Operational Taxonomic Units Research Articles

Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free

BackgroundDNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads.ResultsWe show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12–15 h of sequencing.ConclusionOur results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Response of soil fungal-community structure and function to land conversion to agriculture in desert grassland.

Land conversion to agriculture is an important factor affecting soil ecological processes in the desert grasslands of northern China. However, soil fungal-community structure and function in response to Land conversion remain unclear. In this study, desert grassland, artificial shrubland, and land conversion were investigated in the western part of the Mu Us Sandland (Yanchi, Ningxia; Dingbian, Shaanxi). We found that land conversion significantly increased soil total carbon, nitrogen, and phosphorus, and available phosphorous and potassium contents. In the early stage of conversion to agricultural (April), soil fungal operational taxonomic units and abundance-based coverage estimator were lower than those of dessert grasslands and shrubland plots and had significant correlations with pH, electric conductivity, and available phosphorus and potassium. The dominant phyla strongly correlated with soil physicochemical properties. Concomitantly, the relative abundance of Glomeromycota was significantly lower, and the complexity of the network in the land conversion plots was lower than that in the shrubland plots. In the late stage of land conversion (September), soil fungal operational taxonomic units and abundance-based coverage estimator were lower in the conversion plots than in the desert grassland plots, with more complex network relationships compared to the desert grassland or shrubland plots. Symbiotrophic groups, a functional group of desert grassland soil fungi, can be used as a predictor of environmental change; in addition, land conversion decreases the relative abundance of arbuscular mycorrhizal functional groups. Our study highlights the response of soil fungal communities and functions to human disturbances in desert grasslands. Considering the potential of land conversion to agriculture to influence soil secondary salinization, there is a need for continued observation of soil ecological health over the time continuum of land conversion to agriculture.

Eyeing DNA barcoding for species identification of fish larvae.

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.

Three-dimensional conservation planning of fish biodiversity metrics to achieve the deep-sea 30×30 conservation target.

Accelerating rate of human impact and environmental change severely affects marine biodiversity and increases the urgency to implement the Convention on Biological Diversity (CBD) 30×30 plan for conserving 30% of sea areas by 2030. However, area-based conservation targets are complex to identify in a 3-dimensional (3D) ocean where deep-sea features such as seamounts have been seldom studied mostly due to challenging methodologies to implement at great depths. Yet, the use of emerging technologies, such as environmental DNA combined with modern modeling frameworks, could help address the problem. We collected environmental DNA, echosounder acoustic, and video data at 15 seamounts and deep island slopes across the Coral Sea. We modeled 7 fish community metrics and the abundances of 45 individual species and molecular operational taxonomic units (MOTUs) in benthic and pelagic waters (down to 600-m deep) with boosted regression trees and generalized joint attribute models to describe biodiversity on seamounts and deep slopes and identify 3D protection solutions for achieving the CBD area target in New Caledonia (1.4millionkm2). We prioritized the identified conservation units in a 3D space, based on various biodiversity targets, to meet the goal of protecting at least 30% of the spatial domain, with a focus on areas with high biodiversity. The relationship between biodiversity protection targets and the spatial area protected by the solution was linear. The scenario protecting 30% of each biodiversity metric preserved almost 30% of the considered spatial domain and accounted for the 3D distribution of biodiversity. Our study paves the way for the use of combined data collection methodologies to improve biodiversity estimates in 3D structured marine environments for the selection of conservation areas and for the use of biodiversity targets to achieve area-based international targets.

Mitochondrial DNA-based reassessment of Antennablennius Fowler (Blenniidae: Salariini) from the north-western Indian Ocean, with resurrection of A. persicus (Regan)

Seven Operational Taxonomic Units within the blenniid genus Antennablennius are recognised from the north-western Indian Ocean, based on the Cytochrome oxidase-c subunit I gene (CO1). Five of them can be referred to nominal species with certainty. Two additional distinct lineages originate from the Persian Gulf and the Gulf of Oman, and the Socotra Archipelago. They were initially, morphologically, identified as A. adenensis Fraser-Brunner, 1951, yet could hitherto not be referred to this species with certainty, requiring further genetic data. Detailed descriptions accompanied by photographs are provided for six species, including A. adenensis. Antennablennius persicus (Regan, 1905) is resurrected from the synonymy of A. bifilum (Günther, 1861) and re-described based on specimens from the Iranian coast of the Persian Gulf and Gulf of Oman, increasing the number of valid species in the genus to eight. The former differs from the latter species in details of morphology and coloration, supported by genetic data. A revised key to all species of the genus is included. A phylogenetic investigation of the genus based on Maximum Likelihood (ML) and Bayesian Inference (BI) analyses of the CO1 data is presented for the first time.

