Hydrogen stable isotope probing of lipids demonstrates slow rates of microbial growth in soil.

The rate at which microorganisms grow and reproduce is fundamental to our understanding of microbial physiology and ecology. While soil microbiologists routinely quantify soil microbial biomass levels and the growth rates of individual taxa in culture, there is a limited understanding of how quickly microbes actually grow in soil. For this work, we posed the simple question: what are the growth rates of soil microorganisms? In this study, we measure these rates in three distinct soil environments using hydrogen-stable isotope probing of lipids with 2H-enriched water. This technique provides a taxa-agnostic quantification of in situ microbial growth from the degree of 2H enrichment of intact polar lipid compounds ascribed to bacteria and fungi. We find that growth rates in soil are quite slow and correspond to average generation times of 14 to 45 d but are also highly variable at the compound-specific level (4 to 402 d), suggesting differential growth rates among community subsets. We observe that low-biomass microbial communities exhibit more rapid growth rates than high-biomass communities, highlighting that biomass quantity alone does not predict microbial productivity in soil. Furthermore, within a given soil, the rates at which specific lipids are being synthesized do not relate to their quantity, suggesting a general decoupling of microbial abundance and growth in soil microbiomes. More generally, we demonstrate the utility of lipid-stable isotope probing for measuring microbial growth rates in soil and highlight the importance of measuring growth rates to complement more standard analyses of soil microbial communities.

Construction and characterisation of a structured, tuneable, and transparent 3D culture platform for soil bacteria.

We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the carbon catabolism of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as competition and warfare.

Sulfate-reducing bacteria unearthed: ecological functions of the diverse prokaryotic group in terrestrial environments.

Sulfate-reducing prokaryotes (SRPs) are essential microorganisms that play crucial roles in various ecological processes. Even though SRPs have been studied for over a century, there are still gaps in our understanding of their biology. In the past two decades, a significant amount of data on SRP ecology has been accumulated. This review aims to consolidate that information, focusing on SRPs in soils, their relation to the rare biosphere, uncultured sulfate reducers, and their interactions with other organisms in terrestrial ecosystems. SRPs in soils form part of the rare biosphere and contribute to various processes as a low-density population. The data reveal a diverse range of sulfate-reducing taxa intricately involved in terrestrial carbon and sulfur cycles. While some taxa like Desulfitobacterium and Desulfosporosinus are well studied, others are more enigmatic. For example, members of the Acidobacteriota phylum appear to hold significant importance for the terrestrial sulfur cycle. Many aspects of SRP ecology remain mysterious, including sulfate reduction in different bacterial phyla, interactions with bacteria and fungi in soils, and the existence of soil sulfate-reducing archaea. Utilizing metagenomic, metatranscriptomic, and culture-dependent approaches will help uncover the diversity, functional potential, and adaptations of SRPs in the global environment.

Illegal dumping of oil and gas wastewater alters arid soil microbial communities.

The Permian Basin, underlying southeast New Mexico and west Texas, is one of the most productive oil and gas (OG) provinces in the United States. Oil and gas production yields large volumes of wastewater with complex chemistries, and the environmental health risks posed by these OG wastewaters on sensitive desert ecosystems are poorly understood. Starting in November 2017, 39 illegal dumps, as defined by federal and state regulations, of OG wastewater were identified in southeastern New Mexico, releasing ~600,000 L of fluid onto dryland soils. To evaluate the impacts of these releases, we analyzed changes in soil geochemistry and microbial community composition by comparing soils from within OG wastewater dump-affected samples to unaffected zones. We observed significant changes in soil geochemistry for all dump-affected compared with control samples, reflecting the residual salts and hydrocarbons from the OG-wastewater release (e.g., enriched in sodium, chloride, and bromide). Microbial community structure significantly (P < 0.01) differed between dump and control zones, with soils from dump areas having significantly (P < 0.01) lower alpha diversity and differences in phylogenetic composition. Dump-affected soil samples showed an increase in halophilic and halotolerant taxa, including members of the Marinobacteraceae, Halomonadaceae, and Halobacteroidaceae, suggesting that the high salinity of the dumped OG wastewater was exerting a strong selective pressure on microbial community structure. Taxa with high similarity to known hydrocarbon-degrading organisms were also detected in the dump-affected soil samples. Overall, this study demonstrates the potential for OG wastewater exposure to change the geochemistry and microbial community dynamics of arid soils.IMPORTANCEThe long-term environmental health impacts resulting from releases of oil and gas (OG) wastewater, typically brines with varying compositions of ions, hydrocarbons, and other constituents, are understudied. This is especially true for sensitive desert ecosystems, where soil microbes are key primary producers and drivers of nutrient cycling. We found that releases of OG wastewater can lead to shifts in microbial community composition and function toward salt- and hydrocarbon-tolerant taxa that are not typically found in desert soils, thus altering the impacted dryland soil ecosystem. Loss of key microbial taxa, such as those that catalyze organic carbon cycling, increase arid soil fertility, promote plant health, and affect soil moisture retention, could result in cascading effects across the sensitive desert ecosystem. By characterizing environmental changes due to releases of OG wastewater to soils overlying the Permian Basin, we gain further insights into how OG wastewater may alter dryland soil microbial functions and ecosystems.

