Microbial life in preferential flow paths in subsurface clayey till revealed by metataxonomy and metagenomics.

BACKGROUND: Subsurface microorganisms contribute to important ecosystem services, yet little is known about how the composition of these communities is affected by small scale heterogeneity such as in preferential flow paths including biopores and fractures. This study aimed to provide a more complete characterization of microbial communities from preferential flow paths and matrix sediments of a clayey till to a depth of 400 cm by using 16S rRNA gene and fungal ITS2 amplicon sequencing of environmental DNA. Moreover, shotgun metagenomics was applied to samples from fractures located 150 cm below ground surface (bgs) to investigate the bacterial genomic adaptations resulting from fluctuating exposure to nutrients, oxygen and water. RESULTS: The microbial communities changed significantly with depth. In addition, the bacterial/archaeal communities in preferential flow paths were significantly different from those in the adjacent matrix sediments, which was not the case for fungal communities. Preferential flow paths contained higher abundances of 16S rRNA and ITS gene copies than the corresponding matrix sediments and more aerobic bacterial taxa than adjacent matrix sediments at 75 and 150 cm bgs. These findings were linked to higher organic carbon and the connectivity of the flow paths to the topsoil as demonstrated by previous dye tracer experiments. Moreover, bacteria, which were differentially more abundant in the fractures than in the matrix sediment at 150 cm bgs, had higher abundances of carbohydrate active enzymes, and a greater potential for mixotrophic growth. CONCLUSIONS: Our results demonstrate that the preferential flow paths in the subsurface are unique niches that are closely connected to water flow and the fluctuating ground water table. Although no difference in fungal communities were observed between these two niches, hydraulically active flow paths contained a significantly higher abundance in fungal, archaeal and bacterial taxa. Metagenomic analysis suggests that bacteria in tectonic fractures have the genetic potential to respond to fluctuating oxygen levels and can degrade organic carbon, which should result in their increased participation in subsurface carbon cycling. This increased microbial abundance and activity needs to be considered in future research and modelling efforts of the soil subsurface.

The potential of Pseudomonas fluorescens SBW25 to produce viscosin enhances wheat root colonization and shapes root-associated microbial communities in a plant genotype-dependent manner in soil systems.

Microorganisms interact with plant roots through colonization of the root surface, i.e., the rhizoplane or the surrounding soil, i.e., the rhizosphere. Beneficial rhizosphere bacteria such as Pseudomonas spp. can promote plant growth and protect against pathogens by producing a range of bioactive compounds, including specialized metabolites like cyclic lipopeptides (CLPs) known for their biosurfactant and antimicrobial activities. However, the role of CLPs in natural soil systems during bacteria-plant interactions is underexplored. Here, Pseudomonas fluorescens SBW25, producing the CLP viscosin, was used to study the impact of viscosin on bacterial root colonization and microbiome assembly in two cultivars of winter wheat (Heerup and Sheriff). We inoculated germinated wheat seeds with SBW25 wild type or a viscosin-deficient mutant and grew the plants in agricultural soil. After 2 weeks, enhanced root colonization of SBW25 wild type compared to the viscosin-deficient mutant was observed, while no differences were observed between wheat cultivars. In contrast, the impact on root-associated microbial community structure was plant-genotype-specific, and SBW25 wild type specifically reduced the relative abundance of an unclassified oomycete and Phytophthora in Sheriff and Heerup, respectively. This study provides new insights into the natural role of viscosin and specifically highlights the importance of viscosin in wheat root colonization under natural soil conditions and in shaping the root microbial communities associated with different wheat cultivars. Furthermore, it pinpoints the significance of microbial microdiversity, plant genotype, and microbe-microbe interactions when studying colonization of plant roots. IMPORTANCE: Understanding parameters governing microbiome assembly on plant roots is critical for successfully exploiting beneficial plant-microbe interactions for improved plant growth under low-input conditions. While it is well-known from in vitro studies that specialized metabolites are important for plant-microbe interactions, e.g., root colonization, studies on the ecological role under natural soil conditions are limited. This might explain the often-low translational power from laboratory testing to field performance of microbial inoculants. Here, we showed that viscosin synthesis potential results in a differential impact on the microbiome assembly dependent on wheat cultivar, unlinked to colonization potential. Overall, our study provides novel insights into factors governing microbial assembly on plant roots, and how this has a derived but differential effect on the bacterial and protist communities.

Differential impacts of sewage sludge and biochar on phosphorus-related processes: An imaging study of the rhizosphere.