Impact of floods and landslides on rhizosphere bacterial communities: a high-throughput 16S rRNA gene sequencing study.

This study investigates the diversity and composition of soil bacterial communities in the rhizosphere of Attapadi and Nelliyampathy, prominent hill stations in Palakkad district, Kerala, India. The persistent flooding and landslides in 2018 and 2019 significantly impacted agricultural productivity in these regions. Utilizing high-throughput 16S rRNA gene sequencing (Illumina MiSeq), we conducted a comprehensive analysis of soil samples. Correlative assessments between soil parameters and microbial relative abundance at the phylum level revealed noteworthy positive associations. Notably, nitrogen (N) exhibited a positive relation with Crenarchaeota, Chloroflexi, Actinobacteriota, and Acidobacteriota; pH correlated with Firmicutes; organic carbon (OC) with WPS-2; and phosphorous with Proteobacteria. A total of 31,402 operational taxonomic units (OTUs) were identified, with the highest feature counts observed in undisturbed soils from Attapadi (AUD) and Nelliyampathy (NUD) (13,007 and 12,915, respectively). Disturbed soils in Nelliyampathy (ND) and Attapadi (AD) displayed a substantial decline in microbial diversity and composition, harbouring 1409 and 4071 OTUs, respectively. Alpha and beta diversity indices further underscored the more severe impairment of ND soils compared to AD soils. Interestingly, a majority of ND samples were landslide-affected (four out of five), while flood-affected soils accounted for four out of six AD samples. This indicates that landslides exert a more pronounced impact on microbial diversity and composition than floods. The observed decline in microbial count, composition, and diversity, even after 2years of the disaster, raises concerns about potential threats to agricultural output. The findings emphasize the need for corrective measures, including the incorporation of microbial inoculum, to restore soil fertility in post-disaster landscapes.

The Effect of Spatio‐Temporal Sampling and Biological Replication on the Detection of Kelp Forest Fish Communities Using eDNA Metabarcoding

ABSTRACTEnvironmental DNA (eDNA) metabarcoding surveys can support the acquisition of extensive biodiversity data to support ecosystem monitoring and conservation actions. However, the optimization of eDNA metabarcoding project design is essential to capture spatio‐temporal heterogeneity of eDNA signals and maximize diversity detection. In this study, we developed a system‐specific approach to detect fish communities in kelp forests, by analyzing fine‐scale spatio‐temporal patterns in eDNA signals at two sites along the South African coastline, as well as testing the effect of biological replication and pooling of replicates on species detection. At each site, samples were collected at two stations along the shoreline at two depth zones, and this was repeated at two time points (24 h apart). We detected 113 operational taxonomic units (OTUs) across 32 families, but fewer than 20% of OTUs could be assigned to species, indicating that barcode reference libraries need to be drastically improved. We detected significant differences in communities across small spatial scales (< 600 m) and time points, suggesting that to best capture a site's diversity patterns, samples should be collected at multiple points and times within at least 24 h. To detect ~80% of the fish community, including some low abundance species, a minimum of four samples appear sufficient. In addition, a higher number of OTUs (76 vs. 65) were found in individual replicates than in any of the pools. However, pooling samples prior to sequencing can still detect valuable broad‐scale biodiversity patterns for monitoring and can offset the decrease in data resolution with the benefit of accumulating comprehensive data from increased sampling efforts over time. As a pilot investigation into how best to maximize kelp forest‐associated fish communities, this study provides a basis for optimizing sampling design for coastal eDNA‐based surveys in southern Africa and strengthens the development of long‐term eDNA monitoring programs to better support conservation and management actions.

