site). P-site has received nearly double the precipitation than L-site for the last three decades. P-site documented higher soil water content with a significantly higher abundance of Aluminium (Al), Iron (Fe), Phosphorous (P), and Sulphur (S) than L-site. Rhizospheric soil metabolite profiling revealed an increased abundance of acids, carbohydrates, fatty acids, and alcohols in P-site. There was variance in the relative abundance of distinct microbiomes between the sites. A higher abundance of Proteobacteria, Acidobacteriota, Ascomycota, and Mortiellomycota was observed in P-site receiving high precipitation, while Bacteroidota, Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadota, and Basidiomycota were prevalent in L-site. The higher clustering coefficient of the microbial network in P-site suggested that the microbial community structure is highly interconnected and tends to cluster closely. The current study unveils the impact of precipitation variations on the spruce rhizospheric microbial association and opens new avenues for understanding the impact of global change on conifer rizospheric microbial associations. [ABSTRACT FROM AUTHOR]

Burkholderia (sensu lato) is a diverse group of β-Proteobacteria that exists worldwide in various environments. The SBE clade of this group was thought to be mutualistic with stinkbugs. Riptortus–Burkholderia was suggested as an ideal model system for studying insect–microbe symbiosis. To explore the strain-level diversity of Burkholderia at the individual and population levels of Riptortus stinkbugs (Hemiptera: Alydidae), and to uncover the factors affecting the Burkholderia community, large-scale sampling of two Riptortus species and deep sequencing data (16S amplicon) were used in the present study. Our results showed that: (1) the proportions of facultative symbiotic bacteria Burkholderia were very high, with an average proportion of 87.1% in the samples; (2) only six out of 1373 Burkholderia amplicon sequence variants (ASVs) did not belong to the SBE clade, accounting for only 0.03% of Burkholderia; (3) a relatively small number of Burkholderia ASVs had a large number of sequences, with 22, 54, and 107 ASVs accounting for more than 1.0%, 0.1%, and 0.01% of the total Burkholderia sequences, respectively; (4) multiple Burkholderia ASVs were present in most Riptortus individuals, but there was one dominant or two codominant ASVs, and codominance was more likely to occur when the genetic distance between the two codominant ASVs was small; and (5) the beta diversity of Burkholderia was significantly different between the two host species (PerMANOVA: both Jaccard and Bray–Curtis, p < 0.001) and among localities (PerMANOVA: both Jaccard and Bray–Curtis, p < 0.001). Two-way PerMANOVA also indicated that both the host (Bray–Curtis, p = 0.020; Jaccard, p = 0.001) and geographical location (Bray–Curtis, p = 0.041; Jaccard, p = 0.045) influence Burkholderia communities; furthermore, Mantel tests showed that the Burkholderia communities were significantly correlated with the geographical distance of sample locations (R = 0.056, p = 0.001). Together, our findings demonstrate the fine-scale diversity of Burkholderia symbionts and suggest a region- and host-dependent pattern of Burkholderia in Riptortus stinkbugs. [ABSTRACT FROM AUTHOR]

Plant-associated microbial communities shift in composition as a result of environmental perturbations, such as drought. It has been shown that Actinobacteria are enriched in plant roots and rhizospheres during drought stress. However, the correlations between microbiome dynamics and plant response to drought are poorly understood. Here we apply a combination of bacterial community composition analysis and plant metabolite profiling in Sorghum bicolor roots, rhizospheres, and soil during drought and drought recovery to investigate potential contributions of host metabolism to shifts in bacterial composition. Our results provide a detailed view of metabolic shifts across the plant root during drought and show that the response to rewatering differs between root and soil; additionally, we identify drought-responsive metabolites that are highly correlated with the observed changes in Actinobacteria abundance. Furthermore, we find that pipecolic acid is a drought-enriched metabolite in sorghum roots, and that exogenous application of pipecolic acid inhibits root growth. Finally, we show that this activity functions independent of the systemic acquired resistance pathway and has the potential to impact Actinobacterial taxa within the root microbiome.