Recycling of phosphorus (P) from waste streams in agriculture is essential to reduce the negative environmental effects of surplus P and the unsustainable mining of geological P resources. Sewage sludge (SS) is an important P source; however, several issues are associated with the handling and application of SS in agriculture. Thus, post-treatments such as pyrolysis of SS into biochar (BC) could address some of these issues. Here we elucidate how patches of SS in soil interact with the living roots of wheat and affect important P-related rhizosphere processes compared to their BC counterparts. Wheat plants were grown in rhizoboxes with sandy loam soil, and 1 cm Ø patches with either SS or BC placed 10 cm below the seed. A negative control (CK) was included. Planar optode pH sensors were used to visualize spatiotemporal pH changes during 40 days of plant growth, diffusive gradients in thin films (DGT) were applied to map labile P, and zymography was used to visualize the spatial distribution of acid (ACP) and alkaline (ALP) phosphatase activity. In addition, bulk soil measurements of available P, pH, and ACP activity were conducted. Finally, the relative abundance of bacterial P-cycling genes (phoD, phoX, phnK) was determined in the patch area rhizosphere. Labile P was only observed in the area of the SS patches, and SS further triggered root proliferation and increased the activity of ACP and ALP in interaction with the roots. In contrast, BC seemed to be inert, had no visible effect on root growth, and even reduced ACP and ALP activity in the patch area. Furthermore, there was a lower relative abundance of phoD and phnK genes in the BC rhizosphere compared to the CK. Hence, optimization of BC properties is needed to increase the short-term efficiency of BC from SS as a P fertilizer.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

Activity of root-knot nematodes associated with composition of a nematode-attached microbiome and the surrounding soil microbiota.

We investigated if activity of the pre-infective juveniles (J2s) of root-knot nematodes is linked to the recruitment of a specific microbiome on the nematode surface and/or to the composition of the surrounding microbiota. For this, we determined the J2 activity (active vs. non-motile, which referred to dead and immobile J2s) upon a 3-day incubation in soil suspensions and studied the composition of bacteria, protists, and fungi present on the nematode surface and in the suspensions using amplicon sequencing of the 16S/18S rRNA genes, and ITS region. We also amended suspensions with Pseudomonas protegens strain CHA0 to study its effects on J2 activity and microbial composition. The J2 activity was suppressed in soil suspensions, but increased when suspensions were amended with P. protegens CHA0. The active and non-motile J2s differed in the composition of surface-attached bacteria, which was altered by the presence of P. protegens CHA0 in the soil suspensions. The bacterial genera Algoriphagus, Pedobacter, and Bdellovibrio were enriched on active J2s and may have protected the J2s against antagonists. The incubation time appeared short for attachment of fungi and protists. Altogether, our study is a step forward in disentangling the complex nematode-microbe interactions in soil for more successful nematode control.

Four novel Curtobacterium phages isolated from environmental samples.

Despite Curtobacterium spp. often being associated with the plant phyllosphere, i.e., the areal region of different plant species, only one phage targeting a member of the genus Curtobacterium has been isolated so far. In this study, we isolated four novel plaque-forming Curtobacterium phages, Reje, Penoan, Parvaparticeps, and Pize, with two novel Curtobacterium strains as propagation hosts. Based on the low nucleotide intergenomic similarity (<32.4%) between these four phages and any phage with a genome sequence in the NCBI database, we propose the establishment of the four genera, "Rejevirus", "Pizevirus", "Penoanvirus", and "Parvaparticepsvirus", all in the class of Caudoviricetes.

Three novel Erwinia billingiae phages isolated from organic waste represent three new genera.

Despite the ecological significance of viral communities, phages remain insufficiently studied. Current genomic databases lack high-quality phage genome sequences linked to specific bacteria. Bacteria of the genus Erwinia are known to colonize the phyllosphere of plants, both as commensals and as pathogens. We isolated three Erwinia billingiae phages-Zoomie, Pecta, and Snitter-from organic household waste. Based on sequence similarity to their closest relatives, we propose that they represent three new genera: "Pectavirus" within the family Zobellviridae, "Snittervirus" in the subfamily Tempevirinae, family Drexlerviridae, and "Zoomievirus" within the family Autographiviridae, which, together with the genus Limelightvirus, may constitute a new subfamily.

Draft Genome Sequences of Phytase Active Endophytic and Epiphytic Erwinia gerundensis Isolated from Wheat (Triticum aestivum) Seeds.

Here, we provide draft genome sequences of an epiphytic strain (HEP01) and an endophytic strain (HEN01) of Erwinia gerundensis, isolated from wheat (Triticum aestivum) seeds. Genome sizes of HEP01 and HEN01 were 3,771,322 bp and 3,750,048 bp, respectively. HEP01 and HEN01 carried one plasmid each with sizes of 565,617 bp and 576,781 bp, respectively. Both showed phenotypic phytase activity.

Enzyme activity profiling for physiological phenotyping within functional phenomics: plant growth and stress responses.