Molecular phylogeny and species diversity of the genus Dichaetophora Duda and related taxa (Diptera: Drosophilidae)

Our intensive surveys of wild drosophilids in East and Southeast Asia discovered a great species diversity (more than 100 putatively new species) of the genus Dichaetophora, which is currently comprised of 67 formally described species assigned into five species groups, i.e., agbo, tenuicauda, acutissima, sinensis and trilobita. In the present study, we delimited species from a huge amount of samples of Dichaetophora and allied taxa (the genus Mulgravea and the subgenus Dudaica of Drosophila) collected from a wide range of the Oriental and east Palearctic regions. We first sorted all specimens into morpho-species, and representative specimen(s) selected from each morpho-species were subjected to barcoding of COI (the cytochrome c oxidase subunit I gene) sequences. The applied ASAP (Assemble Species by Automatic Partitioning) analysis estimated a total of 166 to 168 MOTUs (molecular operational taxonomic units). Integrating this result with morphological evidence from re-examined, detailed structures of male terminalia, we recognized a total of 144 (109 new and 35 known) species in our sample. Out of them, 83 species representing the supraspecific taxa of Dichaetophora, Mulgravea and Dudaica were selected, along with 33 species from major genera and subgenera of Drosophila in the tribe Drosophilini, as in-group and four species from the tribe Colocasiomyini as out-group for phylogenetic reconstruction based on 12 nuclear gene markers. In the trees constructed by the maximum likelihood and Bayesian inference methods, the three focal taxa (i.e., Dichaetophora, Mulgravea and Dudaica) formed a clade provisionally called the “pan-Dichaetophora”. Within this large clade, the agbo, tenuicauda, sinensis and trilobita groups of Dichaetophora, Mulgravea and Dudaica were recovered as monophyletic groups, but Dichaetophora and its acutissima group were regarded as paraphyletic. In addition, two clusters were recognized among ungrouped species of Dichaetophora. Thus, the present study has uncovered some issues concerning the taxonomy of the pan-Dichaetophora. Such issues will be addressed elsewhere in the phylogenetic reclassification of the pan-Dichaetophora, along with descriptions/redescriptions of a large number of new/known species delimited in the present study.

Mother’s milk microbiota is associated with the developing gut microbial consortia in very-low-birth-weight infants

Mother's milk contains diverse bacterial communities, although their impact on microbial colonization in very-low-birth-weight (VLBW, <1,500 g) infants remains unknown. Here, we examine relationships between the microbiota in preterm mother's milk and the VLBW infant gut across initial hospitalization (n= 94 mother-infant dyads, 422 milk-stool pairs). Shared zero-radius operational taxonomic units (zOTUs) between milk-stool pairs account for ∼30%-40% of zOTUs in the VLBW infant's gut. We show dose-response relationships between intakes of several genera from milk and their concentrations in the infant's gut. These relationships and those related to microbial sharing change temporally and are modified by in-hospital feeding practices (especially direct breastfeeding) and maternal-infant antibiotic use. Correlations also exist between milk and stool microbial consortia, suggesting that multiple milk microbes may influence overall gut communities together. These results highlight that the mother's milk microbiota may shape the gut colonization of VLBW infants by delivering specific bacteria and through intricate microbial interactions.

Impacts of Soil Compaction and Phosphorus Levels on the Dynamics of Phosphate-Solubilizing and Nitrogen-Fixing Bacteria in the Peanut Rhizosphere

