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Over the past few decades, regulatory RNAs, such as small RNAs (sRNAs), have received increasing attention in the context of host-microbe interactions due to their diverse roles in controlling various biological processes in eukaryotes. In addition, studies have identified an increasing number of sRNAs with novel functions across a wide range of bacteria. What is not well understood is why cells regulate gene expression through post-transcriptional mechanisms rather than at the initiation of transcription. The finding of a multitude of sRNAs and their identified associated targets has allowed further investigation into the role of sRNAs in mediating gene regulation. These foundational data allow for further development of hypotheses concerning how a precise control of gene activity is accomplished through the combination of transcriptional and post-transcriptional regulation. Recently, sRNAs have been reported to participate in interkingdom communication and signalling where sRNAs originating from one kingdom are able to target or control gene expression in another kingdom. For example, small RNAs of fungal pathogens that silence plant genes and vice-versa plant sRNAs that mediate bacterial gene expression. However, there is currently a lack of evidence regarding sRNA-based inter-kingdom signalling across more than two interacting organisms. A habitat that provides an excellent opportunity to investigate interconnectivity is the plant rhizosphere, a multifaceted ecosystem where plants and associated soil microbes are known to interact. In this paper, we discuss how the interconnectivity of bacteria, fungi, and plants within the rhizosphere may be mediated by bacterial sRNAs with a particular focus on disease suppressive and non-suppressive soils. We discuss the potential roles sRNAs may play in the below-ground world and identify potential areas of future research, particularly in reference to the regulation of plant immunity genes by bacterial and fungal communities in disease-suppressive and non-disease-suppressive soils.

Denitrification is an important global N cycle process. The gene encoding NosZ that converts nitrous oxide (N2O) to N2 has been widely used as a biomarker to study denitrifying communities. However, conventional PCR primers target a limited range of the genetically diverse Clade I nosZ, and the amplicons are too long for sequencing on current NGS platforms. To address these issues, we developed a new PCR primer set that amplifies a 355-bp region of Clade I nosZ and captures broader taxonomic coverage than conventional primers in in silico tests. When compared with the widely used nosZF_nosZR_Rich_2003 set using the same soil samples and the same sequencing depth, the new set retrieved genes from four times more unique species, with consistently higher general diversity-based metrics. The new primer set performed well with different sequencing platforms (Ion Torrent and Illumina), and among a wide variety of soils from polar to tropical, desert to agricultural, and surface to a very low biomass subsoil, with significant differences in denitrifying community diversity and composition. This new primer set for Clade I together with the primers recently reported for Clade II by Chee-Sanford et al. (J Microbiol Meth 172:105908, 2020) provides a more comprehensive assessment of denitrifier gene hosts, their ecological patterns, and the degree of novelty in retrieved gene sequences.

Background Process and function that underlie the assembly of a rhizosphere microbial community may be strongly linked to the maintenance of plant health. However, their assembly processes and functional changes in the deterioration of soilborne disease remain unclear. Here, we investigated features of rhizosphere microbiomes related to Fusarium wilt disease and assessed their assembly by comparison pair of diseased/healthy sequencing data. The untargeted metabolomics was employed to explore potential community assembly drivers, and shotgun metagenome sequencing was used to reveal the mechanisms of metabolite-mediated process after soil conditioning. Results Results showed the deterministic assembly process associated with diseased rhizosphere microbiomes, and this process was significantly correlated to five metabolites (tocopherol acetate, citrulline, galactitol, octadecylglycerol, and behenic acid). Application of the metabolites resulted in a deterministic assembly of microbiome with the high morbidity of watermelon. Furthermore, metabolite conditioning was found to weaken the function of autotoxin degradation undertaken by specific bacterial group (Bradyrhizobium, Streptomyces, Variovorax, Pseudomonas, and Sphingomonas) while promoting the metabolism of small-molecule sugars and acids initiated from another bacterial group (Anaeromyxobacter, Bdellovibrio, Conexibacter, Flavobacterium, and Gemmatimonas). Conclusion These findings strongly suggest that shifts in a metabolite-mediated microbial community assembly process underpin the deterministic establishment of soilborne Fusarium wilt disease and reveal avenues for future research focusing on ameliorating crop loss due to this pathogen.

Soil microbial respiration is an important source of uncertainty in projecting future climate and carbon (C) cycle feedbacks. However, its feedbacks to climate warming and underlying microbial mechanisms are still poorly understood. Here we show that the temperature sensitivity of soil microbial respiration (Q10) in a temperate grassland ecosystem persistently decreases by 12.0 ± 3.7% across 7 years of warming. Also, the shifts of microbial communities play critical roles in regulating thermal adaptation of soil respiration. Incorporating microbial functional gene abundance data into a microbially-enabled ecosystem model significantly improves the modeling performance of soil microbial respiration by 5–19%, and reduces model parametric uncertainty by 55–71%. In addition, modeling analyses show that the microbial thermal adaptation can lead to considerably less heterotrophic respiration (11.6 ± 7.5%), and hence less soil C loss. If such microbially mediated dampening effects occur generally across different spatial and temporal scales, the potential positive feedback of soil microbial respiration in response to climate warming may be less than previously predicted., Mechanisms and consequences of the acclimation of soil respiration to warming are unclear. Here, the authors combine soil respiration, metagenomics, and functional gene results from a 7-year grassland warming experiment to a microbial-enzyme decomposition model, showing functional gene information to lower uncertainty and improve fit.

Atmospheric nitrous oxide (N2O) is a potent greenhouse gas thought to be mainly derived from microbial metabolism as part of the denitrification pathway. Here, we report that in unexplored peat soils of Central and South America, N2O production can be driven by abiotic reactions (≤ 98 %) highly competitive to their enzymatic counterparts. Extracted soil iron positively correlated with in-situ abiotic N2O production determined by isotopic tracers. Moreover, we found that microbial N2O reduction accompanied abiotic production, essentially closing a coupled abiotic-biotic N2O cycle. Anaerobic N2O consumption occurred ubiquitously (pH 6.4-3.7), with proportions of diverse clade II N2O-reducers increasing with consumption rates. Our findings show denitrification in tropical peat soils is not a purely biological process, but rather a “mosaic” of abiotic and biotic reduction reactions. We predict hydrological and temperature fluctuations differentially affect abiotic and biotic drivers and further contribute to the high N2O flux variation in the region.