Methyl halide transferase-based gas reporters for quantification of filamentous bacteria in microdroplet emulsions.

The application of microfluidic techniques in experimental and environmental studies is a rapidly emerging field. Water-in-oil microdroplets can serve readily as controllable micro-vessels for studies that require spatial structure. In many applications, it is useful to monitor cell growth without breaking or disrupting the microdroplets. To this end, optical reporters based on color, fluorescence, or luminescence have been developed. However, optical reporters suffer from limitations when used in microdroplets such as inaccurate readings due to strong background interference or limited sensitivity during early growth stages. In addition, optical detection is typically not amenable to filamentous or biofilm-producing organisms that have significant nonlinear changes in opacity and light scattering during growth. To overcome such limitations, we show that volatile methyl halide gases produced by reporter cells expressing a methyl halide transferase (MHT) can serve as an alternative nonoptical detection approach suitable for microdroplets. In this study, an MHT-labeled Streptomyces venezuelae reporter strain was constructed and characterized. Protocols were established for the encapsulation and incubation of S. venezuelae in microdroplets. We observed the complete life cycle for S. venezuelae including the vegetative expansion of mycelia, mycelial fragmentation, and late-stage sporulation. Methyl bromide (MeBr) production was detected by gas chromatography-mass spectrometry (GC-MS) from S. venezuelae gas reporters incubated in either liquid suspension or microdroplets and used to quantitatively estimate bacterial density. Overall, using MeBr production as a means of quantifying bacterial growth provided a 100- to 1,000-fold increase in sensitivity over optical or fluorescence measurements of a comparable reporter strain expressing fluorescent proteins. IMPORTANCE Quantitative measurement of bacterial growth in microdroplets in situ is desirable but challenging. Current optical reporter systems suffer from limitations when applied to filamentous or biofilm-producing organisms. In this study, we demonstrate that volatile methyl halide gas production can serve as a quantitative nonoptical growth assay for filamentous bacteria encapsulated in microdroplets. We constructed an S. venezuelae gas reporter strain and observed a complete life cycle for encapsulated S. venezuelae in microdroplets, establishing microdroplets as an alternative growth environment for Streptomyces spp. that can provide spatial structure. We detected MeBr production from both liquid suspension and microdroplets with a 100- to 1,000-fold increase in signal-to-noise ratio compared to optical assays. Importantly, we could reliably detect bacteria with densities down to 106 CFU/mL. The combination of quantitative gas reporting and microdroplet systems provides a valuable approach to studying fastidious organisms that require spatial structure such as those found typically in soils.

Challenges in upscaling laboratory studies to ecosystems in soil microbiology research.

Soil microbiology has entered into the big data era, but the challenges in bridging laboratory-, field-, and model-based studies of ecosystem functions still remain. Indeed, the limitation of factors in laboratory experiments disregards interactions of a broad range of in situ environmental drivers leading to frequent contradictions between laboratory- and field-based studies, which may consequently mislead model development and projections. Upscaling soil microbiology research from laboratory to ecosystems represents one of the grand challenges facing environmental scientists, but with great potential to inform policymakers toward climate-smart and resource-efficient ecosystems. The upscaling is not only a scale problem, but also requires disentangling functional relationships and processes on each level. We point to three potential reasons for the gaps between laboratory- and field-based studies (i.e., spatiotemporal dynamics, sampling disturbances, and plant-soil-microbial feedbacks), and three key issues of caution when bridging observations and model predictions (i.e., across-scale effect, complex-process coupling, and multi-factor regulation). Field-based studies only cover a limited range of environmental variation that must be supplemented by laboratory and mesocosm manipulative studies when revealing the underlying mechanisms. The knowledge gaps in upscaling soil microbiology from laboratory to ecosystems should motivate interdisciplinary collaboration across experimental, observational, theoretic, and modeling research.