Abstract Background Cereal diseases caused by insect-transmitted viruses are challenging to forecast and control because of their intermittent outbreak patterns, which are usually attributed to increased population densities of vector insects due to cereal crop rotations and indiscriminate use of pesticides, and lack of resistance in commercial varieties. Root microbiomes are known to significantly affect plant health, but there are significant knowledge gaps concerning epidemics of cereal virus diseases at the microbiome-wide scale under a variety of environmental and biological factors. Results Here, we characterize the diversity and composition of rice (Oryza sativa) root-associated bacterial communities after infection by an insect-transmitted reovirus, rice black-streaked dwarf virus (RBSDV, genus Fijivirus, family Spinareoviridae), by sequencing the bacterial 16S rRNA gene amplified fragments from 1240 samples collected at a consecutive 3-year field experiment. The disease incidences gradually decreased from 2017 to 2019 in both Langfang (LF) and Kaifeng (KF). BRSDV infection significantly impacted the bacterial community in the rice rhizosphere, but this effect was highly susceptible to both the rice-intrinsic and external conditions. A greater correlation between the bacterial community in the rice rhizosphere and those in the root endosphere was found after virus infection, implying a potential relationship between the rice-intrinsic conditions and the rhizosphere bacterial community. The discrepant metabolites in rhizosphere soil were strongly and significantly correlated with the variation of rhizosphere bacterial communities. Glycerophosphates, amino acids, steroid esters, and triterpenoids were the metabolites most closely associated with the bacterial communities, and they mainly linked to the taxa of Proteobacteria, especially Rhodocyclaceae, Burkholderiaceae, and Xanthomonadales. In addition, the greenhouse pot experiments demonstrated that bulk soil microbiota significantly influenced the rhizosphere and endosphere communities and also regulated the RBSDV-mediated variation of rhizosphere bacterial communities. Conclusions Overall, this study reveals unprecedented spatiotemporal dynamics in rhizosphere bacterial communities triggered by RBSDV infection with potential implications for disease intermittent outbreaks. The finding has promising implications for future studies exploring virus-mediated plant-microbiome interactions. Video Abstract

Abstract The deterioration of fibre-reinforced sprayed concrete was studied in the Oslofjord subsea tunnel (Norway). At sites with intrusion of saline groundwater resulting in biofilm growth, the concrete exhibited significant concrete deterioration and steel fibre corrosion. Using amplicon sequencing and shotgun metagenomics, the microbial taxa and surveyed potential microbial mechanisms of concrete degradation at two sites over five years were identified. The concrete beneath the biofilm was investigated with polarised light microscopy, scanning electron microscopy and X-ray diffraction. The oxic environment in the tunnel favoured aerobic oxidation processes in nitrogen, sulfur and metal biogeochemical cycling as evidenced by large abundances of metagenome-assembled genomes (MAGs) with potential for oxidation of nitrogen, sulfur, manganese and iron, observed mild acidification of the concrete, and the presence of manganese- and iron oxides. These results suggest that autotrophic microbial populations involved in the cycling of several elements contributed to the corrosion of steel fibres and acidification causing concrete deterioration.

Abstract Background Clustering of sequences into operational taxonomic units (OTUs) and denoising methods are a mainstream stopgap to taxonomically classifying large numbers of 16S rRNA gene sequences. Environment-specific reference databases generally yield optimal taxonomic assignment. Results We developed SpeciateIT, a novel taxonomic classification tool which rapidly and accurately classifies individual amplicon sequences ( https://github.com/Ravel-Laboratory/speciateIT ). We also present vSpeciateDB, a custom reference database for the taxonomic classification of 16S rRNA gene amplicon sequences from vaginal microbiota. We show that SpeciateIT requires minimal computational resources relative to other algorithms and, when combined with vSpeciateDB, affords accurate species level classification in an environment-specific manner. Conclusions Herein, two resources with new and practical importance are described. The novel classification algorithm, SpeciateIT, is based on 7th order Markov chain models and allows for fast and accurate per-sequence taxonomic assignments (as little as 10 min for 107 sequences). vSpeciateDB, a meticulously tailored reference database, stands as a vital and pragmatic contribution. Its significance lies in the superiority of this environment-specific database to provide more species-resolution over its universal counterparts.

To understand pathogenic mechanisms and suggest molecular biomarker candidates for the disease status of edible red algae, Pyropia, we analyzed relationships between disease symptom severity, changes in microbial communities, and the metabolic dysregulation of the red algae with 16S and 18S ribosomal RNA gene amplicon sequencing and mass spectrometry-based metabolomics. Our observations of disease symptoms and microbial communities indicated that Pythium was the major pathogen. As the disease progressed, the prokaryotic diversity and composition of the microbial communities were clearly distinct from diseased thalli compared with healthy and weakly diseased thalli. The results of the microbial correlation network analysis showed the co-occurrence of known pathogenic microbes in the diseased thalli. Similarly, the results of the metabolomic analysis showed that the relative abundance of annotated metabolites differed in the diseased thalli compared with the healthy and weakly diseased thalli. In particular, three purine metabolism-related metabolites—hypoxanthine, guanine, and inosine—increased significantly in the diseased thalli. All the affected metabolites in abundance regulation, including the three metabolites, are known to be related to biotic stress and defense mechanisms. This study contributes to filling the gaps in research on molecular and microbial events, especially the links between pathogenic and non-pathogenic microbes, metabolites, and symptom severity in Pyropia. Additionally, the results indicate that the observed prokaryotic diversity reduction and co-occurrence of the known microbial pathogens are new non-target indicators that could be beneficial in cases where no known pathogens indicate pathogenicity. Dysregulated purine metabolism-related metabolites and other stress- and defense-related metabolites are potential molecular indicators of disease status of Pyropia. [ABSTRACT FROM AUTHOR]