Soil properties, including soil compaction and the nutrient content, influence the composition and functions of rhizosphere microbial communities. There is limited information on how soil compaction and phosphorus application affect phosphate-solubilizing (PSB) and nitrogen-fixing bacteria (NFB). This study aimed to examine the responses of PSB and NFB in the rhizosphere of peanut (Arachis hypogaea L.) plants under varying soil compaction and phosphorus application levels. To address this, pot experiments were conducted to assess the composition and assembly processes of rhizosphere PSB and NFB in peanut cultivar Hua Yu 22 under two soil compaction levels (T1, 1.25 g/cm3 compaction, and T2, 1.00 g/cm3 compaction) and two phosphorus (P) levels (P0, no P applied, and P1, 1.2 mM P/kg soil applied). The results showed that PSB community shifts were closely correlated with the content of soil available phosphorus, soil acid phosphatase activity, soil nitrogenase activity, and soil compaction. Additionally, the content of soil available phosphorus and soil compaction were correlated with changes in operational taxonomic units of NFB. A network analysis revealed that the complexities of PSB were significantly higher than those of NFB. A stronger negative relationship was identified among NFB communities. The assembly of PSB communities was primarily driven by drift processes, whereas NFB communities were influenced by a combination of homogenizing selection and drift. Both PSB and NFB community compositions were significantly affected by phosphorus limitations and soil compaction. These findings enhance our understanding of the impacts of soil compaction and phosphorus application on PSB and NFB communities, with implications for optimizing peanut crop production. Our results will provide reference for crop cultivation in compacted and low-phosphorus soils. The important phosphate-solubilizing and nitrogen-fixing bacteria screened in the interaction network in this study will become candidate microbial agents for alleviating soil compaction and low phosphorus levels.

Fetal echogenic bowel may be related to intestinal microbiota: A prospective cohort study.

The purpose of the current study was to determine the difference in intestinal microbiota after delivery between healthy fetuses and fetuses with hyperechogenic bowel during the second trimester and the relationship between fetal echogenic bowel and microbiota. Fourteen healthy fetuses (control group), 13 fetuses with echogenic bowel (EB group), and seven fetuses with echogenic bowel and other abnormalities (C-EB group) were selected. The first meconium after delivery was collected for 16S rRNA sequencing. A total of 1 222 131 high-quality sequences were generated after sequencing optimization of all samples. Each sample contained an average of 35 945 high-quality sequences and 2036 operational taxonomic units (OTUs). There was no significant difference in the Shannon, Simpson index among the three groups. At the genus level, the abundance of Escherichia coli/Shigella in the EB and C-EB groups was significantly lower than the control group, while the abundance of Staphylococcus, Methylobactrium, and Curvibacter in the EB group was significantly higher than the other groups. There was a difference in abundance of Gammaproteobacteria, Fusobacteria, Enterobacteriaceae, and E. coli in the EB and C-EB groups. The formation of echogenic bowel may be related to the microbiota.

Changes in the microbial community structure and diversity during the electricity generation process of a microbial fuel cell with algal-film cathode

Abstract A two-chamber microbial fuel cell (MFC) with algal-film cathode was constructed. It showed good electric-generating performance with three electric-generating stages: start-up, development, and stable. An average output voltage reached ~0.412 V during the stable period. A maximum power density during continuous operation was 19.76 mW/m2. Bacterial samples were collected from the anode in the three stages (A1, A2, and A3), and their community structure and diversity were analyzed using Illumina MiSeq high-throughput sequencing technology. A total of 4238 operational taxonomic units were identified based on the number of taxa. At the phylum level, Proteobacteria and Bacteroidetes played a dominant role in the three stages and increased significantly during electricity generation. Compared with A1, the relative abundances of Proteobacteria in A2 and A3 increased by 23.30% and 32.06%, respectively, whereas those of Bacteroidetes in A2 and A3 increased by 5.56% and 14.50%, respectively. At the genus level, there were differences in the composition of bacterial communities among the three stages. Acinetobacter and Chlorobium became the dominant genera in A2, replacing Nitrospira and norank_f__Saprospiraceae in A1, and Sphingobacterium and Ochrobactrum became the dominant genera in A3. According to the sample cluster and principal component analyses, A1 was clustered into one class, and A2 and A3 were clustered into a second class. This work revealed bacterial community succession at the anode of an algal-film cathode MFC during the electricity generation process, which provides a theoretical basis for the subsequent promotion of electricity generation by algal-film cathode MFCs.

