Molecular Profiling on Surface-Disinfected Tomato Seeds Reveals High Diversity of Cultivation-Recalcitrant Endophytic Bacteria with Low Shares of Spore-Forming Firmicutes Abstract Seeds are known to harbor diverse microorganisms offering protective effects on them with the prospects of quick root colonization at germination, selective recruitment as endophytes, and possible vertical transmission. The study was undertaken to assess the gross seed-internal bacterial community in tomato and to confirm if spore-forming Firmicutes constituted major seed endophytes adopting cultivation versus molecular approach on surface-sterilized seeds. Testing the initial seed wash solutions of “Arka Vikas” and “Arka Abha” cultivars showed > 1000 bacterial cfu per dry seed, largely Bacillus spp. Tissue homogenates from surface-disinfected seeds did not show any cultivable bacteria on enriched media for 1–2 weeks, while 16S rRNA V3-V4 taxonomic profiling revealed a huge bacterial diversity (10–16 phyla per cultivar). Proteobacteria formed the dominant phylum (65.7–69.6% OTUs) followed by Firmicutes, Actinobacteria, Bacteroidetes, and a notable share of Euryarchaeota (1.1–3.1%). Five more phyla appeared common to both cultivars in minor shares (Acidobacteria, Planctomycetes, Chloroflexi, Spirochaetes, Verrucomicrobia) with the ten phyla together constituting 99.6–99.9% OTUs. Class level and family level, the cultivars displayed elevated bacterial diversity, but similar taxonomic profiles. Arka Vikas and Arka Abha showed 114 and 107 genera, respectively, with 63 common genera constituting 96–97% OTUs. Psychrobacter formed the dominant genus. Bacillus and related genera constituted only negligible OTU share (0.16–0.28%). KEGG functional analysis showed metabolism as the major bacterial community role. One-month-old in vitro seedlings showed the activation of some originally uncultivable bacteria uninfluenced by the OTU share. The study reveals a high diversity of cultivation-recalcitrant endophytic bacteria prevailing in tomato seeds with possible vertical transmission and significant roles in plant biology.

Hysteretic response of Microbial Eukaryotic Communities to Gradually Decreased Nutrient Concentrations in Eutrophic Water Abstract External environments to microbial eukaryotic communities often change gradually with time. However, whether the responses of microbial eukaryotic communities to these gradually changed environments are continuous or hysteretic and the mechanisms underlying these responses are largely unknown. Here, we used a microcosm to investigate the temporal variation of microbial eukaryotic communities with the gradually decreased nutrient concentrations (nitrogen and phosphorus). We found the differences of microbial eukaryotic community composition and species richness between the control and treatment groups were low during the days 0 to 12, although the nutrient concentrations decreased rapidly during this period in treatment group. However, these differences were clear during the days 14 to 18, although the nutrient concentrations decreased slowly during this period in treatment group. The mechanisms for these results are that the strong homogenous selection (perhaps due to the biotic factors) during the days 8 to 10 in treatment group might enhance the stability of microbial eukaryotic communities. However, the continuously decreased nutrient concentrations weakened the homogenous selection and promoted the strength of environmental filtering, and therefore resulted in the distinct change of microbial eukaryotic communities during the days 14 to 18 in treatment group. Fungi, Chlorophyta and Chrysophyta which associated with the nutrient removal played important roles in this hysteretic change of microbial eukaryotic communities. Overall, our findings suggest that disentangling the non-linear response of communities to gradual environmental changes is essential for understanding ecosystem restoration and degradation in future.

Environment Shapes the Intra-species Diversity of Bacillus subtilis Isolates Abstract Cosmopolitan bacteria are those that are found practically everywhere in the world. One of them is Bacillus subtilis, which can travel around the world through dust storms rising from various deserts. Upon landing, bacterial survival is determined by the ability to adjust to the heterogonous environments and bacteria isolated from extremely different environments, such as desert and riverbank soil, are expected to be less related due to the environmental pressure of each region. However, little is known about the influence of soil and habitat on B. subtilis evolution. Here, we show that desert and riverbank B. subtilis strains differ in genetic relatedness and physiological traits, such as biofilm morphology and utilisation of carbon sources. Desert strains showed more diversity at the genetic level and were able to utilise more carbon sources than riverbank strains which were highly genetically conserved. Biofilm morphologies of desert and riverbank strains generally segregated and both groups formed different morphology clusters despite the astonishing diversity observed among riverbank strains. We also show that relatedness of B. subtilis strains does not decrease with distance inside the same habitat, which, together with diversity data implies that the difference in environmental selection pressures plays a fundamental role in the evolution of this species.