Fungal spores are common airborne allergens, and fungal richness has been implicated in allergic disease. Amplicon sequencing of environmental DNA from air samples is a promising method to estimate fungal spore richness with semi-quantification of hundreds of taxa and can be combined with quantitative PCR to derive abundance estimates. However, it remains unclear how the choice of air sampling method influences these estimates. This study compared active sampling with a portable impactor and passive sampling with a passive trap over different durations to estimate fungal spore richness and the abundance of allergenic taxa. Air sampling was conducted indoors and outdoors at 12 residences, including repeated measurements with a portable impactor and passive traps with 1-day and 7-day durations. ITS2 amplicon sequence data were transformed to spore equivalents estimated by quantitative PCR, repeated active samples were combined, and abundance-based rarefaction was performed to standardize sample coverage for estimation of genus-level richness and spore abundance. Rarefied fungal richness was similar between methods indoors but higher for passive traps with a 7-day duration outdoors. Rarefied abundance of allergenic genera was similar between methods but some genera had lower abundance for passive traps with a 1-day duration, which differed indoors and outdoors indicating stochasticity in the collection of spores on collocated samplers. This study found that similar estimates of fungal spore richness and abundance of allergenic taxa can be obtained using a portable impactor or a passive trap within one day and that increased passive sample duration provides limited additional information. [ABSTRACT FROM AUTHOR]

Microbial endophytes live asymptomatically within a host plant and contribute positively to its fitness. Endophytes associate ubiquitously with all plants examined so far and form an integral part of the host for processes such as nutrient uptake and stress response and traits like disease resistance. Well-characterized host–endophyte interaction models exist, but little is known about the microbiome of the monocot plant host, palm. In this study, we identified the endophytic fungal community associated with mule palm leaves with an objective to test their antagonist effects on lethal pathogens of palms. The leaflets of mule palms were sampled across five sites in Central and South Florida, and fungal endophytes were identified using culture-dependent (CD) and amplicon sequence-based culture-independent (CI) methods. Operational taxonomic units (OTUs) corresponding to phylum Ascomycota were identified by both approaches, whereas Basidiomycota were only detected using the CI method. Among the ascomycetes, a small (22%) overlap in the OTUs at the genus level was observed between the two methods, whereas the majority (64%) of OTUs were detected using the CI approach. In vitro cocultivation of endophytes with palm pathogens was observed to produce three distinct interaction phenotypes: inhibition zone, niche establishment, and overlapping growth. Eight endophytes capable of inhibiting fungal pathogen growth in vitro were also identified, suggesting a biocontrol potential for endophyte-derived secondary metabolites. A better understanding of the composition, assembly, and ecology of palm-associated microbial communities would eventually lead to healthy and resilient palms. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Resource use and diet specialisation of Madagascan dung beetles have been little studied especially concerning the possible associations between specific dung beetle and lemur species. Pilot studies have demonstrated that amplicon sequencing is a promising tool for the lemur inventories. In the present contribution, we report the results of the gut content analysis of three endemic Madagascan dung beetles species: Helictopleurus clouei (Harold), Epilissus apotolamproides (Lebis) and Nanos dubitatus (Lebis). Amplicon metagenomics revealed trophic associations of these species with Eulemur sanfordi (Archbold), Eu. fulvus (É.Geoffroy Saint-Hilaire) and Cheirogaleus crossleyi (Grandidier), respectively. The reads of other mammal species, revealed by the analysis, including putative contaminations, are discussed.

Abstract The colonization of alien plants in new habitats is typically facilitated by microorganisms present in the soil environment. However, the diversity and structure of the archaeal, bacterial, and fungal communities in the latitudinal spread of alien plants remain unclear. In this study, the rhizosphere and bulk soil of Chromolaena odorata were collected from five latitudes in Pu’ er city, Yunnan Province, followed by amplicon sequencing of the soil archaeal, bacterial, and fungal communities. Alpha and beta diversity results revealed that the richness indices and the structures of the archaeal, bacterial, and fungal communities significantly differed along the latitudinal gradient. Additionally, significant differences were observed in the bacterial Shannon index, as well as in the structures of the bacterial and fungal communities between the rhizosphere and bulk soils. Due to the small spatial scale, trends of latitudinal variation in the archaeal, bacterial, and fungal communities were not pronounced. Total potassium, total phosphorus, available nitrogen, available potassium and total nitrogen were the important driving factors affecting the soil microbial community structure. Compared with those in bulk soil, co-occurrence networks in rhizosphere microbial networks presented lower complexity but greater modularity and positive connections. Among the main functional fungi, arbuscular mycorrhizae and soil saprotrophs were more abundant in the bulk soil. The significant differences in the soil microbes between rhizosphere and bulk soils further underscore the impact of C. odorata invasion on soil environments. The significant differences in the soil microbiota along latitudinal gradients, along with specific driving factors, demonstrate distinct nutrient preferences among archaea, bacteria, and fungi and indicate complex microbial responses to soil nutrient elements following the invasion of C. odorata.