Atmospheric nitrous oxide (N

The susceptibility of soil organic carbon (SOC) in tundra to microbial decomposition under warmer climate scenarios potentially threatens a massive positive feedback to climate change, but the underlying mechanisms of stable SOC decomposition remain elusive. Herein, Alaskan tundra soils from three depths (a fibric O horizon with litter and course roots, an O horizon with decomposing litter and roots, and a mineral-organic mix, laying just above the permafrost) were incubated. Resulting respiration data were assimilated into a 3-pool model to derive decomposition kinetic parameters for fast, slow, and passive SOC pools. Bacterial, archaeal, and fungal taxa and microbial functional genes were profiled throughout the 3-year incubation. Correlation analyses and a Random Forest approach revealed associations between model parameters and microbial community profiles, taxa, and traits. There were more associations between the microbial community data and the SOC decomposition parameters of slow and passive SOC pools than those of the fast SOC pool. Also, microbial community profiles were better predictors of model parameters in deeper soils, which had higher mineral contents and relatively greater quantities of old SOC than in surface soils. Overall, our analyses revealed the functional potential of microbial communities to decompose tundra SOC through a suite of specialized genes and taxa. These results portray divergent strategies by which microbial communities access SOC pools across varying depths, lending mechanistic insights into the vulnerability of what is considered stable SOC in tundra regions.

Fusarium wilt disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense race 4, is a serious soil-borne fungal disease that currently threatens worldwide banana production. No single agricultural practice has yet been developed to effectively manage this disease. In the present study, greenhouse experiments were conducted to evaluate the effect of an integrated biofertilizer application after ammonia fumigation to enhance control of Fusarium wilt disease in severely infected banana mono-cropped soils. Quantitative PCR and high-throughput sequencing were used to characterise soil microbial community structure and the results from both two-season experimental studies showed that biofertilizer application after ammonia fumigation significantly reduced the incidence of banana Panama disease by approximately 55% and promoted the plant biomass, compared to the control application of cow manure to non-fumigated soil. Ammonia fumigation significantly reduced total fungal and F. oxysporum abundances and bacterial and fungal diversities. Biofertilizer application after fumigation further depleted the abundance of the pathogen. Biofertilizer application and fumigation altered the soil microbial community composition with the bacterial community responding first to fumigation, while the fungal community responded first to fertilization. Although Bacillus, including our inoculated strain, was not enriched after biofertilization, putative beneficial microbes such as Paenibacillus, Virgibacillus, Nitrosomonas, and Nitrobacter, were significantly enriched by ammonia fumigation and biofertilizer application, and were significantly correlated with disease suppression or increased plant biomass. Furthermore, fumigation and biofertilization significantly increased the soil pH and nutrient contents, with concomitant effects on the microbial community. Overall, the observed disease suppression and increased plant biomass resulting from both soil fumigation and biofertilization after banana cropping can be attributed to the reduced abundance of F. oxysporum and general suppression from altered soil properties that may enable the establishment of a beneficial soil microbiome.

Air carries a diverse load of particulate microscopic biological matter in suspension, either aerosolized or aggregated with dust particles, the aerobiome, which is dispersed by winds from sources to sinks. The aerobiome is known to contain microbes, including pathogens, as well as debris or small-sized propagules from plants and animals, but its variability and composition has not been studied comprehensibly. To gain a dynamic insight into the aerobiome existing over a mixed-use dryland setting, we conducted a biologically comprehensive, year-long survey of its composition and dynamics for particles less than 10 μm in diameter based on quantitative analyses of DNA content coupled to genomic sequencing. Airborne biological loads were more dependent on seasonal events than on meteorological conditions and only weakly correlated with dust loads. Core aerobiome species could be understood as a mixture of high elevation (e.g. Microbacteriaceae, Micrococcaceae, Deinococci), and local plant and soil sources (e.g. Sphingomonas, Streptomyces, Acinetobacter). Despite the mixed used of the land surrounding the sampling site, taxa that contributed to high load events were largely traceable to proximal agricultural practices like cotton and livestock farming. This included not only the predominance of specific crop plant signals over those of native vegetation, but also that of their pathogens (bacterial, viral and eukaryotic). Faecal bacterial loads were also seasonally important, possibly sourced in intensive animal husbandry or manure fertilization activity, and this microbial load was enriched in tetracycline resistance genes. The presence of the native opportunistic pathogen, Coccidioides spp., by contrast, was detected only with highly sensitive techniques, and only rarely. We conclude that agricultural activity exerts a much stronger influence that the native vegetation as a mass loss factor to the land system and as an input to dryland aerobiomes, including in the dispersal of plant, animal and human pathogens and their genetic resistance characteristics.

Background In a warmer world, microbial decomposition of previously frozen organic carbon (C) is one of the most likely positive climate feedbacks of permafrost regions to the atmosphere. However, mechanistic understanding of microbial mediation on chemically recalcitrant C instability is limited; thus, it is crucial to identify and evaluate active decomposers of chemically recalcitrant C, which is essential for predicting C-cycle feedbacks and their relative strength of influence on climate change. Using stable isotope probing of the active layer of Arctic tundra soils after depleting soil labile C through a 975-day laboratory incubation, the identity of microbial decomposers of lignin and, their responses to warming were revealed. Results The β-Proteobacteria genus Burkholderia accounted for 95.1% of total abundance of potential lignin decomposers. Consistently, Burkholderia isolated from our tundra soils could grow with lignin as the sole C source. A 2.2 °C increase of warming considerably increased total abundance and functional capacities of all potential lignin decomposers. In addition to Burkholderia, α-Proteobacteria capable of lignin decomposition (e.g. Bradyrhizobium and Methylobacterium genera) were stimulated by warming by 82-fold. Those community changes collectively doubled the priming effect, i.e., decomposition of existing C after fresh C input to soil. Consequently, warming aggravates soil C instability, as verified by microbially enabled climate-C modeling. Conclusions Our findings are alarming, which demonstrate that accelerated C decomposition under warming conditions will make tundra soils a larger biospheric C source than anticipated.

