Conserved and reproducible bacterial communities associate with extraradical hyphae of arbuscular mycorrhizal fungi

Extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) extend from plant roots into the soil environment and interact with soil microbial communities. Evidence of positive and negative interactions between AMF and soil bacteria point to functionally important ERH-associated communities. To characterize communities associated with ERH and test controls on their establishment and composition, we utilized an in-growth core system containing a live soil–sand mixture that allowed manual extraction of ERH for 16S rRNA gene amplicon profiling. Across experiments and soils, consistent enrichment of members of the Betaproteobacteriales, Myxococcales, Fibrobacterales, Cytophagales, Chloroflexales, and Cellvibrionales was observed on ERH samples, while variation among samples from different soils was observed primarily at lower taxonomic ranks. The ERH-associated community was conserved between two fungal species assayed, Glomus versiforme and Rhizophagus irregularis, though R. irregularis exerted a stronger selection and showed greater enrichment for taxa in the Alphaproteobacteria and Gammaproteobacteria. A distinct community established within 14 days of hyphal access to the soil, while temporal patterns of establishment and turnover varied between taxonomic groups. Identification of a conserved ERH-associated community is consistent with the concept of an AMF microbiome and can aid the characterization of facilitative and antagonistic interactions influencing the plant-fungal symbiosis.Subject terms: Symbiosis, Microbiome     

Dual inoculation of dark septate endophytes and Trichoderma viride drives plant performance and rhizosphere microbiome adaptations of Astragalus mongholicus to drought

Rhizosphere microbiome adapts their structural compositions to water scarcity and have the potential to mitigate drought stress of plants. To unlock this potential, it is crucial to understand community responses to drought in the interplay between soil properties, water management and exogenous microbes interference. Inoculation with dark septate endophytes (DSE) (Acrocalymma vagum, Paraboeremia putaminum) and Trichoderma viride on Astragalus mongholicus grown in the non-sterile soil was exposed to drought. Rhizosphere microbiome were assessed by Illumina MiSeq sequencing of the 16S and ITS2 rRNA genes. Inoculation positively affected plant growth depending on DSE species and water regime. Ascomycota, Proteobacteria, Actinobacteria, Chloroflexi and Firmicutes were the dominant phyla. The effects of dual inoculation on bacterial community were greater than those on fungal community, and combination of P. putaminum and T. viride exerted a stronger impact on the microbiome under drought stress. The observed changes in soil factors caused by inoculation could be explained by the variations in microbiome composition. Rhizosphere microbiome mediated by inoculation exhibited distinct preferences for various growth parameters. These findings suggest that dual inoculation of DSE and T. viride enriched beneficial microbiota, altered soil nutrient status and might contribute to enhance the cultivation of medicinal plants in dryland agriculture.     

Quantitative Microbial Ecology through Stable Isotope Probing

Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon''s density shift relative to that taxon''s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in ¹⁸O and ¹³C composition after exposure to [¹⁸O]water or [¹³C]glucose. The addition of glucose increased the assimilation of ¹⁸O into DNA from [¹⁸O]water. However, the increase in ¹⁸O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.     

Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea

The hydrolysis of urea as a source of ammonia has been proposed as a mechanism for the nitrification of ammonia-oxidizing bacteria (AOB) in acidic soil. The growth of Nitrososphaera viennensis on urea suggests that the ureolysis of ammonia-oxidizing archaea (AOA) might occur in natural environments. In this study, ¹⁵N isotope tracing indicates that ammonia oxidation occurred upon the addition of urea at a concentration similar to the in situ ammonium content of tea orchard soil (pH 3.75) and forest soil (pH 5.4) and was inhibited by acetylene. Nitrification activity was significantly stimulated by urea fertilization and coupled well with abundance changes in archaeal amoA genes in acidic soils. Pyrosequencing of 16S rRNA genes at whole microbial community level demonstrates the active growth of AOA in urea-amended soils. Molecular fingerprinting further shows that changes in denaturing gradient gel electrophoresis fingerprint patterns of archaeal amoA genes are paralleled by nitrification activity changes. However, bacterial amoA and 16S rRNA genes of AOB were not detected. The results strongly suggest that archaeal ammonia oxidation is supported by hydrolysis of urea and that AOA, from the marine Group 1.1a-associated lineage, dominate nitrification in two acidic soils tested.     