Peanuts (Arachis hypogaea) are of significant agricultural importance due to their versatile uses, providing a valuable source of edible oil and protein-rich food products and serving in industrial applications. Peanuts are susceptible to various diseases, one of them being net syndrome, a poorly understood threat triggered by an undefined causal agent, with symptoms including black coloring and lesions of the peanut pod that affect product quality. In this study, amplicon sequencing was harnessed to explore the fungal and bacterial populations associated with the syndrome. Using this approach, we identified a specific Fusarium sp. amplicon sequence variant that demonstrated a significant correlation with the presence of net syndrome symptoms in both young and mature peanuts. This finding was further validated by Koch's postulates testing, including isolation, cultivation, and application of this strain to corroborate its involvement in net syndrome in peanuts. Our findings contribute to a better understanding of the interactions between peanuts and their microbial communities and to identification of the previously undetermined agent involved in net syndrome and the etiology of the disease.

Tumorigenic agrobacteria are widespread plant pathogens causing crown gall and cane gall diseases in various agricultural crops. These pathogens genetically transform the host plant and thus form an ecological niche (galls) in which specific metabolites (i.e., opines) are produced. Opines provide the pathogen with multiple competitive advantages, but they can also be utilized by other bacteria colonizing galls. To gain a thorough understanding of disease processes and ecology, it is necessary to consider the pathogen in the context of its microbial environment within the diseased plant (i.e., the pathobiome). Therefore, in this study, we investigated the bacterial pathobiome associated with aerial crown gall tumors (gallobiome) on rhododendron. For this purpose, a combination of cultivation-dependent and cultivation-independent approaches were applied, which also involved development of a novel amplicon sequencing approach targeting the recA housekeeping gene. The 16S rRNA and recA gene amplicon sequencing clearly indicated that Rhizobium rhododendri and the group of Agrobacterium spp. primarily belonging to the so-called “rubi” clade were the dominant members of bacterial microbiota in rhododendron galls. While the tumor-inducing (Ti) plasmid-harboring R. rhododendri strains are causative agents of crown gall disease, Agrobacterium spp. strains isolated in this study were nonpathogenic and carried genes for the catabolism of opines, enabling these bacteria to efficiently colonize tumor tissue. Taken together, our results clearly showed that the tumorigenic R. rhododendri and nonpathogenic opine-catabolizing Agrobacterium spp. were the key players within the bacterial microbiota associated with aerial crown gall tumors on rhododendron. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Abstract Cryoconites are the deposits on the surface of glaciers that create specific ecological niches for the development of microorganism communities. The sediment material can vary in origin, structure, and nutrient content, creating local variations in the growth conditions. An additional factor of variability is the location of the glaciers, as they are found in different climatic zones in the high mountain regions and closer to the poles. Here, using the analysis of amplicon sequencing of the 16S rRNA gene, we studied the taxonomic composition of the prokaryotic communities from glaciers from remote regions, including the Arctic (Mushketova on the Severnaya Zemlya, IGAN in Polar Ural), Antarctic (Pimpirev on the Livingstone Island) and Central Caucasus (Skhelda and Garabashi) and connected it with the variation of the physicochemical characteristics of the substrate: pH, carbon, nitrogen, macro- and microelements. The cryoconite microbiomes were comprised of specific for this environment phyla (mostly Pseudomonadota, Cyanobacteria, Bacteroidota, Acidobacteriota, and Actinobacteriota), but each glacier had a unique taxonomic imprint. The core microbiome between regions was composed of only a few ASVs, among which the most likely globally distributed ones attributed to Polaromonas sp., Rhodoferax sp., Cryobacterium sp., and Hymenobacter frigidus. The WGSNA defined clusters of co-occurring ASVs between microbiomes, that significantly change their abundance corresponding with the variation of chemical parameters of cryoconites, but do not fully coincide with their regional separation. Thus, our work demonstrates that the chemical characteristics of the sediment material can explain the variation in the cryoconite prokaryotic community which is not always linked to geographic isolation.