Soil-borne plant diseases are increasingly causing devastating losses in agricultural production. The development of a more refined model for disease prediction can aid in reducing crop losses through the use of preventative control measures or soil fallowing for a planting season. The emergence of high-throughput DNA sequencing technology has provided unprecedented insight into the microbial composition of diseased versus healthy soils. However, a single independent case study rarely yields a general conclusion predictive of the disease in a particular soil. Here, we attempt to account for the differences among various studies and plant varieties using a machine-learning approach based on 24 independent bacterial data sets comprising 758 samples and 22 independent fungal data sets comprising 279 samples of healthy or Fusarium wilt-diseased soils from eight different countries. We found that soil bacterial and fungal communities were both clearly separated between diseased and healthy soil samples that originated from six crops across nine countries or regions. Alpha diversity was consistently greater in the fungal community of healthy soils. While diseased soil microbiomes harbored higher abundances of Xanthomonadaceae, Bacillaceae, Gibberella, and Fusarium oxysporum, the healthy soil microbiome contained more Streptomyces Mirabilis, Bradyrhizobiaceae, Comamonadaceae, Mortierella, and nonpathogenic fungi of Fusarium. Furthermore, a random forest method identified 45 bacterial OTUs and 40 fungal OTUs that categorized the health status of the soil with an accuracy >80%. We conclude that these models can be applied to predict the potential for occurrence of F. oxysporum wilt by revealing key biological indicators and features common to the wilt-diseased soil microbiome.

Soil microbial respiration is an important source of uncertainty in projecting future climate and carbon (C) cycle feedbacks. Despite intensive studies for two decades, the magnitude, direction, and duration of such feedbacks are uncertain, and their underlying microbial mechanisms are still poorly understood. Here we examined the responses of soil respiration and microbial community structure to long-term experimental warming in a temperate grassland ecosystem. Our results indicated that the temperature sensitivity of soil microbial respiration (i.e.,Q10) persistently decreased by 12.0±3.7% across 7 years of warming. Integrated metagenomic and functional analyses showed that microbial community adaptation played critical roles in regulating respiratory acclimation. Incorporating microbial functional gene abundance data into a microbially-enabled ecosystem model significantly improved the modeling performance of soil microbial respiration by 5–19%, compared to the traditional non-microbial model. Model parametric uncertainty was also reduced by 55–71% when gene abundances were used. In addition, our modeling analyses suggested that decreased temperature sensitivity could lead to considerably less heterotrophic respiration (11.6±7.5%), and hence less soil C loss. If such microbially mediated dampening effects occur generally across different spatial and temporal scales, the potential positive feedback of soil microbial respiration in response to climate warming may be less than previously predicted.

Additional file 1: Supplementary Figure 1. Distribution of 16S rRNA gene copy numbers along buoyant density of the samples in warmed and control samples. Supplementary Figure 2. The average relative abundance of (a) Burkholderia and (b) Alphaproteobacteria among 13C-labelled DNA. Supplementary Figure 3. The Napierian logarithm of relative abundances of Burkholderia at different time points of the experiment, as revealed by 16S rRNA gene amplicon sequencing. Supplementary Figure 4.Z-P plot showing the distribution of OTUs based on their topological roles. Supplementary Figure 5. Growth curves of Burkholderia strains AK3 and AK1 in defined BMM medium with alkaline lignin as the sole C substrate (n=3). Supplementary Figure 6. Predicted lignin decomposition pathway in Burkholderia AK1 and AK3. Supplementary Figure 7. (a) The number of 13C-labelled 16S rRNA gene copies of Bradyrhizobium; (b) The number of 13C-labelled 16S rRNA gene copies of Methylobacterium. Supplementary Figure 8. Relative abundances of active α-Proteobacteria at different time points during the experiment, as revealed by 16S rRNA gene amplicon sequencing.

The effect of genetic markers and reference databases on analyses of fungal communities were estimated using fungal large subunit (LSU) and internal transcribed spacer (ITS) amplicon datasets in consecutive years of rhizosphere samples from three candidate biofuel crops, corn (Zea mays), switchgrass (Panicum virgatum), and miscanthus (Miscanthus × giganteus). These two marker genes were selected to contrast possible differences in biological conclusions. In addition, two ITS schemes based on two ITS reference databases were used to assess differences due to reference database composition. A taxonomy-supervised method was invoked using the Ribosomal Database Project naive Bayesian classifier that accesses all three databases. The UNITE classification scheme had the highest number of classified taxa in the raw classification result; however, it also had the highest proportion of unknown taxa (sequences that were classified to “unclassified,” “unidentified,” incertae sedis or, in the case of Warcup, to matches containing two unique names). After removal of these unknown taxa, LSU had the highest classification rate followed by Warcup and UNITE. As expected, the communities resolved using the two ITS databases, based on the same sequences, were relatively more similar than those from the lower-coverage LSU classification scheme. The choice of marker gene or even the same reads with different classification databases revealed different community patterns due to database coverage, e.g., the relative abundance of the most abundant groups changed or were only detected in one or two of the classification schemes, such as for Mortierella, Fusarium, and Phoma. No marked difference in fungal beta-diversity was identified among the three methods. Differentiation between the three biofuel crops and between the drought and normal rainfall years was apparent, regardless of method. Though classification rates, taxonomic conflicts, and coverage differences within the high-abundance fungal groups varied according to classification scheme, there was no overall impact on beta diversity among the three methods.

Enhancement of soil fertility and mitigation of atmospheric greenhouse gases are potential benefits of biochar amendments with proximate links to microbiological processes, yet the impact of biochar on the soil microbial community is poorly resolved. Here, we assessed changes in bacterial community composition and microbial assemblage patterns with biochar amendment in two soils with contrasting fertility under two cropping systems; perennial grass and annual sweet corn rotation at two time periods. Overall, soil type exerted the greatest effect on the soil microbial community, followed by sampling time and cropping system which exerted a greater effect on the microbial community than biochar treatment. The influence of biochar on community composition was most pronounced in the highly weathered, low fertility Oxisol, than the high fertility Mollisol. We further investigated microbial assemblage architecture using random-matrix theory based molecular ecological network analyses. Microbial assemblage complexity increased with biochar amendment, with the largest impact associated with the Oxisol, accompanied by more negative OTU co-variations, indicating enhanced competition and/or niche partitioning in concert with an increase in the number of putative “keystone species”. Network analysis suggests that biochar addition, especially in the highly weathered, low fertility Oxisol, confers higher-level organization, competition, and complexity to the soil microbiome that may result in higher resistance to change due to environmental perturbation and thereby increase system sustainability.