Penicillium rhizophilum, a novel species in the section Exilicaulis isolated from the rhizosphere of sugarcane in Southwest Iran.

During a survey of species diversity of Penicillium and Talaromyces in sugarcane (Saccharum officinarum) rhizosphere in the Khuzestan province of Iran [1], 195 strains were examined, from which 187 belonged to Penicillium (11 species) and eight to Talaromyces (one species). In the present study, three strains of Penicillium belonging to section Exilicaulis series Restricta, identified as P. restrictum by Ansari et al. [1], were subjected to a phylogenetic study. The multilocus phylogeny of partial ß-tubulin, calmodulin and RNA polymerase II second largest subunit genes enabled the recognition of one new phylogenetic species that is here formally described as Penicillium rhizophilum sp. nov. This species is phylogenetically distinct in series Restricta, but it does not show significant morphological differences from other species previously classified in the series. Therefore, we here placed bias on the phylogenetic species concept. The holotype of Penicillium rhizophilum sp. nov. is IRAN 18169F and the ex-type culture is LA30T (=IRAN 4042CT=CBS 149737T).

A new spike-in-based method for quantitative metabarcoding of soil fungi and bacteria.

Metabarcoding is a powerful tool to characterize biodiversity in biological samples. The interpretation of taxonomic profiles from metabarcoding data has been hindered by their compositional nature. Several strategies have been proposed to transform compositional data into quantitative data, but they have intrinsic limitations. Here, I propose a workflow based on bacterial and fungal cellular internal standards (spike-ins) for absolute quantification of the microbiota in soil samples. These standards were added to the samples before DNA extraction in amounts estimated after qPCRs, to target around 1-2% coverage in the sequencing run. In bacteria, proportions of spike-in reads in the sequencing run were very similar (< 2-fold change) to those predicted by the qPCR assessment, and for fungi they differed up to 40-fold. The low variation among replicates highlights the reproducibility of the method. Estimates based on multiple bacterial spike-ins were highly correlated (r = 0.99). Procrustes analysis evidenced significant biological effects on the community composition when normalizing compositional data. A protocol based on qPCR estimation of input amounts of cellular spikes is proposed as a cheap and reliable strategy for quantitative metabarcoding of biological samples.

Hopanoids Confer Robustness to Physicochemical Variability in the Niche of the Plant Symbiont Bradyrhizobium diazoefficiens.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the Bradyrhizobium clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in symbioses with success similar to that of the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the growth defects of extended-hopanoid-deficient mutants can be at least partially compensated for by the physicochemical environment, specifically, by optimal osmotic and divalent cation concentrations. Through biophysical measurements of lipid packing and membrane permeability, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in-culture and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environments within root nodules. To improve the legume-rhizobium symbiosis through either bioengineering or strain selection, it will be important to consider the full life cycle of rhizobia, from soil to symbiosis. IMPORTANCE Rhizobia, such as B. diazoefficiens, play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has received increased attention as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long-term integration of rhizobia into the soil community has been less successful. This work represents a small step toward improving the legume-rhizobium symbiosis by identifying a cellular component-hopanoid lipids-that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

Responses of the Soil Bacterial Community, Resistome, and Mobilome to a Decade of Annual Exposure to Macrolide Antibiotics.

Biosolids that are applied to agricultural soil as an organic fertilizer are frequently contaminated with pharmaceutical residues that have persisted during wastewater treatment and partitioned into the organic phase. Macrolide antibiotics, which serve as a critically important human medicine, have been detected within biosolids. To determine the impacts of macrolide antibiotics on soil bacteria, every year for a decade, a series of replicated field plots received an application of a mixture of erythromycin, clarithromycin, and azithromycin at a realistic (0.1 mg kg soil-1) or an unrealistically high (10 mg kg soil-1) dose or were left untreated. The effects of repeated antibiotic exposure on the soil bacterial community, resistome, mobilome, and integron gene cassette content were evaluated by 16S rRNA and integron gene cassette amplicon sequencing, as well as whole-metagenome sequencing. At the unrealistically high dose, the overall diversity of the resistome and mobilome was altered, as 21 clinically important antibiotic resistance genes predicted to encode resistance to 10 different antibiotic drug classes were increased and 20 mobile genetic element variants (tnpA, intI1, tnpAN, and IS91) were increased. In contrast, at the realistic dose, no effect was observed on the overall diversity of the soil bacterial community, resistome, mobilome, or integron gene cassette-carrying genes. Overall, these results suggest that macrolide antibiotics entrained into soil at concentrations anticipated with biosolid applications would not result in major changes to these endpoints. IMPORTANCE Biosolids, produced from the treatment of sewage sludge, are rich in plant nutrients and are a valuable alternative to inorganic fertilizer when applied to agricultural soil. However, the use of biosolids in agriculture, which are frequently contaminated with pharmaceuticals, such as macrolide antibiotics, may pose a risk to human health by selecting for antibiotic resistance genes that could be transferred to plant-based food destined for human consumption. The consequences of long-term, repeated macrolide antibiotic exposure on the diversity of the soil bacterial community, resistome, and mobilome were evaluated. At unrealistically high concentrations, macrolide antibiotics alter the overall diversity of the resistome and mobilome, enriching for antibiotic resistance genes and mobile genetic elements of concern to human health. However, at realistic antibiotic concentrations, no effect on these endpoints was observed, suggesting that current biosolids land management practices are unlikely to pose a risk to human health due to macrolide antibiotic contamination alone.