Abstract Background The rhizosphere microbiome displays structural and functional dynamism driven by plant, microbial, and environmental factors. While such plasticity is a well-evidenced determinant of host health, individual and community-level microbial activity within the rhizosphere remain poorly understood, due in part to the insufficient taxonomic resolution achieved through traditional marker gene amplicon sequencing. This limitation necessitates more advanced approaches (e.g., long-read sequencing) to derive ecological inferences with practical application. To this end, the present study coupled synthetic long-read technology with avidity sequencing to investigate eukaryotic and prokaryotic microbiome dynamics within the soybean (Glycine max) rhizosphere under field conditions. Results Synthetic long-read sequencing permitted de novo reconstruction of the entire 18S-ITS1-ITS2 region of the eukaryotic rRNA operon as well as all nine hypervariable regions of the 16S rRNA gene. All full-length, mapped eukaryotic amplicon sequence variants displayed genus-level classification, and 44.77% achieved species-level classification. The resultant eukaryotic microbiome encompassed five kingdoms (19 genera) of protists in addition to fungi – a depth unattainable with conventional short-read methods. In the prokaryotic fraction, every full-length, mapped amplicon sequence variant was resolved at the species level, and 23.13% at the strain level. Thirteen species of Bradyrhizobium were thereby distinguished in the prokaryotic microbiome, with strain-level identification of the two Bradyrhizobium species most reported to nodulate soybean. Moreover, the applied methodology delineated structural and compositional dynamism in response to experimental parameters (i.e., growth stage, cultivar, and biostimulant application), unveiled a saprotroph-rich core microbiome, provided empirical evidence for host selection of mutualistic taxa, and identified key microbial co-occurrence network members likely associated with edaphic and agronomic properties. Conclusions This study is the first to combine synthetic long-read technology and avidity sequencing to profile both eukaryotic and prokaryotic fractions of a plant-associated microbiome. Findings herein provide an unparalleled taxonomic resolution of the soybean rhizosphere microbiota and represent significant biological and technological advancements in crop microbiome research.

Abstract Background Seed endophytes have a significant impact on plant health and fitness. They can be inherited and passed on to the next plant generation. However, the impact of breeding on their composition in seeds is less understood. Here, we studied the indigenous seed microbiome of a recently domesticated perennial grain crop (Intermediate wheatgrass, Thinopyrum intermedium L.) that promises great potential for harnessing microorganisms to enhance crop performance by a multiphasic approach, including amplicon and strain libraries, as well as molecular and physiological assays. Results Intermediate wheatgrass seeds harvested from four field sites in Europe over three consecutive years were dominated by Proteobacteria (88%), followed by Firmicutes (10%). Pantoea was the most abundant genus and Pantoea agglomerans was identified as the only core taxon present in all samples. While bacterial diversity and species richness were similar across all accessions, the relative abundance varied especially in terms of low abundant and rare taxa. Seeds from four different breeding cycles (TLI C3, C5, C704, C801) showed significant differences in bacterial community composition and abundance. We found a decrease in the relative abundance of the functional genes nirK and nifH as well as a drop in bacterial diversity and richness. This was associated with a loss of amplicon sequence variants (ASVs) in Actinobacteria, Alphaproteobacteria, and Bacilli, which could be partially compensated in offspring seeds, which have been cultivated at a new site. Interestingly, only a subset assigned to potentially beneficial bacteria, e.g. Pantoea, Kosakonia, and Pseudomonas, was transmitted to the next plant generation or shared with offspring seeds. Conclusion Overall, this study advances our understanding of the assembly and transmission of endophytic seed microorganisms in perennial intermediate wheatgrass and highlights the importance of considering the plant microbiome in future breeding programs.

ABSTRACT: Notoginseng Radix et Rhizoma (Sanqi in Chinese) is a precious traditional Chinese herbal medicine. It has the effect of dispersing blood stasis and stopping bleeding, reducing swelling and fixing pain. However, it tends to contaminate with harmful fungi during storage, which may make it much less effective. In order to understand the fungal contamination of Notoginseng Radix et Rhizoma and master its composition of the exogenous fungi. The surface fungi of Notoginseng Radix et Rhizoma samples collected from six Chinese provinces and districts were investigated by using dilution plate method. Detection of aflatoxins by UPLC-MS/MS. The results showed that Penicillium citrinum was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.1 to No.4. Aspergillus flavus, which produces aflatoxin, was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.5 and No.6. In addition, kinds of mycotoxin were assayed which were produced by three of those identified A. flavus. All three fungi strains produced aflatoxin B1 (AFB1) and one strain HBSQ1-5 additionally produced other three kinds of mycotoxin, AFB2, AFG1 and AFG2. It is the results implied that it will be very important to take serious cautions when using Notoginseng Radix et Rhizoma. As well as, understanding the composition of the exogenous fungi of Notoginseng Radix et Rhizoma and the strains of toxin-producing fungi, which can play an important role in guiding the storage of Notoginseng Radix et Rhizoma..