Agricultural soils with (M+) or without (M−) organic amendment were collected from four long-term field experiments to investigate soil fungal community composition and its relationship to the soil ionome by employing both the sequencing of fungal internal transcription spacer (ITS) fragments and inductively coupled plasma mass spectrometry (ICP-MS). Fungal community composition was primarily impacted by physical location while organic amendment triggered community shifts in the same direction in all four sites. Overall, the fungal community was strongly correlated to soil pH that, conversely, impacted soil ion availability. Fungal community dissimilarity was strongly correlated to soil ionome (ionic profile) variability. Network analysis has been conducted to explore the biotic interactions in soil ecosystem, in which species (nodes) are connected by pairwise interactions (links). The results revealed that organic amendment led to a higher number of correlated nodes to soil ions, links, modules (a group of nodes more densely connected to each other than to nodes outside the group), and positive links within and between modules. Moreover, specific fungal modules were independently correlated with soil ions, suggesting that each module represents a functional guild or collection of similar fungal ecotypes. Module size (number of nodes in a module) exhibited no apparent influence on the scale of these correlations. The increase in cooperative/synergistic interactions with organic amendment suggests that application results in a better-organized and more efficient community with enhanced potential soil fungal interactions mediated by alterations in the soil ionome. Overall, this study indicates that these less commonly measured soil ions play an important role and may be used to reveal previously undetermined drivers of the soil microbial community.

Biofuel-cropping systems, projected for large land areas, can potentially change their soil microbiome and the ecosystem services they catalyze. We determined the bacterial community composition and relevant soil properties for samples collected after 6 crop years at 0–10 cm, 10–25 cm, 25–50 cm, and 50–100 cm under corn, switchgrass, Miscanthus, and restored prairie, as well as 0–10 cm under six additional candidate biofuel crops in replicate side-by side plots. Deep sequencing of the 16S rRNA-V4 region established that soil bacterial communities were significantly differentiated by depth as determined by proportional OTU abundance and composition, UniFrac distance, and taxonomic and indicator analyses. The cropping system significantly impacted bacterial community composition within the top three layers, with corn and switchgrass communities the most different within the 0–25 cm and 25–50 cm depths, respectively. The effects of crop type and depth co-mingled, likely attributed to differences in rooting depth and biomass among crops. Individual phyla demonstrated varying patterns with depth, with significant proportional decreases of Proteobacteria, Actinobacteria, Planctomycetes, and Bacteroidetes but proportional increases of Firmicutes from shallow to deep soils. The Acidobacteria, Verrucomicrobia, and Chloroflexi peaked in abundance in the middle layers, whereas Thaumarchaeota decreased in abundance. Importantly, some classes within the Acidobacteria, Verrucomicrobia, and Firmicutes followed contrasting patterns with depth suggesting that they have different ecological specializations. Poplar, followed by soils with perennial crops contained the most C in the surface soils, with data indicating that these differences will become more pronounced with time.

Worldwide, banana production is severely hindered by Fusarium wilt, a devastating disease caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). With no widely adopted efficient method of control or prevention, the emergence of a new Foc variant, tropical race 4 (Foc TR4), has led to the widespread destruction of plantations in Cavendish-producing areas. Recently, banana Fusarium wilt has been controlled by the consecutive application of biofertilizer (BIO) in newly reclaimed fields. In this study we examine the temporal effects of BIO versus compost application in newly converted banana fields on the composition and abundance of the rhizosphere bacterial and fungal communities and the survival of the biocontrol inoculant, Bacillus amyloliquefaciens NJN-6. Our findings show that BIO-amended rhizosphere soils increased the abundance of bacteria while decreasing fungal abundance. This corresponded to higher bacterial richness and diversity in the BIO amendment, while no trends were observed with the fungal community. Rhizosphere soil bacterial and fungal community composition were significantly different between BIO and compost amendment and treatment, not time, exhibited the largest impact. Other potential taxa involved in disease suppression were also identified, such as increased abundances of Sphingobium, Dyadobacter, and Cryptococcus and lower abundances of Fusarium, Ralstonia, and Burkholderia. Overall, decreased abundances of F. oxysporum and a lack of variability in the abundance of the biocontrol agent NJN-6 over three years contributed to disease suppression, in combination with alterations in fungal and bacterial composition and abundance, pointing to the sustainability of BIO as an amendment for disease suppression.

Soil-borne diseases, especially those caused by fungal pathogens, lead to profound annual yield losses. One key example for such a disease is Fusarium wilt disease in banana. In some soils, plants do not show disease symptoms, even if the disease-causing pathogens are present. However, the underlying agents that make soils suppressive against Fusarium wilt remain elusive. In this study, we aimed to determine the underlying microbial agents governing soil disease-suppressiveness. We traced the shift of microbiomes during the invasion of disease-causing Fusarium oxysporum f. sp. cubense in disease-suppressive and disease-conducive soils. We found distinct microbiome structures in the suppressive and conducive soils after pathogen invasion. The alpha diversity indices increased (or did not significantly change) and decreased, respectively, in the suppressive and conducive soils, indicating that the shift pattern of the microbiome with pathogen invasion was notably different between the suppressive and conductive soils. Microbiome networks were more complex with higher numbers of links and revealed more negative links, especially between bacterial taxa and the disease-causing Fusarium, in suppressive soils than in conducive soils. We identified the bacterial genera Chryseolinea, Terrimonas, and Ohtaekwangia as key groups that likely confer suppressiveness against disease-causing Fusarium. Overall, our study provides the first insights into agents potentially underlying the disease suppressiveness of soils against Fusarium wilt pathogen invasion. The results of this study may help to guide efforts for targeted cultivation and application of these potential biocontrol agents, which might lead to the development of effective biocontrol agents against Fusarium wilt disease.