Extracellular DNAses Facilitate Antagonism and Coexistence in Bacterial Competitor-Sensing Interference Competition.

Over the last 4 decades, the rate of discovery of novel antibiotics has decreased drastically, ending the era of fortuitous antibiotic discovery. A better understanding of the biology of bacteriogenic toxins potentially helps to prospect for new antibiotics. To initiate this line of research, we quantified antagonists from two different sites at two different depths of soil and found the relative number of antagonists to correlate with the bacterial load and carbon-to-nitrogen (C/N) ratio of the soil. Consecutive studies show the importance of antagonist interactions between soil isolates and the lack of a predicted role for nutrient availability and, therefore, support an in situ role in offense for the production of toxins in environments of high bacterial loads. In addition, the production of extracellular DNAses (exDNases) and the ability to antagonize correlate strongly. Using an in domum-developed probabilistic cellular automaton model, we studied the consequences of exDNase production for both coexistence and diversity within a dynamic equilibrium. Our model demonstrates that exDNase-producing isolates involved in amensal interactions act to stabilize a community, leading to increased coexistence within a competitor-sensing interference competition environment. Our results signify that the environmental and biological cues that control natural-product formation are important for understanding antagonism and community dynamics, structure, and function, permitting the development of directed searches and the use of these insights for drug discovery. IMPORTANCE Ever since the first observation of antagonism by microorganisms by Ernest Duchesne (E. Duchesne, Contribution à l'étude de la concurrence vitale chez les microorganisms. Antagonism entre les moisissures et les microbes, These pour obtenir le grade de docteur en medicine, Lyon, France, 1897), many scientists successfully identified and applied bacteriogenic bioactive compounds from soils to cure infection. Unfortunately, overuse of antibiotics and the emergence of clinical antibiotic resistance, combined with a lack of discovery, have hampered our ability to combat infections. A deeper understanding of the biology of toxins and the cues leading to their production may elevate the success rate of the much-needed discovery of novel antibiotics. We initiated this line of research and discovered that bacterial reciprocal antagonism is associated with exDNase production in isolates from environments with high bacterial loads, while diversity may increase in environments of lower bacterial loads.

Acetylenotrophic and Diazotrophic Bradyrhizobium sp. Strain I71 from TCE-Contaminated Soils.

Acetylene (C2H2) is a molecule rarely found in nature, with very few known natural sources, but acetylenotrophic microorganisms can use acetylene as their primary carbon and energy source. As of 2018 there were 15 known strains of aerobic and anaerobic acetylenotrophs; however, we hypothesize there may yet be unrecognized diversity of acetylenotrophs in nature. This study expands the known diversity of acetylenotrophs by isolating the aerobic acetylenotroph, Bradyrhizobium sp. strain I71, from trichloroethylene (TCE)-contaminated soils. Strain I71 is a member of the class Alphaproteobacteria and exhibits acetylenotrophic and diazotrophic activities, the only two enzymatic reactions known to transform acetylene. This unique capability in the isolated strain may increase the genus' economic impact beyond agriculture as acetylenotrophy is closely linked to bioremediation of chlorinated contaminants. Computational analyses indicate that the Bradyrhizobium sp. strain I71 genome contains 522 unique genes compared to close relatives. Moreover, applying a novel hidden Markov model of known acetylene hydratase (AH) enzymes identified a putative AH enzyme. Protein annotation with I-TASSER software predicted the AH from the microbe Syntrophotalea acetylenica as the closest structural and functional analog. Furthermore, the putative AH was flanked by horizontal gene transfer (HGT) elements, like that of AH in anaerobic acetylenotrophs, suggesting an unknown source of acetylene or acetylenic substrate in the environment that is selecting for the presence of AH. IMPORTANCE The isolation of Bradyrhizobium strain I71 expands the distribution of acetylene-consuming microbes to include a group of economically important microorganisms. Members of Bradyrhizobium are well studied for their abilities to improve plant health and increase crop yields by providing bioavailable nitrogen. Additionally, acetylene-consuming microbes have been shown to work in tandem with other microbes to degrade soil contaminants. Based on genome, cultivation, and protein prediction analysis, the ability to consume acetylene is likely not widespread within the genus Bradyrhizobium. These findings suggest that the suite of phenotypic capabilities of strain I71 may be unique and make it a good candidate for further study in several research avenues.