Diversity and Structure of Soil Bacterial Communities in the Area of Non-ferrous Metal Plant Revealed by 16S rRNA Gene Retrieval

The goals of the study were to assess the diversity and structure of the bacterial communities within the soil depth along a gradient of heavy metal (HM) contamination and to identify indigenous bacterial species in the agricultural area of a non-ferrous metal plant KCM 2000 Group (Plovdiv) by using 16S rRNA gene retrieval. 16S rRNA gene clone libraries were constructed for nine samples, which were collected from two soil depths in June 2020. From each library, up to 100 clones were analysed and grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP). The representatives of the OTUs were sequenced, followed by phylogenetic analysis. The results revealed that phyla Proteobacteria (11.11–71.43%) and Actinomycetota (3.57–33%) were the most abundant. Surface soils (12 phyla and 15 classes) were more diverse than subsurface ones (7 phyla and 12 classes). The lowest diversity at both phylum and class levels was calculated for the moderately contaminated soils from the two studied depths. Thirteen 16S rDNA sequences were identified to a species level, and they belonged to Proteobacteria, Actinomycetota and Firmicutes. This study highlighted that both HM contamination and soil depth caused shifts in diversity and structure of soil bacterial communities.

Dysbiosis of the gut microbiota in glioblastoma patients and potential biomarkers for risk assessment

BackgroundThe significant death rate of glioblastoma is well-known around the world. The link between gut microbiota and glioma is becoming more studied. The goal of this study was to look at the relationships between intestinal flora and glioblastoma, and to provide a new perspective for the diagnosis as well as treatment of glioblastoma. MethodsFecal samples from 80 participants with glioblastoma (n = 40) and healthy individuals (n = 40) in this study were collected as well as analyzed utilizing 16S rRNA gene amplicon sequencing in order to characterize the gut microbial community. ResultsEach group has its own microbial community, and the microbial environment of glioblastoma patients had lower richness and evenness. The structure of gut microbiota community in glioblastoma patients showed profound changes, which includes the increase of pathogens in Fusobacteria and Bacteroidetes, and the reduction of probiotic bacteria in Firmicutes, Actinobacteria and Verrucomicrobia. Meanwhile, the significant correlations and clustering of OTUS (operational taxonomic units) in glioblastoma patients were discovered, and a biomarker panel (Fusobacterium, Escherichia/Shigella, Ruminococcus gnavus group, Lachnospira, Akkermansia, Parasutterella) had been used to discriminate the patients with glioblastoma from the healthy subjects (AUC: 0.80). Furthermore, the glioblastoma group exhibited multiple disturbed pathways through KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, particularly in genetic information processing. Moreover, the prediction of phenotypic characteristics of microbiome proposed that the glioblastoma patients might have more Gram-negative bacteria and opportunistic pathogens than the healthy controls. ConclusionsWhen compared to healthy people, glioblastoma sufferers have a different host-microbe interaction. Furthermore, certain types of intestinal flora could be regarded as biomarkers and drug targets for the diagnosis as well as treatment of glioblastomas.

Massive expansion of the pig gut virome based on global metagenomic mining

The pig gut virome plays a vital role in the gut microbial ecosystem of pigs. However, a comprehensive understanding of their diversity and a reference database for the virome are currently lacking. To address this gap, we established a Pig Virome Database (PVD) that comprised of 5,566,804 viral contig sequences from 4650 publicly available gut metagenomic samples using a pipeline designated “metav”. By clustering sequences, we identified 48,299 viral operational taxonomic units (vOTUs) genomes of at least medium quality, of which 92.83% of which were not found in existing major databases. The majority of vOTUs were identified as Caudoviricetes (72.21%). The PVD database contained a total of 2,362,631 protein-coding genes across the above medium-quality vOTUs genomes that can be used to explore the functional potential of the pig gut virome. These findings highlight the extensive diversity of viruses in the pig gut and provide a pivotal reference dataset for forthcoming research concerning the pig gut virome.