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or ¹³C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of ¹⁴CO₂ from ¹⁴C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307 bp from aerobic soil and 212 bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and ¹³C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of ¹³C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the ¹³C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.     

Bioorganic Fertilizer Enhances Soil Suppressive Capacity against Bacterial Wilt of Tomato

Tomato bacterial wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases. Many strategies have been taken to improve soil suppressiveness against this destructive disease, but limited success has been achieved. In this study, a novel bioorganic fertilizer revealed a higher suppressive ability against bacterial wilt compared with several soil management methods in the field over four growing seasons from March 2011 to July 2013. The application of the bioorganic fertilizer significantly (P<0.05) reduced disease incidence of tomato and increased fruit yields in four independent trials. The association among the level of disease incidence, soil physicochemical and biological properties was investigated. The soil treated with the bioorganic fertilizer increased soil pH value, electric conductivity, organic carbon, NH₄ ⁺-N, NO₃ ^--N and available K content, microbial activities and microbial biomass carbon content, which were positively related with soil suppressiveness. Bacterial and actinomycete populations assessed using classical plate counts were highest, whereas R. solanacearum and fungal populations were lowest in soil applied with the bioorganic fertilizer. Microbial community diversity and richness were assessed using denaturing gel gradient electrophoresis profile analysis. The soil treated with the bioorganic fertilizer exhibited higher bacterial community diversity but lower fungal community diversity. Redundancy analysis showed that bacterial community diversity and richness negatively related with bacterial wilt suppressiveness, while fungal community richness positively correlated with R. solanacearum population. We concluded that the alteration of soil physicochemical and biological properties in soil treated with the bioorganic fertilizer induced the soil suppressiveness against tomato bacterial wilt.     

Effect of monospecific and mixed Cunninghamia lanceolata plantations on microbial community and two functional genes involved in nitrogen cycling

Conversion of native broadleaf forest (NF) and introduction of broadleaf species into monospecific Cunninghamia lanceolata plantations are silvicultural practices driven by the increasing demand for timber production. This study was conducted to evaluate the impacts of successive planting of C. lanceolata and mixed plantations (C. lanceolata-Michelia macclurei, CFM; C. lanceolata-Alnus cremastogyne, CFA; C. lanceolata-Kalopanax septemlobus, CFK) on microbial community diversity. Microbial biomass (MBC) was assessed using chloroform fumigation-extraction. Using denaturing gradient gel electrophoresis (DGGE), we examined the biodiversity within eubacterial (16S rRNA gene) and fungal (28S rRNA gene) species and two genes involved in N cycling: nifH and amoA. Microbial community diversities and microbial biomass decreased as NF was substituted by successive plantings of C. lanceolata plantations, whereas the trend reversed after introducing the broadleaf, M. macclurei, into pure C. lanceolata plantations. A strong positive correlation between MBC changes and total organic C (TOC), total organic N (TON), available N and extractable C (C_ext) were seen, which suggests that MBC was tightly coupled with the content of soil organic matter. The Shannon index showed that bacterial diversity and two functional genes (nifH and amoA) diversities associated with monospecific C. lanceolata plantations were lower than that of NF or mixed C. lanceolata plantations, such as CFM and CFA, whereas the opposite was seen for fungal diversity. Bacterial diversity was positively correlated with pH, TOC, TON, C_ext and NH ₄ ⁺ -N; while fungal diversity was positively correlated with C/N ratio and negatively correlated with pH. Both nitrogen fixing and ammonia oxidizing bacterial diversities were positively correlated with pH. Thus, soil pH was not only significantly positively correlated with bacterial diversity (r?=?0.502, P?<?0.05), nifH gene diversity (r?=?0.564, P?<?0.01) and amoA gene diversity (r?=?0.659, P?<?0.001), but also negatively correlated with fungal diversity (r?=?? 0.505, P?<?0.05), which seemed to be responsible for the discrimination of the soil microbial communities among these plantations. These findings suggest that different silvicultural practices have significant impacts on the soil microbial community through influences on soil chemical properties.     