High-throughput profiling of key enzyme activities of carbon, nitrogen, and antioxidant metabolism is emerging as a valuable approach to integrate cell physiological phenotyping into a holistic functional phenomics approach. However, the analyses of the large datasets generated by this method represent a bottleneck, often keeping researchers from exploiting the full potential of their studies. We address these limitations through the exemplary application of a set of data evaluation and visualization tools within a case study. This includes the introduction of multivariate statistical analyses that can easily be implemented in similar studies, allowing researchers to extract more valuable information to identify enzymatic biosignatures. Through a literature meta-analysis, we demonstrate how enzyme activity profiling has already provided functional information on the mechanisms regulating plant development and response mechanisms to abiotic stress and pathogen attack. The high robustness of the distinct enzymatic biosignatures observed during developmental processes and under stress conditions underpins the enormous potential of enzyme activity profiling for future applications in both basic and applied research. Enzyme activity profiling will complement molecular -omics approaches to contribute to the mechanistic understanding required to narrow the genotype-to-phenotype knowledge gap and to identify predictive biomarkers for plant breeding to develop climate-resilient crops.

Deep-rooted perennial crops differ in capacity to stabilize C inputs in deep soil layers.

Comprehensive climate change mitigation necessitates soil carbon (C) storage in cultivated terrestrial ecosystems. Deep-rooted perennial crops may help to turn agricultural soils into efficient C sinks, especially in deeper soil layers. Here, we compared C allocation and potential stabilization to 150 cm depth from two functionally distinct deep-rooted perennials, i.e., lucerne (Medicago sativa L.) and intermediate wheatgrass (kernza; Thinopyrum intermedium), representing legume and non-legume crops, respectively. Belowground C input and stabilization was decoupled from nitrogen (N) fertilizer rate in kernza (100 and 200 kg mineral N ha-1), with no direct link between increasing mineral N fertilization, rhizodeposited C, and microbial C stabilization. Further, both crops displayed a high ability to bring C to deeper soil layers and remarkably, the N2-fixing lucerne showed greater potential to induce microbial C stabilization than the non-legume kernza. Lucerne stimulated greater microbial biomass and abundance of N cycling genes in rhizosphere soil, likely linked to greater amino acid rhizodeposition, hence underlining the importance of coupled C and N for microbial C stabilization efficiency. Inclusion of legumes in perennial cropping systems is not only key for improved productivity at low fertilizer N inputs, but also appears critical for enhancing soil C stabilization, in particular in N limited deep subsoils.

Diversity and Structure of Bacterial Communities in Different Rhizocompartments (Rhizoplane, Rhizosphere, and Bulk) at Flag Leaf Emergence in Four Winter Wheat Varieties.

Understanding basic interactions at the plant-soil interphase is critical if we are to exploit natural microbial communities for improved crop resilience. We report here 16S amplicon sequencing data from 3 rhizocompartments of 4 wheat cultivars grown under controlled greenhouse conditions. We observed that rhizocompartments and cultivar affect the community composition.

Succession of the wheat seed-associated microbiome as affected by soil fertility level and introduction of Penicillium and Bacillus inoculants in the field.

During germination, the seed releases nutrient-rich exudates into the spermosphere, thereby fostering competition between resident microorganisms. However, insight into the composition and temporal dynamics of seed-associated bacterial communities under field conditions is currently lacking. This field study determined the temporal changes from 11 to 31 days after sowing in the composition of seed-associated bacterial communities of winter wheat as affected by long-term soil fertilization history, and by introduction of the plant growth-promoting microbial inoculants Penicillium bilaiae and Bacillus simplex. The temporal dynamics were the most important factor affecting the composition of the seed-associated communities. An increase in the relative abundance of genes involved in organic nitrogen metabolism (ureC and gdhA), and in ammonium oxidation (amoA), suggested increased mineralization of plant-derived nitrogen compounds over time. Dynamics of the phosphorus cycling genes ppt, ppx and cphy indicated inorganic phosphorus and polyphosphate cycling, as well as phytate hydrolysis by the seed-associated bacteria early after germination. Later, an increase in genes for utilization of organic phosphorus sources (phoD, phoX and phnK) indicated phosphorus limitation. The results indicate that community temporal dynamics are partly driven by changed availability of major nutrients, and reveal no functional consequences of the added inoculants during seed germination.

Back to our roots: exploring the role of root morphology as a mediator of beneficial plant-microbe interactions.