A consortium of key bacterial taxa plays critical roles in the composting process. In order to elucidate the identity and mechanisms by which specific bacterial species drive high-efficiency composting, the succession of key bacterial consortia and extracellular enzymes produced during the composting process were monitored in composting piles with varying initial C/N ratios. Results showed that C/N ratios of 25 and 35 enhanced composting efficiency through elevated temperatures, higher germination indices, enhanced cellulose and hemicellulose degradation, and higher cellulase and dehydrogenase activities. The activities of cellulase and β-glucosidase, cellulase and protease, and cellulase and β-glucosidase exhibited significant relationships with bacterial community composition within the mesophilic, thermophilic, and mature phases, respectively. Putative key taxa, linked to a higher composting efficiency, such as Nonomuraea, Desemzia, Cellulosimicrobium, Virgibacillus, Clostridium, and Achromobacter, exhibited significantly positive relationships with extracellular enzyme activities, suggesting a significant contribution to these taxa to the development of composting maturity.

With the likelihood that changes in global climate will adversely affect the soil C reservoir in the northern circumpolar permafrost zone, an understanding of the potential role of diazotrophic communities in enhancing biological N2 fixation, which constrains both plant production and microbial decomposition in tundra soils, is important in elucidating the responses of soil microbial communities to global climate change. A recent study showed that the composition of the diazotrophic community in a tundra soil exhibited no change under a short-term (1.5-year) winter warming experiment. However, it remains crucial to examine whether the lack of diazotrophic community responses to warming is persistent over a longer time period as a possibly important mechanism in stabilizing tundra soil C. Through a detailed characterization of the effects of winter warming on diazotrophic communities, we showed that a long-term (5-year) winter warming substantially enhanced diazotrophic abundance and altered community composition, though soil depth had a stronger influence on diazotrophic community composition than warming. These changes were best explained by changes in soil moisture, soil thaw duration, and plant biomass. These results provide crucial insights into the potential factors that may impact future C and N availability in tundra regions., Tundra ecosystems are typically carbon (C) rich but nitrogen (N) limited. Since biological N2 fixation is the major source of biologically available N, the soil N2-fixing (i.e., diazotrophic) community serves as an essential N supplier to the tundra ecosystem. Recent climate warming has induced deeper permafrost thaw and adversely affected C sequestration, which is modulated by N availability. Therefore, it is crucial to examine the responses of diazotrophic communities to warming across the depths of tundra soils. Herein, we carried out one of the deepest sequencing efforts of nitrogenase gene (nifH) to investigate how 5 years of experimental winter warming affects Alaskan soil diazotrophic community composition and abundance spanning both the organic and mineral layers. Although soil depth had a stronger influence on diazotrophic community composition than warming, warming significantly (P

Accumulating evidence indicates that the functional rather than taxonomic composition of microbial communities is closely correlated to local environmental factors. While composting is a widely accepted practice, specific knowledge of how fungal functional groups interact during the composting process remains limited. To address this, the impact of the initial C/N ratio of composting material on fungal community was analyzed in order to reveal the succession of functional diversity. Compared with the raw materials, the final composting product significantly reduced the relative abundances of plant and animal pathogens. Abundances of plant and animal pathogens, as well as dung saprotrophs, were negatively correlated with compost maturity, while abundances of wood saprotrophs exhibited positive correlations. Specific OTUs that showing highly abundant in each treatment were expected to compete for environmental preferences (niches) and/or interact with each other in positive (facilitative) ways. OTU2 (wood saprotroph) exhibiting the highest occurrence was negatively related to OTU7 (animal pathogen) and OTU4 (plant pathogen) during the mesophilic phase. Taken together, high-efficiency composting is represented as pattern variations of fungal community with a process of gradual decline of plant and animal pathogens as well as dung saprotrophs.

The capacity for soils to perform vital functions, such as agricultural production, is dependent on numerous properties. Their simultaneous effect on soil biota is of interest to assess impacts of agricultural management. Using a soil quality index (SQI) based on a priori assumptions of eight soil physico-chemical properties that promote plant growth and microbial biomass, we sought to: 1) investigate the effect of land use on SQI; and 2) test a relationship between SQI and the composition of microbial functional genes. In 29 soils under four distinct land uses (cotton, wheat, pasture and native vegetation) gene composition was most distinct in cotton. The SQI followed the gradient cotton

The complex structural and functional responses of agricultural soil microbial communities to the addition of carbonaceous compounds such as biochar remain poorly understood. This severely limits the predictive ability for both the potential enhancement of soil fertility and greenhouse gas mitigation. In this study, we utilized shotgun metagenomics in order to decipher changes in the microbial community in soil microcosms after 14 days of incubation at 23°C, which contained soils from biochar-amended and control plots cultivated with Napier grass. Our analyses revealed that biochar-amended soil microbiomes exhibited significant shifts in both community composition and predicted metabolism. Key metabolic pathways related to carbon turnover, such as the utilization of plant-derived carbohydrates as well as denitrification, were enriched under biochar amendment. These community shifts were in part associated with increased soil carbon, such as labile and aromatic carbon compounds, which was likely stimulated by the increased available nutrients associated with biochar amendment. These findings indicate that the soil microbiome response to the combination of biochar addition and to incubation conditions confers enhanced nutrient cycling and a small decrease in CO2 emissions and potentially mitigates nitrous oxide emissions. IMPORTANCE The incorporation of biochar into soil is a promising management strategy for sustainable agriculture owing to its potential to sequester carbon and improve soil fertility. Expanding the addition of biochar to large-scale agriculture hinges on its lasting beneficial effects on the microbial community. However, there exists a significant knowledge gap regarding the specific role that biochar plays in altering the key biological soil processes that influence plant growth and carbon storage in soil. Previous studies that examined the soil microbiome under biochar amendment principally characterized only how the composition alters in response to biochar amendment. In the present study, we shed light on the functional alterations of the microbial community response 2 years after biochar amendment. Our results show that biochar increased the abundance of genes involved in denitrification and carbon turnover and that biochar-amended soil microcosms had a reduction in cumulative CO2 production.