Soil pathogenic fungal groups and soil nutrient cycling under land use practices in Liupanshan Mountain in China

AbstractDifferent land‐use practices in temperate forests strongly affect soil quality and soil microbial communities, whereas the assembly and interactions of soil functional fungal communities provide positive feedback. Therefore, the effects of forest ecosystem degradation on the composition of functional soil fungal community and soil nutrient cycling are of particular importance. We studied forest ecosystems in the Liupanshan Mountains in the northwestern part of the Loess Plateau and analyzed the relationship of soil fungal community and soil nutrient cycling under different land use practices (natural forest [NF], plantation forest, and farmland [FL]). The results showed that soil pH and electrical conductivity were the highest in FL, whereas the soil carbon cycle index and nitrogen cycle index decreased. The soil total phosphorus content did not change significantly with an increase in available phosphorus content. The change from NF to FL significantly increased the number of operational taxonomic units, diversity, and richness of soil fungal communities. The composition of the soil fungal communities was also strongly influenced by carbon and nitrogen cycle indices. In addition, FL reclamation increased the complexity of the soil microorganism co‐occurrence network, and the interrelationships between soil functional fungal community were enhanced. Pathogenic fungal communities were enriched in FLs, and their relative abundance was significantly regulated by environmental factors such as pH and the ratio of nitrogen to phosphorus. The soil pathogenic fungal community affected carbon and nitrogen cycle indices to varying degrees.

Impact of Lactobacillus- and Bifidobacterium-Based Direct-Fed Microbials on the Performance, Intestinal Morphology, and Fecal Bacterial Populations of Nursery Pigs.

Weaning is a critical stage in the swine production cycle, as young pigs need to adjust to sudden and dramatic changes in their diet and environment. Among the various organ systems affected, the gastrointestinal tract is one of the more severely impacted during this transition. Traditionally, challenges at weaning have been managed by prophylactic use of antibiotics, which not only provides protection against diarrhea and other gut dysfunction but also has growth-promoting effects. With banning or major restrictions on the use of antibiotics for this purpose, various alternative products have been developed as potential replacements, including direct-fed microbials (DFMs) such as probiotics and postbiotics. As their efficiency needs to be improved, a continued effort to gain a deeper understanding of their mechanism of action is necessary. In this context, this report presents a study on the impact of a Lactobacillus-based probiotic (LPr) and a Bifidobacterium-based postbiotic (BPo) when added to the diet during the nursery phase. For animal performance, an effect was observed in the early stages (Day 0 to Day 10), as pigs fed diets supplemented with either DFMs were found to have higher average daily feed intake (ADFI) compared to pigs fed the control diet (p < 0.05). Histological analysis of intestinal morphology on D10 revealed that the ileum of supplemented pigs had a higher villus height/crypt depth ratio (p < 0.05) compared to controls, indicating a benefit of the DFMs for gut health. In an effort to further explore potential mechanisms of action, the effects of the DFMs on gut microbial composition were investigated using fecal microbial communities as a non-invasive representative approach. At the bacterial family level, Lactobacillaceae were found in higher abundance in pigs fed either LPr (D10; p < 0.05) or BPo (D47; p < 0.05). At the Operational Taxonomic Unit (OTU) level, which can be used as a proxy to assess species composition, Ssd-00950 and Ssd-01187 were found in higher abundance in DFM-supplemented pigs on D47 (p < 0.05). Using nucleotide sequence identity, these OTUs were predicted to be putative strains of Congobacterium massiliense and Absicoccus porci, respectively. In contrast, OTU Ssd-00039, which was predicted to be a strain of Streptococcus alactolyticus, was in lower abundance in BPo-supplemented pigs on D47 (p < 0.05). Together, these results indicate that the DFMs tested in this study can impact various aspects of gut function.

Enhanced salt tolerance in Glycyrrhiza uralensis Fisch. via Bacillus subtilis inoculation alters microbial community.