Coordinated Metacommunity Assembly and Spatial Distribution of Multiple Microbial Kingdoms within a Lake Abstract Freshwater planktonic communities comprise a tremendous diversity of microorganisms. This study investigated the distribution patterns of microbial kingdoms (bacteria, fungi, protists, and microbial metazoans) within a lake ecosystem. Water samples were collected from 50 sites along the shoreline in a lake during an early eutrophication period, and MiSeq sequencing was performed with different marker genes. Metacommunity analyses revealed a bimodal occupancy-frequency distribution and a Clementsian gradient persisting throughout all microbial kingdoms, suggesting similar regional processes in all kingdoms. Variation partitioning revealed that environmental characteristics, macrophyte/macroinvertebrate composition, space coordinates, and distance-based Moran’s eigenvector maps (dbMEM) together could explain up to 29% of the community variances in microbial kingdoms. Kingdom synchrony results showed strong couplings between kingdoms (R2 ≥ 0.31), except between Fungi and Metazoa (R2 = 0.09). Another variation partitioning revealed that microbial kingdoms could well explain their community variances up to 73%. Interestingly, the kingdom Protista was best synchronized with the other kingdoms. A correlation network showed that positive associations between kingdoms outnumbered the negative ones and that the kingdom Protista acted as a hub among kingdoms. Module analysis showed that network modules included multi-kingdom associations that were prevalent. Our findings suggest that protists coordinate community assembly and distribution of other kingdoms, and inter-kingdom interactions are a key determinant in shaping their community structures in a freshwater lake.

Uncovering Viable Microbiome in Anaerobic Sludge Digesters by Propidium Monoazide (PMA)-PCR Abstract Use of anaerobic sludge digester is a common practice around the world for solids digestion and methane generation from municipal sewage sludge. Understanding microbial community structure is vital to get better insight into the anaerobic digestion process and to gain better process control. However, selective analysis of viable microorganisms is limited by DNA-based assays. In this study, propidium monoazide (PMA)-PCR with 16S rRNA gene sequencing analysis was used to distinguish live and dead microorganisms based on cell membrane integrity. Microbial community structures of PMA-treated and PMA-untreated anaerobic digester sludge samples were compared. Quantitative PCR revealed that 5–30% of the rRNA genes were derived from inactive or dead cells in anaerobic sludge digesters. This caused a significant decrease in the numbers of operational taxonomic units and Chao1 and Shannon indices compared with that of the PMA-untreated sludge. Microbial community analysis showed that majority of the viable microbiome consisted of Euryarchaeota, Bacteroidetes, Deltaproteobacteria, Chloroflexi, Firmicutes, WWE1, Spirochaetes, Synergistetes, and Caldiserica. On the other hand, after the PMA treatment, numbers of Alphaproteobacteria and Betaproteobacteria declined. These were considered residual microbial members. The network analysis also revealed a relationship among the OTUs belonging to WWE1 and Bacteroidales. PMA-PCR-based 16S rRNA gene sequencing analysis is an effective tool for uncovering viable microbiome in complex environmental samples.

Correction to: Enhanced Growth of Pilin-Deficient Geobacter sulfurreducens Mutants in Carbon Poor and Electron Donor Limiting Conditions The published version of this article contained an old version of Fig. 2.

Effect of Composting Under Semipermeable Film on the Sewage Sludge Virome Abstract The addition of compost from sewage sludge to soils represents a sustainable option from an environmental and economic point of view, which involves the valorisation of these wastes. However, before their use as a soil amendment, compost has to reach the quality levels according to the normative, including microbial parameters. Viruses are not included in this regulation and they can produce agricultural problems and human diseases if the compost is not well sanitised. In this study, we carried out the analysis of the viral populations during a composting process with sewage sludge at an industrial scale, using semipermeable cover technology. Viral community was characterised by the presence of plant viruses and bacteriophages of enteric bacteria. The phytopathogen viruses were the group with the highest relative abundance in the sewage sludge sample and at 70 days of the composting process. The diversity of bacterial viruses and their specificity, with respect to the more abundant bacterial taxa throughout the process, highlights the importance of the interrelations between viral and bacterial communities in the control of pathogenic communities. These results suggest the possibility of using them as a tool to predict the effectiveness of the process.