Increases in carbon (C) inputs to soil can replenish soil organic C (SOC) through various mechanisms. However, recent studies have suggested that the increased C input can also stimulate the decomposition of old SOC via priming. Whether the loss of old SOC by priming can override C replenishment has not been rigorously examined. Here we show, through data–model synthesis, that the magnitude of replenishment is greater than that of priming, resulting in a net increase in SOC by a mean of 32% of the added new C. The magnitude of the net increase in SOC is positively correlated with the nitrogen-to-C ratio of the added substrates. Additionally, model evaluation indicates that a two-pool interactive model is a parsimonious model to represent the SOC decomposition with priming and replenishment. Our findings suggest that increasing C input to soils likely promote SOC accumulation despite the enhanced decomposition of old C via priming., The magnitudes of replenishment and priming, two important but opposing fluxes in soil organic carbon (SOC) dynamics, have not been compared. Here the authors show that the magnitude of replenishment is greater than that of priming, resulting in a net SOC accumulation after additional carbon input to soils.

The sequencing chips and kits of the Ion Torrent Personal Genome Machine (PGM), which employs semiconductor technology to measure pH changes in polymerization events, have recently been upgraded. The quality of PGM sequences has not been reassessed, and results have not been compared in the context of a gene-targeted microbial ecology study. To address this, we compared sequence profiles across available PGM chips and chemistries and with 454 pyrosequencing data by determining error types and rates and diazotrophic community structures. The PGM was then used to assess differences in nifH -harboring bacterial community structure among four corn-based cropping systems. Using our suggested filters from mock community analyses, the overall error rates were 0.62, 0.36, and 0.39% per base for chips 318 and 314 with the 400-bp kit and chip 318 with the Hi-Q chemistry, respectively. Compared with the 400-bp kit, the Hi-Q kit reduced indel rates by 28 to 59% and produced one to seven times more reads acceptable for downstream analyses. The PGM produced higher frameshift rates than pyrosequencing that were corrected by the RDP FrameBot tool. Significant differences among platforms were identified, although the diversity indices and overall site-based conclusions remained similar. For the cropping system analyses, a total of 6,182 unique NifH operational taxonomic units at 5% amino acid dissimilarity were obtained. The current crop type, as well as the crop rotation history, significantly influenced the composition of the soil diazotrophic community detected.

The continuous cropping of banana in the same field may result in a serious soil-borne Fusarium wilt disease and a severe yield decline, a phenomenon known as soil sickness. Although soil microorganisms play key roles in maintaining soil health, the alternations of soil microbial community and relationship between these changes and soil sickness under banana monoculture are still unclear. Bacterial and fungal communities in the soil samples collected from banana fields with different monoculture spans were profiled by sequencing of the 16S rRNA genes and internal transcribed spacer using the MiSeq platform to explore the relationship between banana monoculture and Fusarium wilt disease in the present study. The results showed that successive cropping of banana was significantly correlated with the Fusarium wilt disease incidence. Fungal communities responded more obviously and quickly to banana consecutive monoculture than bacterial community. Moreover, a higher fungal richness significantly correlated to a higher banana Fusarium wilt disease incidence but a lower yield. Banana fungal pathogenic genus of Fusarium and Phyllosticta were closely associated with banana yield depletion and disease aggravation. Potential biocontrol agents, such as Funneliformis, Mortierella, Flavobacterium, and Acidobacteria subgroups, exhibited a significant correlation to lower disease occurrence. Further networks analysis revealed that the number of functionally interrelated modules decreased, the composition shifted from bacteria- to fungi-dominated among these modules, and more resources-competitive interactions within networks were observed after banana long-term monoculture. Our results also showed that bacterial and fungal communities were mainly driven by soil organic matter. Overall, the findings indicated that the bacterial and fungal community structures altered significantly after banana long-term monoculture, and the fungal richness, abundance of Fusarium, interactions between and within bacteria and fungi in ecological networks, and soil organic matter were associated with banana soil-borne Fusarium wilt disease.

Predicting N losses in flooded agricultural systems is complex because of the large number of potential N transformation pathways. Although coupled nitrification/denitrification at the flooded sediment near-surface oxic/anoxic interface is a recognized pathway of fertilizer N loss, the role of the newly discovered anaerobic ammonium oxidation (anammox) pathway in agricultural systems is poorly understood. A survey study was implemented to characterize sediment denitrification rates, anammox activity, and fluxes of ammonium and nitrate, and the vertical distribution of N functional genes were measured in Hawaiian taro (Colocasia esculenta) fields under three fertilizer management regimens (conventional, organic, and hybrid) as a model for flooded agroecosystems. Potential denitrification rates and anammox activity were measured using a slurry-based isotope pairing technique, and fluxes were determined by porewater modeling. Although significant numbers of anammox 16S rRNA genes were detected, only negligible anammox activity was found, illustrating the need to quantify not only the presence but also the activity of these bacteria. Quantitative polymerase chain reaction was performed for bacterial amoA, nirS, nosZ, and the 16S rRNA gene at 1-cm soil depth increments. Slurry isotope pairing technique-based potential denitrification rates (4.3-12.3 mmol N 2 m -2 day -1 ) were on the high end of previous anaerobic incubation studies. Flux-derived denitrification rates were up to three orders of magnitude lower than slurry isotope pairing technique rates, suggesting strong nitrification regulation. Organically managed fields exhibited significantly lower slurry and flux-based denitrification rates than conventional and hybrid systems. This study supports the use of alternative fertilizer management techniques in flooded agroecosystems by the mitigation of N losses through denitrification and decreased overall N flux rates.

Characterizing the rhizosphere microbial community composition associated with enhanced crop yield is an important first step towards understanding the role of the microbiota in soil fertility. In the present study, we conducted a two-seasons field experiment in a maize-cabbage cropping system under chemical (CF), organic (OF) and bio-organic (BOF) fertilizer regimes as a model to investigate the combinatory effect of fertilizer treatment and crop type on rhizosphere soil microbiota by targeted sequencing of both the bacterial and fungal communities. The two-seasons sustainable application of bio-organic fertilizer (BOF) containing Trichoderma effectively increased maize and cabbage yields, whereas organic fertilizer (OF) increased but not significant, compared to the application of chemical fertilizer (CF). Both fertilizer treatment and crop type induced a significant effect on soil physiochemical properties and were the major factors that impacted the composition of the rhizosphere soil microbiome. Relative abundances of Trichoderma were significantly enhanced in the BOF treatment, compared to the OF and CF treatments, and exhibited significant positive relationships with crop yield improvement. The application of bio-organic fertilizer may enhance the growth promotion effect of Trichoderma and increase the abundance of potentially beneficial microbial groups such as the genera Cladorrhinum and Massilia, which were found to be highly correlated to increased crop yields. Overall, the influence of bio-organic fertilizer on crop yield is proposed to be through mechanisms by introduction of the target strain NJAU 4742 and stimulation of a potentially beneficial microbial consortia, in combination with alterations in fungal and bacterial composition and abundance, leading to an enhancement in crop yield.