Bulk and Spatially Resolved Extracellular Metabolome of Free-Living Nitrogen Fixation.

Soil nitrogen (N) transformations constrain terrestrial net primary productivity and are driven by the activity of soil microorganisms. Free-living N fixation (FLNF) is an important soil N transformation and key N input to terrestrial systems, but the forms of N contributed to soil by FLNF are poorly understood. To address this knowledge gap, a focus on microorganisms and microbial scale processes is needed that links N-fixing bacteria and their contributed N sources to FLNF process rates. However, studying the activity of soil microorganisms in situ poses inherent challenges, including differences in sampling scale between microorganism and process rates, which can be addressed with culture-based studies and an emphasis on microbial-scale measurements. Culture conditions can differ significantly from soil conditions, so it also important that such studies include multiple culture conditions like liquid and solid media as proxies for soil environments like soil pore water and soil aggregate surfaces. Here we characterized extracellular N-containing metabolites produced by two common, diazotrophic soil bacteria in liquid and solid media, with or without N, across two sampling scales (bulk via GC-MS and spatially resolved via MALDI mass spec imaging). We found extracellular production of inorganic and organic N during FLNF, indicating terrestrial N contributions from FLNF occur in multiple forms not only as ammonium as previously thought. Extracellular metabolite profiles differed between liquid and solid media supporting previous work indicating environmental structure influences microbial function. Metabolite profiles also differed between sampling scales underscoring the need to quantify microbial scale conditions to accurately interpret microbial function. IMPORTANCE Free-living nitrogen-fixing bacteria contribute significantly to terrestrial nitrogen availability; however, the forms of nitrogen contributed by this process are poorly understood. This is in part because of inherent challenges to studying soil microorganisms in situ, such as vast differences in scale between microorganism and ecosystem and complexities of the soil system (e.g., opacity, chemical complexity). Thus, upscaling important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, requires microbial-scale measurements in controlled systems. Our work generated bulk and spatially resolved measurements of nitrogen released during free-living nitrogen fixation under two contrasting growth conditions analogous to soil pores and aggregates. This work allowed us to determine that diverse forms of nitrogen are likely contributed to terrestrial systems by free-living nitrogen bacteria. We also demonstrated that microbial habitat (e.g., liquid versus solid media) alters microbial activity and that measurement of microbial activity is altered by sampling scale (e.g., bulk versus spatially resolved) highlighting the critical importance of quantifying microbial-scale processes to upscaling of ecosystem function.

Characterization of a new Blastobotrys navarrensis strain indicates that it is not a later synonym of Blastobotrys proliferans.

The species Blastobotrys navarrensis Sesma and Ramirez was delineated based on the description of the single strain CBS 139.77T. Based on its phenotypic similarities to Blastobotrys proliferans, B. navarrensis CBS 139.77T was later considered a synonym of B. proliferans. In the present study, we isolated the yeast strain IST 508 (=PYCC 8784=CBS 16671) from the soil surrounding an olive tree in Ferreira do Alentejo, Portugal. The phylogenetic analysis of D1/D2 domain and ITS sequences from strain IST 508 indicates that is closely related to B. navarrensis and B. proliferans. Although strain IST 508 differs from B. navarrensis CBS 139.77T by 14 substitutions and 20 indels (6.6 % divergence) in the ITS sequence, no divergence was detected at the level of D1/D2 domain, mitochondrial small subunit rDNA, and cytochrome oxidase II sequences. On the other hand, strains IST 508 and CBS 139.77 differ from B. proliferans NRRL Y-17577T by eight substitutions (1.4 % divergence) in the D1/D2 domain sequence, by 16 substitutions (2.7 % divergence) in the cytochrome oxidase II sequence, and by 16 substitutions (3.7 % divergence) in the mitochondrial small subunit rDNA sequence. Due to the high number of variable phenotypic tests in B. proliferans and B. navarrensis, strains from the two species are difficult to distinguish. Contrasting with what is described for other Blastobotrys species, no differences were detected at the level of micromorphology between the two species. Nevertheless, based on the molecular differences between the two strains, CBS 139.77 and IST 508, and B. proliferans NRRL Y-17577T and their phylogenetic analysis, strains CBS 139.77 and IST 508 are from B. navarrensis and this species should be considered as an independent species and not a later synonym of B. proliferans. We propose an emended description of B. navarrensis.