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Simple Summary: Inflammatory bowel disease (IBD) has become more common among humans and awareness of its prevalence in animals, including horses, has also increased during the past few decades. Equine IBD is problematic as it decreases the performance of horses and causes diarrhea, weight loss, and pain-related behaviors. Apart from high levels of uncertainty in diagnosing IBD, it is stressful for the horse and expensive for the owner as it typically requires transportation to the horse hospital, fasting, endoscopy, and biopsy assessment. Therefore, there is a need for a non-invasive and affordable method to screen for IBD. Fecal microbiota composition has been shown to change in humans and other animals with IBD; therefore, we used fecal samples to study whether equine IBD is also reflected by changes in the composition of gut microbiota. We show that gut microbiota composition was different and that the abundances of certain bacterial groups were significantly increased or decreased in horses with IBD compared to healthy horses. We further used a machine learning model to predict IBD based on the microbiota composition of the fecal samples. The model produced correct predictions with 100% accuracy in our dataset. We conclude that, in the future, studying the microbiota composition of fecal samples could become an accurate, cheap, and non-invasive method in screening for equine IBD. Gastrointestinal disorders are common and debilitating in horses, but their diagnosis is often difficult and invasive. Fecal samples offer a non-invasive alternative to assessing the gastrointestinal health of horses by providing information about the gut microbiota and inflammation. In this study, we used 16S sequencing to compare the fecal bacterial diversity and composition of 27 healthy horses and 49 horses diagnosed with inflammatory bowel disease (IBD). We also measured fecal calprotectin concentration, a marker of intestinal inflammation, in healthy horses and horses with IBD. We found that microbiota composition differed between healthy horses and horses with IBD, although less than five percent of the variation in microbiota composition was explained by individual health status and age. Several differentially abundant bacterial taxa associated with IBD, age, or body condition were depleted from the most dominant Firmicutes phylum and enriched with the Bacteroidota phylum. An artificial neural network model predicted the probability of IBD among the test samples with 100% accuracy. Our study is the first to demonstrate the association between gut microbiota composition and chronic forms of IBD in horses and highlights the potential of using fecal samples as a non-invasive source of biomarkers for equine IBD. [ABSTRACT FROM AUTHOR]

The colonization of alien plants in new habitats is typically facilitated by microorganisms present in the soil environment. However, the diversity and structure of the archaeal, bacterial, and fungal communities in the latitudinal spread of alien plants remain unclear. In this study, the rhizosphere and bulk soil of Chromolaena odorata were collected from five latitudes in Pu' er city, Yunnan Province, followed by amplicon sequencing of the soil archaeal, bacterial, and fungal communities. Alpha and beta diversity results revealed that the richness indices and the structures of the archaeal, bacterial, and fungal communities significantly differed along the latitudinal gradient. Additionally, significant differences were observed in the bacterial Shannon index, as well as in the structures of the bacterial and fungal communities between the rhizosphere and bulk soils. Due to the small spatial scale, trends of latitudinal variation in the archaeal, bacterial, and fungal communities were not pronounced. Total potassium, total phosphorus, available nitrogen, available potassium and total nitrogen were the important driving factors affecting the soil microbial community structure. Compared with those in bulk soil, co-occurrence networks in rhizosphere microbial networks presented lower complexity but greater modularity and positive connections. Among the main functional fungi, arbuscular mycorrhizae and soil saprotrophs were more abundant in the bulk soil. The significant differences in the soil microbes between rhizosphere and bulk soils further underscore the impact of C. odorata invasion on soil environments. The significant differences in the soil microbiota along latitudinal gradients, along with specific driving factors, demonstrate distinct nutrient preferences among archaea, bacteria, and fungi and indicate complex microbial responses to soil nutrient elements following the invasion of C. odorata. [ABSTRACT FROM AUTHOR]