We analyzed the microbial community that developed after 4 years of testing different soil-crop management systems in the savannah–forest transition zone of Eastern Ghana where management systems can rapidly alter stored soil carbon as well as soil fertility. The agricultural managements were: (i) the local practice of fallow regrowth of native elephant grass ( Pennisetum purpureum ) followed by biomass burning before planting maize in the spring, (ii) the same practice but without burning and the maize receiving mineral nitrogen fertilizer, (iii) a winter crop of a legume, pigeon pea ( Cajanus cajan ), followed by maize, (iv) vegetation free winter period (bare fallow) followed by maize, and (v) unmanaged elephant grass-shrub vegetation. The mean soil organic carbon (SOC) contents of the soils after 4 years were: 1.29, 1.67, 1.54, 0.80 and 1.34%, respectively, differences that should affect resources for the microbial community. From about 290,000 sequences obtained by pyrosequencing the SSU rRNA gene, canonical correspondence analysis showed that SOC was the most important factor that explained differences in microbial community structure among treatments. This analysis as well as phylogenetic ecological network construction indicated that members of the Acidobacteria GP4 and GP6 were more abundant in soils with relatively high SOC whereas Acidobacteria GP1, GP7, and Actinobacteria were more prevalent in soil with lower SOC. Burning of winter fallow vegetation led to an increase in Bacillales, especially those belonging to spore-forming genera. Of the managements, pigeon-pea cultivation during the winter period promoted a higher microbial diversity and also sequestered more SOC, presumably improving soil structure, fertility, and resiliency.

The fate of fertilizer nitrogen (N) in flooded agroecosystems is difficult to predict given the multitude of potential N transformation pathways. In particular, rhizosphere effects are known to play a significant role in N cycling, but are especially difficult to quantify in large emergent macrophytes. To address these issues, we utilized a whole core 15 NH 4 + perfusion technique with porewater equilibrators for the extraction of 14+15 N–NO 3 − , NH 4 + , and N 2 . Sub-surface denitrification was found to be an important N loss pathway in wetland sediments vegetated with aerenchymatous taro ( Colocasia esculenta ) versus bare sediments. Driven by hypothesized thermo-osmotic mechanisms linked to photosynthesis, diurnal O 2 transport into the sub-surface stimulated nitrification–denitrification in the extensive root rhizosphere. Porewater denitrification rates were also positively influenced by airflow across leaf surfaces. Depth-integrated porewater denitrification rates in this system were very high, ranging from 23 to 845 μmol N 2 m −2 h −1 . The N cycling functional genes nosZ and amoA were found at high abundances throughout the sub-surface with nirS dominating nitrite reduction in these sediments. Overall we were able to account for >82% of added 15 NH 4 + in the vegetated cores over a ten-day incubation through both plant incorporation and surface/sub-surface coupled nitrification–denitrification. In summary, these results suggested (1) that oxygen flux through the taro stem and root system into the flooded sediment may be an important driver of nitrification and coupled denitrification in these systems, and (2) that oxygen flux is mediated by air movement (wind) and the diurnal light-cycle related to photosynthesis.

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.

The significance of anaerobic ammonium oxidation (anammox) for nitrogen removal in deep continental margin sediments was studied with 15N amendments to suboxic sediments collected from 2800–3100-m water depth at eight sites in the Cascadia Basin (eastern North Pacific Ocean). Consistent with earlier data from deep continental margin sediments, pore-water distributions of inorganic N indicated NH z removal from suboxic zone sediments, likely due to reaction with nitrate. Anammox rates estimated from suboxic sediment incubations with 15N-labled substrates ranged between 0.065 and 1.7 nmol N mL21 h21 (wet sediment), which suggested that anammox was responsible for the observed NH z removal. Anammox and denitrification rates derived from NH z and NO { pore-water profiles were 32–82 mmol N m 22 d21 and 50–110 m mol Nm 22 d21, respectively. The average contribution of anammox to total N2 production was 40% (15N-amended sediment incubations) to 42% (flux from pore-water inorganic N), which does not support earlier reports that suggested that the relative importance of anammox increased with water depth and thereby should dominate over denitrification at depths greater than 1000 m.

Enzyme catalyzed reactions are generally considered the rate-limiting step in organic matter degradation and may be significantly influenced by the structure and composition of plant communities. Changes in these rates have the potential to effect long-term peat accumulation and influence the topography of a wetland ecosystem. To determine habitat influences on enzyme activities, we examined slough and sawgrass plots within enriched and reference phosphorus (P) sites in the Everglades. Assays were performed for the enzymes involved in carbon (C), nitrogen (N), and P cycling and lignin depolymerization. Enzyme activities were normalized and analyzed in terms of a resource allocation strategy. Plant composition was found to significantly alter the allocation of enzymatic resources due to varying substrate complexities. Potential decomposition in the slough was less influenced by lignin than in the sawgrass habitats. Additionally, an index relating hydrolytic and oxidative enzymes was significantly greater in the slough habitats, whereas C/N ratios were significantly lower. These indices suggest more favorable decomposition conditions and thus slower peat accretion within the slough communities, which may contribute to the development of elevation differences within the sawgrass ridge and slough topography of the Everglades.