Responses of tundra soil microbial communities to half a decade of experimental warming at two critical depths.

Northern-latitude tundra soils harbor substantial carbon (C) stocks that are highly susceptible to microbial degradation with rising global temperatures. Understanding the magnitude and direction (e.g., C release or sequestration) of the microbial responses to warming is necessary to accurately model climate change. In this study, Alaskan tundra soils were subjected to experimental in situ warming by ∼1.1 °C above ambient temperature, and the microbial communities were evaluated using metagenomics after 4.5 years, at 2 depths: 15 to 25 cm (active layer at outset of the experiment) and 45 to 55 cm (transition zone at the permafrost/active layer boundary at the outset of the experiment). In contrast to small or insignificant shifts after 1.5 years of warming, 4.5 years of warming resulted in significant changes to the abundances of functional traits and the corresponding taxa relative to control plots (no warming), and microbial shifts differed qualitatively between the two soil depths. At 15 to 25 cm, increased abundances of carbohydrate utilization genes were observed that correlated with (increased) measured ecosystem carbon respiration. At the 45- to 55-cm layer, increased methanogenesis potential was observed, which corresponded with a 3-fold increase in abundance of a single archaeal clade of the Methanosarcinales order, increased annual thaw duration (45.3 vs. 79.3 days), and increased CH4 emissions. Collectively, these data demonstrate that the microbial responses to warming in tundra soil are rapid and markedly different between the 2 critical soil layers evaluated, and identify potential biomarkers for the corresponding microbial processes that could be important in modeling.

Fire and land use impact soil properties in a Mediterranean dry sclerophyll woodland.

Fire directly impacts soil properties responsible for soil function and can result in soil degradation. Across the globe, climate change-induced droughts and elevated temperatures are exacerbating fire regime severity, breadth, and frequency, thus posing a threat to soil function and dependent ecosystem services. In Australia, the 2019-2020 fire season consumed nearly 50% of Kangaroo Island, South Australia, burning both dry sclerophyll woodland and adjacent historically cleared and grazed pastureland. Due to exacerbated fire regime elements, e.g., intensity and area affected, and interactions with historical land use, post-fire recovery of soil function was uncertain. This study assessed the impacts of a) the 2019-2020 fire event in Western River, Kangaroo Island on dry sclerophyll woodland and b) the interaction between this fire event and historical clearing and grazing on post-fire function of the soil. To do so, the following physicochemical and biological soil properties were analysed: labile active carbon, total carbon, total nitrogen, carbon to nitrogen ratio (C/N), pH, electrical conductivity, soil water repellency, aggregate stability, microbial community composition, and microbial diversity. Our results showed that the fire was of high severity, causing a reduction in nutrient content, an extreme rise in pH, and significant modifications to fungal communities in burnt compared to unburnt dry sclerophyll woodland. Furthermore, clearing and grazing raised post-fire soil nutrient levels and soil microbial diversity but reduced soil C/N and the abundance of ectomycorrhizal fungi in burnt pastureland compared to burnt woodland soils. This study highlights the role of management and fire severity in post-fire outcomes and emphasizes the need for comprehensive soil function assessments to evaluate the impacts of disturbance on soil. Taking direct measure of soil properties, as done here, will improve future assessments of fire season impacts and post-fire recovery in fire-prone landscapes.

Beneficial Cyanosphere Heterotrophs Accelerate Establishment of Cyanobacterial Biocrust.