Microbiome assembly critically impacts the ability of hosts to access beneficial symbiont functions. Fungus-farming termites have co-evolved with a fungal cultivar as a primary food source and complex gut microbiomes, which collectively perform complementary degradation of plant biomass. A large subset of the bacterial community residing within termite guts are inherited (vertically transmitted) from parental colonies, while the fungal symbiont is, in most termite species, acquired from the environment (horizontally transmitted). It has remained unknown how the gut microbiota sustains incipient colonies prior to the acquisition of the fungal cultivar, and how, if at all, bacterial contributions are modulated by fungus garden establishment. Here, we test the latter by determining the composition and predicted functions of the gut microbiome using metabarcoding and shotgun metagenomics, respectively. We focus our functional predictions on bacterial carbohydrate-active enzyme and nitrogen cycling genes and verify compositional patterns of the former through enzyme activity assays. Our findings reveal that the vast majority of microbial functions are encoded in the inherited microbiome, and that the establishment of fungal gardens incurs only minor modulations of predicted bacterial capacities for carbohydrate and nitrogen metabolism. While we cannot rule out that other symbiont functions are gained post-fungus garden establishment, our findings suggest that fungus-farming termite hosts are equipped with a near-complete set of gut microbiome functions at the earliest stages of colony life. This inherited, incipient bacterial microbiome likely contributes to the high extent of functional specificity and coevolution observed between termite hosts, gut microbiomes, and the fungal cultivar. [ABSTRACT FROM AUTHOR]

The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread. [ABSTRACT FROM AUTHOR]

Improving the understanding of the causes and effects of anthropogenic hybridization is fundamental to ensure species conservation, particularly in the case of hybridization between wild species and their domestic relatives. Knowledge is missing for many species also because of a lack of appropriate tools for hybrid identification. Here, coupling genotype and phenotype analysis, we carried out an extensive investigation of ongoing hybridization in Alpine ibex Capra ibex, a mountain ungulate of conservation concern from a genetic perspective. By genotyping 63 diagnostic and 465 neutral SNPs, 20 suspected hybrids and 126 Alpine ibex without suspicious phenotype, representing 8 populations across a major part of the species distribution, we found evidence for ongoing hybridization between Alpine ibex and domestic goat. We identified different levels of hybridization including backcrosses into both Alpine ibex and domestic goat. Our results suggest a lack of reproductive barriers between the two species and good survival and reproductive success of the hybrids. Hybridization was locally intense, like a hybrid swarm, but not spread across the rest of the species distribution. Most of the hybrids were discovered in two locations in the north‐west of Italy, while random sampling of individuals from different areas did not provide evidence of recent hybridization. Our method, based on amplicon sequencing of 63 diagnostic SNPs specifically developed for this purpose, allowed us to identify hybrids and backcrosses up to the fourth to fifth generations and was suitable for genetic samples of different quality, although with varying levels of certainty regarding the exact number of generations passed since hybridization. Based on the paired analysis of genotype and phenotype, we provide guidelines for the first identification of hybrids in the field and suggest a procedure for the reliable identification of hybrids. [ABSTRACT FROM AUTHOR]

Although established biotechnological applications of microalgae e.g., the production of high-value metabolites is based on axenic cultures, exploitation of the mutualistic consortia of microalgae and bacteria quickly comes to foreground, especially in bioremediation and wastewater treatment. This trend shifts the focus from genomic research of certain microalgal species to metagenomic studies of interactions between microalgae and bacteria in natural communities and in artificial consortia. Dissection of the genetic determinants of the robustness and productivity of the consortia become a hot research direction, too. Admirable contribution to this topic had been made by high-throughput sequencing (HTS), while recent breakthrough in this field was entailed by the advent and rapid development of the 3rd generation nanopore sequencing which becomes increasingly accurate while providing unprecedented sequencing performance. Recent progress of the Oxford Nanopore Technologies (ONT) enabled both classical metagenomic analysis of microalgal-bacterial communities based on whole metagenome sequencing as well as taxonomic and genetic profiling based on the amplicon sequencing. The parallel emergence of novel bioinformatic algorithms for processing the metagenomic datasets opened new opportunities for the analysis of structure and physiology of microalgal-bacterial communities. From the practical perspective, the new HTS techniques became a time- and labor-savers in discovery of new microalgae with a high potential for the accumulation of valuable metabolites, biodegradation of hazardous micropollutants, and biosequestration of nutrients from waste streams. Search for prokaryotic species boosting the biotechnological potential of eukaryotic microalgae via mutualistic interactions with them is another important goal. The insights from the both short-read and long-read metagenomics will form a solid foundation for the rational design of microalgal-bacterial consortia for biotechnology. In this review, we briefly outline the benefits of the long-read sequencing for structural and functional investigation of algal-bacterial consortia and summarize recent reports on using this approach for achieving the biotechnology-related goals. [ABSTRACT FROM AUTHOR]