Rhizosphere soil bacterial communities and nitrogen cycling affected by deciduous and evergreen tree species

Deciduous and evergreen trees differ in their responses to drought and nitrogen (N) demand. Whether or not these functional types affect the role of the bacterial community in the N cycle during drought remains uncertain. Two deciduous tree species (Alnus cremastogyne, an N₂‐fixing species, and Liquidambar formosana) and two evergreen trees (Cunninghamia lanceolata and Pinus massoniana) were used to assess factors in controlling rhizosphere soil bacterial community and N cycling functions. Photosynthetic rates and biomass production of plants, 16S rRNA sequencing and N‐cycling‐related genes of rhizosphere soil were measured. The relative abundance of the phyla Actinobacteria and Firmicutes was higher, and that of Proteobacteria, Acidobacteria, and Gemmatimondaetes was lower in rhizosphere soil of deciduous trees than that of evergreen. Beta‐diversity of bacterial community also significantly differed between the two types of trees. Deciduous trees showed significantly higher net photosynthetic rates and biomass production than evergreen species both at well water condition and short‐term drought. Root biomass was the most important factor in driving soil bacterial community and N‐cycling functions than total biomass and aboveground biomass. Furthermore, 44 bacteria genera with a decreasing response and 46 taxa showed an increased response along the root biomass gradient. Regarding N‐cycle‐related functional genes, copy numbers of ammonia‐oxidizing bacteria (AOB) and autotrophic ammonia‐oxidizing archaea (AOA), N₂ fixation gene (nifH), and denitrification genes (nirK, nirS) were significantly higher in the soil of deciduous trees than in that of the evergreen. Structural equation models explained 50.2%, 47.6%, 48.6%, 49.4%, and 37.3% of the variability in copy numbers of nifH, AOB, AOA, nirK, and nirS, respectively, and revealed that root biomass had significant positive effects on copy numbers of all N‐cycle functional genes. In conclusion, root biomass played key roles in affecting bacterial community structure and soil N cycling. Our findings have important implications for our understanding of plants control over bacterial community and N‐cycling function in artificial forest ecosystems.     

Relationship between genetic variability in Rhizophagus irregularis and tolerance to saline conditions

Reclamation of saline soils produced by extraction of bitumen from oil sands is challenging. The main objective of this study was to select a salt-tolerant arbuscular mycorrhizal (AM) fungal isolate that could, in the future, be used to pre-inoculate plants used in reclamation of saline substrates produced by oil sand industry. To achieve this, the effects of NaCl, Na₂SO₄, and saline release water from composite tailings (CT) on hyphal growth of two AM fungal isolates from non-saline (Rhizophagus irregularis DAOM 181602, Rhizophagus sp. DAOM 227023) and three isolates of R. irregularis isolated from saline or sodic soils (DAOM 234181, DAOM241558, and DAOM241559) were tested in vitro. Pre-symbiotic hyphal growth of the five isolates, in absence of a host plant, decreased with increasing salt stress and no spores germinated in CT. The symbiotic extraradical phase of the four isolates of R. irregularis developed well in saline media compared to the Rhizophagus sp. Nevertheless, fungal development of the four R. irregularis isolates differed in saline media indicating phenotypic variations between isolates.     

Verrucomicrobia Are Candidates for Polysaccharide-Degrading Bacterioplankton in an Arctic Fjord of Svalbard