Biological soil crusts (biocrusts) are communities of microbes that inhabit the surface of arid soils and provide essential services to dryland ecosystems. While resistant to extreme environmental conditions, biocrusts are susceptible to anthropogenic disturbances that can deprive ecosystems of these valuable services for decades. Until recently, culture-based efforts to produce inoculum for cyanobacterial biocrust restoration in the southwestern United States focused on producing and inoculating the most abundant primary producers and biocrust pioneers, Microcoleus vaginatus and members of the family Coleofasciculaceae (also called Microcoleus steenstrupii complex). The discovery that a unique microbial community characterized by diazotrophs, known as the cyanosphere, is intimately associated with M. vaginatus suggests a symbiotic division of labor in which nutrients are traded between phototrophs and heterotrophs. To probe the potential use of such cyanosphere members in the restoration of biocrusts, we performed coinoculations of soil substrates with cyanosphere constituents. This resulted in cyanobacterial growth that was more rapid than that seen for inoculations with the cyanobacterium alone. Additionally, we found that the mere addition of beneficial heterotrophs enhanced the formation of a cohesive biocrust without the need for additional phototrophic biomass within native soils that contain trace amounts of biocrust cyanobacteria. Our findings support the hitherto-unknown role of beneficial heterotrophic bacteria in the establishment and growth of biocrusts and allow us to make recommendations concerning biocrust restoration efforts based on the presence of remnant biocrust communities in disturbed areas. Future biocrust restoration efforts should consider cyanobacteria and their beneficial heterotrophic community as inoculants. IMPORTANCE The advancement of biocrust restoration methods for cyanobacterial biocrusts has been largely achieved through trial and error. Successes and failures could not always be traced back to particular factors. The investigation and application of foundational microbial interactions existing within biocrust communities constitute a crucial step toward informed and repeatable biocrust restoration methods.

Manure-Based Amendments Influence Surface-Associated Bacteria and Markers of Antibiotic Resistance on Radishes Grown in Soils with Different Textures.

A controlled greenhouse study was performed to determine the effect of manure or compost amendments, derived during or in the absence of antibiotic treatment of beef and dairy cattle, on radish taproot-associated microbiota and indicators of antibiotic resistance when grown in different soil textures. Bacterial beta diversity, determined by 16S rRNA gene amplicon sequencing, bifurcated according to soil texture (P < 0.001, R = 0.501). There was a striking cross-effect in which raw manure from antibiotic-treated and antibiotic-free beef and dairy cattle added to loamy sand (LS) elevated relative (16S rRNA gene-normalized) (by 0.9 to 1.9 log10) and absolute (per-radish) (by 1.1 to 3.0 log10) abundances of intI1 (an integrase gene and indicator of mobile multiantibiotic resistance) on radishes at harvest compared to chemical fertilizer-only control conditions (P < 0.001). Radishes tended to carry fewer copies of intI1 and sul1 when grown in silty clay loam than LS. Composting reduced relative abundance of intI1 on LS-grown radishes (0.6 to 2.4 log10 decrease versus corresponding raw manure; P < 0.001). Effects of antibiotic use were rarely discernible. Heterotrophic plate count bacteria capable of growth on media containing tetracycline, vancomycin, sulfamethazine, or erythromycin tended to increase on radishes grown in turned composted antibiotic-treated dairy or beef control (no antibiotics) manures relative to the corresponding raw manure in LS (0.8- to 2.3-log10 increase; P < 0.001), suggesting that composting sometimes enriches cultivable bacteria with phenotypic resistance. This study demonstrates that combined effects of soil texture and manure-based amendments influence the microbiota of radish surfaces and markers of antibiotic resistance, illuminating future research directions for reducing agricultural sources of antibiotic resistance.IMPORTANCE In working toward a comprehensive strategy to combat the spread of antibiotic resistance, potential farm-to-fork routes of dissemination are gaining attention. The effects of preharvest factors on the microbiota and corresponding antibiotic resistance indicators on the surfaces of produce commonly eaten raw is of special interest. Here, we conducted a controlled greenhouse study, using radishes as a root vegetable grown in direct contact with soil, and compared the effects of manure-based soil amendments, antibiotic use in the cattle from which the manure was sourced, composting of the manure, and soil texture, with chemical fertilizer only as a control. We noted significant effects of amendment type and soil texture on the composition of the microbiota and genes used as indicators of antibiotic resistance on radish surfaces. The findings take a step toward identifying agricultural practices that aid in reducing carriage of antibiotic resistance and corresponding risks to consumers.

Hyperlocal Variation in Soil Iron and the Rhizosphere Bacterial Community Determines Dollar Spot Development in Amenity Turfgrass.