Eutrophication caused by anthropogenic nutrient inputs is one of the greatest threats to the integrity of freshwater wetlands. The resultant changes in organic carbon cycling and nutrient mineralization may be expressed through increased decomposition rates, which are ultimately dependent on the metabolism of the resident microbial community. Specifically, microbial nutrient acquisition is controlled through the activity of enzymes, which are in turn influenced by local biogeochemical conditions. This study examines enzyme activities along distinct North-South P gradients within four distinct hydrologic units of the Florida Everglades. The results indicate that nutrient enriched sites exhibit lower N and P limitations on microbially constrained C mineralization, in addition to enhanced cellulose decomposition rates. Nutrient loading resulted in decreased microbial mobilization of resources for P mineralization, resulting in greater energetic allocation for C mineralization. Additionally, N appears to become less limiting to C mineralization in the enriched sites within Everglades National Park, the least P enriched area within the Everglades. A simple two component model, incorporating total P and the relationship between the enzymes involved in C and P mineralization accounted for between 46 and 92% of the variability in measured cellulose decomposition rates and thus demonstrates the significant influence that P loading plays in these systems. These results also suggest there is an environmental threshold TP concentration below which changes in enzyme-based resource allocation will not occur.

Panama disease caused by Fusarium oxysporum f. sp. cubense infection on banana is devastating banana plantations worldwide. Biological control has been proposed to suppress Panama disease, though the stability and survival of bio-control microorganisms in field setting is largely unknown. In order to develop a bio-control strategy for this disease, 16S rRNA gene sequencing was used to assess the microbial community of a disease-suppressive soil. Bacillus was identified as the dominant bacterial group in the suppressive soil. For this reason, B. amyloliquefaciens NJN-6 isolated from the suppressive soil was selected as a potential bio-control agent. A bioorganic fertilizer (BIO), formulated by combining this isolate with compost, was applied in nursery pots to assess the bio-control of Panama disease. Results showed that BIO significantly decreased disease incidence by 68.5%, resulting in a doubled yield. Moreover, bacterial community structure was significantly correlated to disease incidence and yield and Bacillus colonization was negatively correlated with pathogen abundance and disease incidence, but positively correlated to yield. In total, the application of BIO altered the rhizo-bacterial community by establishing beneficial strains that dominated the microbial community and decreased pathogen colonization in the banana rhizosphere, which plays an important role in the management of Panama disease.

Panama disease caused by Fusarium oxysporum f. sp. cubense infection on banana is devastating banana plantations worldwide. Biological control has been proposed to suppress Panama disease, though the stability and survival of bio-control microorganisms in field setting is largely unknown. In order to develop a bio-control strategy for this disease, 16S rRNA gene sequencing was used to assess the microbial community of a disease-suppressive soil. Bacillus was identified as the dominant bacterial group in the suppressive soil. For this reason, B. amyloliquefaciens NJN-6 isolated from the suppressive soil was selected as a potential bio-control agent. A bioorganic fertilizer (BIO), formulated by combining this isolate with compost, was applied in nursery pots to assess the bio-control of Panama disease. Results showed that BIO significantly decreased disease incidence by 68.5%, resulting in a doubled yield. Moreover, bacterial community structure was significantly correlated to disease incidence and yield and Bacillus colonization was negatively correlated with pathogen abundance and disease incidence, but positively correlated to yield. In total, the application of BIO altered the rhizo-bacterial community by establishing beneficial strains that dominated the microbial community and decreased pathogen colonization in the banana rhizosphere, which plays an important role in the management of Panama disease.

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of ∼700-bp 16S rRNA gene sequences with >96% homology to the “ Candidatus Scalindua” group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to “ Ca . Scalindua,” “ Candidatus Brocadia,” and “ Candidatus Kuenenia.” This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils 'suppressive' or 'non-suppressive' for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria , Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. © 2014 Penton et al.

We analyzed the microbial community that developed after 4 years of testing different soil-crop management systems in the savannah-forest transition zone of Eastern Ghana where management systems can rapidly alter stored soil carbon as well as soil fertility. The agricultural managements were: (i) the local practice of fallow regrowth of native elephant grass (Pennis e tum purpureum) followed by biomass burning before planting maize in the spring, (ii) the same practice but without burning and the maize receiving mineral nitrogen fertilizer, (iii) a winter crop of a legume, pigeon pea (Cajanus cajan), followed by maize, (iv) vegetation free winter period (bare fallow) followed by maize, and (v) unmanaged elephant grass-shrub vegetation. The mean soil organic carbon (SOC) contents of the soils after 4 years were: 1.29, 1.67, 1.54, 0.80 and 1.34%, respectively, differences that should affect resources for the microbial community.From about 290,000 sequences obtained by pyrosequencing the SSU rRNA gene, canonical correspondence analysis showed that SOC was the most important factor that explained differences in microbial community structure among treatments. This analysis as well as phylogenetic ecological network construction indicated that members of the Acidobacteria GP4 and GP6 were more abundant in soils with relatively high SOC whereas Acidobacteria GP1, GP7, and Actinobacteria were more prevalent in soil with lower SOC. Burning of winter fallow vegetation led to an increase in Bacillales, especially those belonging to spore-forming genera. Of the managements, pigeon-pea cultivation during the winter period promoted a higher microbial diversity and also sequestered more SOC, presumably improving soil structure, fertility, and resiliency. © 2013 Elsevier Ltd.

Targeting sequencing to genes involved in key environmental processes, i.e., ecofunctional genes, provides an opportunity to sample nature's gene guilds to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. The overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknown diversity. To illustrate current issues surrounding amplicon read processing we provide examples for three ecofunctional gene groups. A combination of in silico, environmental and cultured strain sequences was used to test new primers targeting the dioxin and dibenzofuran degrading genes dxnA1, dbfA1, and carAa. The majority of obtained environmental sequences were classified into novel sequence clusters, illustrating the discovery value of the approach. For the nitrite reductase step in denitrification, the well-known nirK primers exhibited deficiencies in reference database coverage, illustrating the need to refine primer-binding sites and/or to design multiple primers, while nirS primers exhibited bias against five phyla. Amino acid-based OTU clustering of these two N-cycle genes from soil samples yielded only 114 unique nirK and 45 unique nirS genus-level groupings, likely a reflection of constricted primer coverage. Finally, supervised and non-supervised OTU analysis methods were compared using the nifH gene of nitrogen fixation, with generally similar outcomes, but the clustering (non-supervised) method yielded higher diversity estimates and stronger site-based differences. High throughput amplicon sequencing can provide inexpensive and rapid access to nature's related sequences by circumventing the culturing barrier, but each unique gene requires individual considerations in terms of primer design and sequence processing and classification.