In Arctic marine bacterial communities, members of the phylum Verrucomicrobia are consistently detected, although not typically abundant, in 16S rRNA gene clone libraries and pyrotag surveys of the marine water column and in sediments. In an Arctic fjord (Smeerenburgfjord) of Svalbard, members of the Verrucomicrobia, together with Flavobacteria and smaller proportions of Alpha- and Gammaproteobacteria, constituted the most frequently detected bacterioplankton community members in 16S rRNA gene-based clone library analyses of the water column. Parallel measurements in the water column of the activities of six endo-acting polysaccharide hydrolases showed that chondroitin sulfate, laminarin, and xylan hydrolysis accounted for most of the activity. Several Verrucomicrobia water column phylotypes were affiliated with previously sequenced, glycoside hydrolase-rich genomes of individual Verrucomicrobia cells that bound fluorescently labeled laminarin and xylan and therefore constituted candidates for laminarin and xylan hydrolysis. In sediments, the bacterial community was dominated by different lineages of Verrucomicrobia, Bacteroidetes, and Proteobacteria but also included members of multiple phylum-level lineages not observed in the water column. This community hydrolyzed laminarin, xylan, chondroitin sulfate, and three additional polysaccharide substrates at high rates. Comparisons with data from the same fjord in the previous summer showed that the bacterial community in Smeerenburgfjord changed in composition, most conspicuously in the changing detection frequency of Verrucomicrobia in the water column. Nonetheless, in both years the community hydrolyzed the same polysaccharide substrates.     

Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.     

Soil nitrogen cycling rates in low arctic shrub tundra are enhanced by litter feedbacks

Shrub growth has increased across the Arctic in recent decades and is strongly limited by soil nitrogen (N) availability. In order to understand the role of N in controlling shrub growth, we compared N-cycling in tall birch (Betula glandulosa) and surrounding dwarf birch hummock vegetation on similar soils in a Canadian low arctic site. Stable isotope tracer analysis revealed N pools and cycling rates were ～3 times larger and faster in the tall birch ecosystem in the late growing season, just prior to leaf senescence. Gross NH ₄ ⁺ -N production rates in these ecosystems correlated positively with larger pools and production rates of dissolved soil C and N, higher quality litter inputs and lower soil C. Analyses of the soil microbial community in both ecosystems indicated similar fungal dominance (epifluorescence microscopy) and similar compositions of the principal fungal or bacterial phylotypes (denaturing gradient gel electrophoresis). Together, these results strongly suggest that vegetation feedbacks associated with larger inputs of higher quality litter promote rapid soil N-cycling and enhanced shrub growth in tall birch tundra. We conclude that these litter-related feedbacks during summer may be as important as snow-shrub feedbacks in maintaining and promoting differences in shrub growth across the arctic landscape.     

Different Bacterial Populations Associated with the Roots and Rhizosphere of Rice Incorporate Plant-Derived Carbon

Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH₄, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with ¹³CO₂ for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with ¹³C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the “Spartobacteria” and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (∼20%) than did those in the rhizosphere (∼4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment.     

Growth and photosynthetic responses in Jatropha curcas L. seedlings of different provenances to watering regimes

Seedlings from four provenances of Jatropha curcas were subjected to 80, 50, and 30% of soil field capacity in potted experiments in order to study their responses to water availability. Our results showed that with the decline of soil water availability, plant growth, biomass accumulation, net photosynthetic rate, stomatal conductance (g_s), and transpiration rate (E) decreased, whereas leaf carbon isotope composition (δ¹³C), leaf pigment contents, and stomatal limitation value increased, while maximal quantum yield of PSII photochemistry was not affected. Our findings proved that stomatal limitation to photosynthesis dominated in J. curcas under low water availability. The increase of δ¹³C should be attributed to the decrease in g_s and E under the lowest water supply. J. curcas could adapt to low water availability by adjusting its plant size, stomata closure, reduction of E, increasing δ¹³C, and leaf pigment contents. Moreover, effects of provenance and the interaction with the watering regime were detected in growth and many physiological parameters. The provenance from xeric habitats showed stronger plasticity in the plant size than that from other provenances under drought. The variations may be used as criteria for variety/provenance selection and improvement of J. curcas performance.     

Bacterial Succession in a Petroleum Land Treatment Unit

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C₁₀ to C₃₂ range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.     