Small-Scale Spatial Heterogeneity of Photosynthetic Fluorescence Associated with Biological Soil Crust Succession in the Tengger Desert, China Abstract In dryland regions, biological soil crusts (BSCs) have numerous important ecosystem functions. Crust species and functions are, however, highly spatially heterogeneous and remain poorly understood at a range of scales. In this study, chlorophyll fluorescence imaging was used to quantify millimeter-scale patterns in the distribution and activity of photosynthetic organisms in BSCs of different successional stages (including cyanobacterial, lichen, moss three main successional stages and three intermixed transitional stages) from the Tengger Desert, China. Chlorophyll fluorescence images derived from the Imaging PAM (Pulse Amplitude Modulation) showed that with the succession from cyanobacterial to lichen and to moss crusts, crust photosynthetic efficiency (including the maximum and effective photosynthetic efficiency, respectively) and fluorescence coverage increased significantly (P < 0.05), and that increasing photosynthetically active radiation (PAR) reduced the effective photosynthetic efficiency (Yield). The distribution of photosynthetic organisms in crusts determined Fv/Fm (ratio of variable fluorescence to maximum fluorescence) frequency pattern, although the photosynthetic heterogeneity (SHI index) was not significantly different (P > 0.05) between cyanobacterial and moss crusts, and showed a unimodal pattern of Fv/Fm values. In contrast, photosynthetic heterogeneity was significantly higher in lichen, cyanobacteria-moss and lichen-moss crusts (P < 0.05), with a bimodal pattern of Fv/Fm values. Point pattern analysis showed that the distribution pattern of chlorophyll fluorescence varied at different spatial scales and also among the different crust types. These new results provide a detailed (millimeter-scale) insight into crust photosynthetic mechanisms and spatial distribution patterns associated with their community types. Collectively, this information provides an improved theoretical basis for crust maintenance and management in dryland regions.

Tracking Replicate Divergence in Microbial Community Composition and Function in Experimental Microcosms Abstract The study of microbial community functions necessitates replicating microbial communities. Variation in community development over time renders this an imperfect process. Thus, anticipating the likely degree of variation among replicate communities may aid in experimental design. We examined divergence in replicate community composition and function among 128 naturally assembled starting communities obtained from soils, each replicated three times, following a 30-day microcosm incubation period. Bacterial and fungal communities diverged in both composition and function among replicates, but remained much more similar to each other than to communities from different starting inocula. Variation in bacterial community composition among replicates was, however, correlated with variation in dissolved organic carbon production. A smaller-scale experiment testing nine starting communities showed that divergence was similar whether replicates were incubated on sterile or non-sterile pine litter, suggesting the impact of a pre-existing community on replicate divergence is minor. However, replicates in this experiment which were incubated for 114 days diverged more than those incubated for 30 days, suggesting experiments that run over long time periods will likely see greater variation among replicate community composition. These results suggest that while replicates diverge at a community level, such divergence is unlikely to severely impede the study of community function.

Edaphic Factors Influence the Distribution of Arbuscular Mycorrhizal Fungi Along an Altitudinal Gradient of a Tropical Mountain Abstract Changes in relief in montane areas, with increasing altitude, provide different biotic and abiotic conditions, acting on the species of arbuscular mycorrhizal fungi (AMF). The objective of this work was to determine the influence of altitude, edaphic factors, and vegetation on the AMF species in a mountainous area. The list of AMF species was obtained from morphological identification of the spores, with 72 species recovered from field samples and trap cultures. Lower levels of Shannon’s diversity occurred only at lower altitude; however, there was no difference in AMF richness. The structure of the AMF assembly between the two highest altitudes was similar and differed in relation to the lower altitude. There was variation in the distribution of AMF species, which was related to soil texture and chemical factors along the altitude gradient. Some species, genera, and families were indicative of a certain altitude, showing the preference of fungi for certain environmental conditions, which may aid in decisions to conserve montane ecosystems.