Plant breeding for belowground traits that have a positive impact on the rhizosphere microbiome is a promising strategy to sustainably improve crop yields. Root architecture and morphology are understudied plant breeding targets despite their potential to significantly shape microbial community structure and function in the rhizosphere. In this review, we explore the relationship between various root architectural and morphological traits and rhizosphere interactions, focusing on the potential of root diameter to impact the rhizosphere microbiome structure and function while discussing the potential biological and ecological mechanisms underpinning this process. In addition, we propose three future research avenues to drive this research area in an effort to unravel the effect of belowground traits on rhizosphere microbiology. This knowledge will pave the way for new plant breeding strategies that can be exploited for sustainable and high-yielding crop cultivars.

Complete Genome Sequence of a Novel Dyadobacter sp. Strain, NIV53, Isolated from 2-Meter Deep Subsurface Sediment.

Here, we provide the complete genome sequence of the subsurface bacterial isolate Dyadobacter sp. strain NIV53, a candidate species from the Spirosomaceae family. The isolate contained one 7,587,604-bp chromosome, with a GC content of 40.4%, and one plasmid, pNIV1, with a size of 12,453 bp.

Effects of Intra- and Interspecific Plant Density on Rhizosphere Bacterial Communities.

There have been very few studies on the effects of plant competition on the rhizosphere bacterial community. To investigate the impacts of intra- and interspecific plant competition, we analyzed the responses of rhizosphere bacterial communities to plant density as determined by 16S rRNA gene targeted sequencing. We included five weedy plant species growing in field soil in monocultures and mixed cultures at three densities in a greenhouse experiment. The rhizosphere bacterial community of each species changed more with density in a mixture of all five plant species than in monocultures, so intra- and interspecific plant competition had different effects on the bacterial community. For the dominant plant competitor, Centaurea cyanus, neither intra- nor interspecific competition had major effects on the composition of its rhizosphere bacterial communities. In contrast, the bacterial communities of the weakest competitor, Trifolium repens, were affected differently by intra- and interspecific competition. During increasing intraspecific competition T. repens maintained a highly specialized bacterial community dominated by Rhizobium; while during interspecific competition, the relative abundance of Rhizobium declined while other nitrogen fixing and potentially plant growth promoting taxa became more abundant. Contrary to previous observations made for soil microbial communities, the bacterial rhizosphere community of the weakest competitor did not become more similar to that of the dominant species. Thus, the process of competition, as well as the plant species themselves, determined the rhizosphere bacterial community. Our results emphasize the role of plant-plant interactions for rhizosphere bacterial communities. These effects may feedback to affect plant-plant interactions, and this is an important hypothesis for future research.

Preferential flow paths shape the structure of bacterial communities in a clayey till depth profile.

Preferential flow paths in subsurface soils serve as transport routes for water, dissolved organic matter and oxygen. Little is known about bacterial communities in flow paths or in subsoils below ∼4 m. We compared communities from preferential flow paths (biopores, fractures and sand lenses) with those in adjacent matrix sediments of clayey till from the plough layer to a depth of 6 m. 16S rRNA gene-targeted community analysis showed bacterial communities of greater abundance and diversity in flow paths than in matrix sediments at all depths. Deep fracture communities contained a higher relative abundance of aerobes and plant material decomposers like Nitrospirae, Acidobacteria and Planctomycetes than adjacent matrix sediments. Similarly, analyses of the relative abundances of archaeal amoA, nirK and dsrB genes indicated transition from aerobic to anaerobic nitrogen and sulphur cycling at greater depth in preferential flow paths than in matrix sediments. Preferential flow paths in the top 260 cm contained more indicator operational taxonomic units from the plough layer community than the matrix sediments. This study indicates that the availability of oxygen and organic matter and downward transport of bacteria shape bacterial communities in preferential flow paths, and suggests that their lifestyles differ from those of bacteria in matrix communities.

The biosurfactant viscosin transiently stimulates n-hexadecane mineralization by a bacterial consortium.

Pseudomonas produces powerful lipopeptide biosurfactants including viscosin, massetolide A, putisolvin, and amphisin, but their ability to stimulate alkane mineralization and their utility for bioremediation have received limited attention. The four Pseudomonas lipopeptides yielded emulsification indices on hexadecane of 20-31% at 90 mg/l, which is comparable to values for the synthetic surfactant Tween 80. Viscosin was the optimal emulsifier and significantly stimulated n-hexadecane mineralization by diesel-degrading bacterial consortia but exclusively during the first 2 days of batch culture experiments. Growth of the consortia, as determined by OD600 measurements and quantification of the alkB marker gene for alkane degradation, was arrested after the first day of the experiment. In contrast, the control consortia continued to grow and reached higher OD600 values and higher alkB copy numbers during the next days. Due to the short-lived stimulation of n-hexadecane mineralization, the stability of viscosin was analyzed, and it was observed that added viscosin was degraded by the bacterial consortium during the first 2 days. Hence, viscosin has a potential as stimulator of alkane degradation, but its utility in bioremediation may be limited by its rapid degradation and growth-inhibiting properties.