Effect of biofumigation and chemical fumigation on soil microbial community structure and control of pepper Phytophthora blight

Soil biofumigation with brassica plant residues has been shown to significantly suppress soilborne pathogen. However, little published data reported the impact of biofumigation on microbial community structure in pepper (Capsicum annuum L.) production systems under field conditions. Biofumigation with rapeseed (Brassica napus ‘Dwarf Essex’) meal and chemical fumigation with dazomet were tested to control the pepper disease caused by Phytophthora capsici. BF treatment showed the lowest disease incidence among these treatments. Effects on soil bacterial and fungal communities were assessed by denaturating gradient gel electrophoresis and the results showed that the biofumigation increased bacterial diversity and decreased fungal diversity. There was a negative correlation between soil bacterial diversity and disease incidence and a positive correlation between soil fungal diversity and disease incidence. Cloning of the microbial community showed that the microbial community structures were altered by biofumigation. Soil was also evaluated for their chemical properties. Biofumigation increased soil content of total N, NO₃ ^?–N, available P and available K. A significant correlation between soil microbial community structures and soil chemical properties was found. Overall, these results indicated that biofumigation reduced disease incidence of pepper through altering soil microbial community structures.     

Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing

Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [¹⁵N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in ¹⁵N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.     

Forest understory plant and soil microbial response to an experimentally induced drought and heat‐pulse event: the importance of maintaining the continuum

Drought duration and intensity are expected to increase with global climate change. How changes in water availability and temperature affect the combined plant–soil–microorganism response remains uncertain. We excavated soil monoliths from a beech (Fagus sylvatica L.) forest, thus keeping the understory plant–microbe communities intact, imposed an extreme climate event, consisting of drought and/or a single heat‐pulse event, and followed microbial community dynamics over a time period of 28 days. During the treatment, we labeled the canopy with ¹³CO₂ with the goal of (i) determining the strength of plant–microbe carbon linkages under control, drought, heat and heat–drought treatments and (ii) characterizing microbial groups that are tightly linked to the plant–soil carbon continuum based on ¹³C‐labeled PLFAs. Additionally, we used 16S rRNA sequencing of bacteria from the Ah horizon to determine the short‐term changes in the active microbial community. The treatments did not sever within‐plant transport over the experiment, and carbon sinks belowground were still active. Based on the relative distribution of labeled carbon to roots and microbial PLFAs, we determined that soil microbes appear to have a stronger carbon sink strength during environmental stress. High‐throughput sequencing of the 16S rRNA revealed multiple trajectories in microbial community shifts within the different treatments. Heat in combination with drought had a clear negative effect on microbial diversity and resulted in a distinct shift in the microbial community structure that also corresponded to the lowest level of label found in the PLFAs. Hence, the strongest changes in microbial abundances occurred in the heat–drought treatment where plants were most severely affected. Our study suggests that many of the shifts in the microbial communities that we might expect from extreme environmental stress will result from the plant–soil–microbial dynamics rather than from direct effects of drought and heat on soil microbes alone.     

Overstorey and juvenile response to thinning and drought in a jarrah (Eucalyptus marginata Donn ex Sm.) forest of southwestern Australia

Aims

Forest thinning is expected to affect tree water use and carbon assimilation, but the related influence from climate variability is little known. Recent forest thinning in the Wungong catchment coincided with a record dry year following the thinning, which provides a rare opportunity to understand the climate influence on the thinning effect.

Methods

A field experiment was conducted to examine changes before and after thinning, especially the rainfall, soil moisture, leaf water status, tissue isotope signature (¹³?C and ¹⁵?N) and N concentration of overstorey and understorey juvenile trees of Eucalyptus marginata (Donn ex Sm.).

Results

Despite the post-thinning drought, surface soil was moister and juvenile jarrah plants were less water stressed, attributable to reduced rain interception and transpiration as a result of less canopy cover. The overstorey was under stress but mainly due to drought rather than by thinning. The concentration of N declined in both tree stems and juvenile leaves along with available N in soil, suggesting a soil N limitation. No treatment effects were detected from leaf relative water content and tissue isotope signature (¹³?C and ¹⁵?N).

Conclusions

The drought effects were superimposed over the thinning effects on overstorey growth, with stemwood δ¹³C being a major indicator of water stress. The water relations and carbon assimilation of understorey juveniles were however dependent more on topsoil moisture, and the wetter soil during the year following thinning enhanced growth activity and hence the depletion of ¹³?C (more negative δ¹³C) in juvenile leaves.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.