Ramularia leaf spot of barley, caused by Ramularia collo-cygni has become a serious disease threat to barley production. By inducing necrotic lesions, the disease expiates the onset of leaf senescence, which can result in significant grain yield and quality losses. In addition to surviving between seasons in trash, stubble and volunteers, R. collo-cygni can be transmitted from seed to emerging seedlings. As a potential additional means of transmission it may also pose a risk for the spread of specific traits such as fungicide resistance. In the present study, we screened Irish barley seed stocks harvested in the years 2015–2017 for the presence of R. collo-cygni using qPCR, and subsequently using an amplicon sequencing assay screened for the presence of alleles in the SDHC gene of R. collo-cygni associated with SDHI resistance. Over the three year period > 60% of the samples screened had detectable levels of R. collo-cygni present. In a subsample of these five alleles associated with resistance (SDHC—N87S, G91R, H145R, R152M and H153R) were detected. The prevalence of the key alleles C-N87S and C-H146R remained stable over the three year period, and was similar to that detected amongst an isolate collection established in 2022. [ABSTRACT FROM AUTHOR]

Rocky habitats, globally distributed ecosystems, harbour diverse biota, including numerous endemic and endangered species. Vascular plants thriving in these environments face challenging abiotic conditions, requiring diverse morphological and physiological adaptations. Their engagement with the surrounding microbiomes is, however, equally vital for their adaptation, fitness, and long‐term survival. Nevertheless, there remains a lack of understanding surrounding this complex interplay within this fascinating biotic ecosystem. Using microscopic observations and metabarcoding analyses, we examined the fungal abundance and diversity in the root system of the rock‐dwelling West Carpathian endemic shrub, Daphne arbuscula (Thymelaeaceae). We explored the diversification of root‐associated fungal communities in relation to microclimatic variations across the studied sites. We revealed extensive colonization of the Daphne roots by diverse taxonomic fungal groups attributed to different ecological guilds, predominantly plant pathogens, dark septate endophytes (DSE), and arbuscular mycorrhizal fungi (AMF). Notably, differences in taxonomic composition and ecological guilds emerged between colder and warmer microenvironments. Apart from omnipresent AMF, warmer sites exhibited a prevalence of plant pathogens, while colder sites were characterized by a dominance of DSE. This mycobiome diversification, most likely triggered by the environment, suggests that D. arbuscula populations in warmer areas may be more vulnerable to fungal diseases, particularly in the context of global climate change. [ABSTRACT FROM AUTHOR]

Amplicon sequencing is an effective and increasingly applied method for studying viral communities in the environment. Here, we present vAMPirus, a user‐friendly, comprehensive, and versatile DNA and RNA virus amplicon sequence analysis program, designed to support investigators in exploring virus amplicon sequencing data and running informed, reproducible analyses. vAMPirus intakes raw virus amplicon libraries and, by default, performs nucleotide‐ and amino acid‐based analyses to produce results such as sequence abundance information, taxonomic classifications, phylogenies and community diversity metrics. The vAMPirus analytical framework leverages 16 different opensource tools and provides optional approaches that can increase the ratio of biological signal‐to‐noise and thereby reveal patterns that would have otherwise been masked. Here, we validate the vAMPirus analytical framework and illustrate its implementation as a general virus amplicon sequencing workflow by recapitulating findings from two previously published double‐stranded DNA virus datasets. As a case study, we also apply the program to explore the diversity and distribution of a coral reef‐associated RNA virus. vAMPirus is streamlined within Nextflow, offering straightforward scalability, standardization and communication of virus lineage‐specific analyses. The vAMPirus framework is designed to be adaptable; community‐driven analytical standards will continue to be incorporated as the field advances. vAMPirus supports researchers in revealing patterns of virus diversity and population dynamics in nature, while promoting study reproducibility and comparability. [ABSTRACT FROM AUTHOR]

The search for active cellulolytic consortia among soil microorganisms is of significant applied interest, but the dynamics of the formation of such communities remain insufficiently studied. To gain insight into the formation of an active cellulolytic community, the experiment was designed to examine the colonization of a sterile substrate (cellulose) by microorganisms from two soil types: sod-podzolic and chernozem. To achieve this, the substrate was placed in the soil and incubated for six months. To assess microbiome dynamics, the experiment employed sequencing of 16S rRNA gene fragment and ITS2 amplicon libraries at four time points. It was demonstrated that, from the second month of the experiment, the prokaryotic component of the communities reached a state of stability, with a community composition specific to each soil type. The results demonstrated no relationship between changes in community diversity and soil respiration. There also was no significant shift in the community diversity throughout the chronosequence. Furthermore, the taxonomic composition of the community shifted towards a decrease in the proportion of Pseudomonadota and an increase in representatives of the Bacteroidota, Bacillota, and Verrucomicrobiota phyla. The network analysis of the community demonstrated that, in contrast to sod-podzolic soil, chernozem is distinguished by a higher modularity, with the formation of taxon-specific groups of microorganisms at each stage of the chronoseries. These differences are attributed to the alterations in the eukaryotic component of the community, particularly in the prevalence of nematodes and predatory fungi, which in turn influenced the cellulolytic community. [ABSTRACT FROM AUTHOR]