Dollar spot, caused by the fungal pathogen Clarireedia spp., is an economically important foliar disease of amenity turfgrass in temperate climates worldwide. This disease often occurs in a highly variable manner, even on a local scale with relatively uniform environmental conditions. The objective of this study was to investigate mechanisms behind this local variation, focusing on contributions of the soil and rhizosphere microbiome. Turfgrass, rhizosphere, and bulk soil samples were collected from within a 256-m2 area of healthy turfgrass, transported to a controlled environment chamber, and inoculated with Clarireedia jacksonii Bacterial communities were profiled by targeting the 16S rRNA gene, and 16 different soil chemical properties were assessed. Despite their initial uniform appearance, the samples differentiated into highly susceptible and moderately susceptible groups following inoculation in the controlled environment chamber. The highly susceptible samples harbored a unique rhizosphere microbiome with suggestively lower relative abundance of putative antibiotic-producing bacterial taxa and higher predicted abundance of genes associated with xenobiotic biodegradation pathways. In addition, stepwise regression revealed that bulk soil iron content was the only significant soil characteristic that positively regressed with decreased dollar spot susceptibility during the peak disease development stage. These findings suggest that localized variation in soil iron induces the plant to select for a particular rhizosphere microbiome that alters the disease outcome. More broadly, further research in this area may indicate how plot-scale variability in soil properties can drive variable plant disease development through alterations in the rhizosphere microbiome.IMPORTANCE Dollar spot is the most economically important disease of amenity turfgrass, and more fungicides are applied targeting dollar spot than any other turfgrass disease. Dollar spot symptoms are small (3 to 5 cm), circular patches that develop in a highly variable manner within plot scale even under seemingly uniform conditions. The mechanism behind this variable development is unknown. This study observed that differences in dollar spot development over a 256-m2 area were associated with differences in bulk soil iron concentration and correlated with a particular rhizosphere microbiome. These findings provide interesting avenues for future research to further characterize the mechanisms behind the highly variable development of dollar spot, which may inform innovative control strategies. Additionally, these results suggest that small changes in soil properties can alter plant activity and hence the plant-associated microbial community, which has important implications for a broad array of agricultural and horticultural plant pathosystems.

A Whole-Cell Biosensor for Detection of 2,4-Diacetylphloroglucinol (DAPG)-Producing Bacteria from Grassland Soil.

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) are ecologically important in the rhizosphere, as they can control phytopathogens and contribute to disease suppression. DAPG can also trigger a systemic resistance response in plants and stimulate root exudation and branching as well as induce plant-beneficial activities in other rhizobacteria. While studies of DAPG-producing Pseudomonas have predominantly focused on rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk soil and grassland, where the level of DAPG producers are predicted to be low. In this study, we constructed a whole-cell biosensor for detection of DAPG and DAPG-producing bacteria from environmental samples. The constructed biosensor contains a phlF response module and either lacZ or lux genes as output modules assembled on a pSEVA plasmid backbone for easy transfer to different host species and to enable easy future genetic modifications. We show that the sensor is highly specific toward DAPG, with a sensitivity in the low nanomolar range (>20 nM). This sensitivity is comparable to the DAPG levels identified in rhizosphere samples by chemical analysis. The biosensor enables guided isolation of DAPG-producing Pseudomonas Using the biosensor, we probed the same grassland soil sampling site to isolate genetically related DAPG-producing Pseudomonas kilonensis strains over a period of 12 months. Next, we used the biosensor to determine the frequency of DAPG-producing pseudomonads within three different grassland soil sites and showed that DAPG producers can constitute part of the Pseudomonas population in the range of 0.35 to 17% at these sites. Finally, we showed that the biosensor enables detection of DAPG produced by non-Pseudomonas species. Our study shows that a whole-cell biosensor for DAPG detection can facilitate isolation of bacteria that produce this important secondary metabolite and provide insight into the population dynamics of DAPG producers in natural grassland soil.IMPORTANCE The interest in bacterial biocontrol agents as biosustainable alternatives to pesticides to increase crop yields has grown. To date, we have a broad knowledge of antimicrobial compounds, such as DAPG, produced by bacteria growing in the rhizosphere surrounding plant roots. However, compared to the rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk and grassland soil. Currently, we are restricted to chemical methods with detection limits and time-consuming PCR-based and probe hybridization approaches to detect DAPG and its respective producer. In this study, we developed a whole-cell biosensor, which can circumvent the labor-intensive screening process as well as increase the sensitivity at which DAPG can be detected. This enables quantification of relative amounts of DAPG producers, which, in turn, increases our understanding of the dynamics and ecology of these producers in natural soil environments.