The widespread prevalence of saline environments poses a significant global environmental challenge. Salt stress, induced by saline soils, disrupts soil microecology and affects the plant-microbe-soil cycling process. Utilizing microbial fungicides stands as a primary strategy to mitigate salt stress-induced damage to plants and soils. This study investigated the influence of Bacillus subtilis (Bs) inoculation on the microbial community, assembly processes, and functional changes in bacteria and fungi in Glycyrrhiza uralensis Fisch. (licorice) seedlings under varying salt stress levels, primarily employing microbiomics techniques. Soil enzyme activities displayed a declining trend with increasing salt stress, which was mitigated by Bs inoculation. Microbiome analysis revealed a significant increase in bacterial and fungal operational taxonomic units, particularly in Ascomycetes and Nitrogen-fixing Bacteria, thereby enhancing soil denitrification. The abundance of Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes in bacteria, as well as Ascomycota in fungi, increased with higher salt stress levels, a process facilitated by Bs inoculation. However, functional predictions indicated a reduction in the relative abundance of Dung Saprotrophs with Bs inoculation. Salt stress disrupted soil assembly processes, showcasing a continuous decline in diffusion limitation with increased salt concentration, where Bs inoculation reached a peak under moderate stress. In summary, this research elucidates the communication mechanism of Bs in enhancing salt tolerance in licorice from a microbiome perspective, contributing to a comprehensive understanding of abiotic and biotic factors.IMPORTANCELicorice is a herb that grows in deserts or saline soils. Enhancing the salt tolerance of licorice is necessary to maintain the quality of cultivated licorice and to ensure the supply of medicinal herbs. In the past, we have demonstrated the effectiveness of inoculation with Bacillus subtilis (Bs) to enhance the salt tolerance of licorice and revealed the key metabolic pathways for the development of salt tolerance through multi-omics. In this study, we used the microbiomics approach to reveal the plant-microbe-soil interactions at the level of inoculation of Bs affecting the dynamics of soil microbial communities from bacterial and fungal perspectives, thus bridging the interactions between biotic and abiotic factors.

Structural and Functional Differences in Small Intestinal and Fecal Microbiota: 16S rRNA Gene Investigation in Rats.

To compare the differences in floral composition and functions between the two types of microbiota, ileal contents and feces were collected from Sprague Dawley (SD) rats fed in a conventional or specific-pathogen free (SPF) environment and rats fed a high-fat diet (HFD), and the V3-V4 region of the 16S ribosomal ribonucleic acid (rRNA) gene in these rats was then amplified and sequenced. Compared with feces, about 60% of the bacterial genera in the ileum were exclusive, with low abundance (operational taxonomic units (OTUs) < 1000). Of bacteria shared between the ileum and feces, a few genera were highly abundant (dominant), whereas most had low abundance (less dominant). The dominant bacteria differed between the ileum and feces. Ileal bacteria showed greater β-diversity, and the distance between in-group samples was nearer than that between paired ileum-feces samples. Moreover, the ileum shared various biomarkers and functions with feces (p < 0.05). The HFD and SPF conditions had a profound influence on α-diversity and abundance but not on the exclusive/shared features or β-diversity of samples. The present findings suggested that, under conventional circumstances, fecal bacteria can represent approximately 40% of the low abundant ileal bacterial genera and that dominant fecal bacteria failed to represent the ileal dominant flora. Moreover, fecal flora diversity does not reflect β-diversity in the ileum.

Profiling of the Citrus Leaf Endophytic Mycobiota Reveals Abundant Pathogen-Related Fungal Groups.

Plant endophytic microbial communities consist of many latent plant pathogens and, also, many pathogen-related species with reduced virulence. Though with a long history of co-evolution, the diversity and composition of the endophytic mycobiota, especially the pathogen-related fungal groups, has been under-investigated in Citrus (C.). Based on the amplicon sequencing of fungal internal transcribed spacer (ITS), the leaf endophytic mycobiota were profiled on citrus varieties from different citrus-producing regions. The pomelo variety shared significantly distinctive leaf mycobiota when compared to the mandarin and sweet orange; these conform to their host genetic relationships. In addition, a data set of 241 citrus-related fungi, including 171 (71%) pathogens and potential pathogens, was summarized from previous studies. Under the criteria of local BLAST (covered ITS nucleotide ≥ 150 bp, sequence identity ≥ 99%), a total of 935 fungal operational taxonomic units (OTUs) were assigned to 62 pathogen-related fungal groups, representing 14.9% of the relative abundance in the whole community. Of which, the top groups consisted of Colletotrichum gloeosporioides (mean relative abundance, 4.3%), Co. citricola and Co. karstii (2.7%), Zasmidium citri-griseum (2.4%), and Z. fructigenum (1.4%). At the genus level, the ratio of the pathogen-related fungal groups in 64% of fungal genera (16 out of 25) exceeded 50%, which are the solely or mainly occurring fungi of their genus in citrus. Our study suggests that the leaf endophytic compartment may be an important place for the growth of latent pathogens.