STUDIES ON THE EFFECTS OF PLANT VARIETY AND ROOT EXUDATE COMPOUNDS ON THE SOIL MICROBIAL COMMUNITY

Plants modify the soil environment through their root system, changing its physical properties and exuding compounds that promote or inhibit the growth of certain microorganisms. Therefore the structure of the soil microbial community is different in the rhizosphere than in the bulk soil. This dissertation introduces three research projects that investigated the effects of specific root exudate compounds on the soil microbial community structure, and plant cultivar specific differences in the rhizosphere microbiota. The progenitor of maize is Balsas teosinte (Zea mays subsp. parviglumis). In a greenhouse experiment we compared the structure and function of its bacterial and fungal rhizosphere community with that of domesticated corn cultivars sweet corn and popping corn by terminal restriction fragment length polymorphism, fatty acid methyl ester analysis, and soil enzyme assays. The results allude to functional and structural differences in the rhizosphere microbial communities of the corn varieties that could lead to useful discoveries on how corn domestication has altered rhizosphere processes. To study how root exudate flavonoids 7,4′-dihydroxyflavone and naringenin influence the soil bacterial community structure we constructed model systems to approximate the flavonoid exudation of Medicago sativa roots. Soil samples from the model systems were subjected to ATP assays and 16S rRNA gene amplicon sequencing. Our results suggest that 7,4′-dihydroxyflavone can interact with a diverse range of soil bacteria, including members of Acidobacteria subdivision 4, Gaiellales, Nocardioidaceae, and Thermomonosporaceae and may have other functions in the rhizosphere in addition to nod-gene induction in the legume–rhizobia symbiosis. Hydroxyproline is the most common amino acid in plant cell wall proteins and serves as an important carbon and nitrogen source for soil bacteria. We treated soil with the L or the D enantiomer of hydroxyproline and collected samples for 16S rRNA gene amplicon sequencing three and seven days after the treatment. The L- and D-hydroxyproline treatments induced very similar responses in the bacterial community structure, but there were several differentially abundant groups. Our results inform about the role of hydroxyproline in shaping the soil microbial community in the rhizosphere and about the catabolism of its enantiomers in the soil

The Effect of Root Exudate 7,4\u27-Dihydroxyflavone and Naringenin on Soil Bacterial Community Structure

Our goal was to investigate how root exudate flavonoids influence the soil bacterial community structure and to identify members of the community that change their relative abundance in response to flavonoid exudation. Using a model system that approximates flavonoid exudation of Medicago sativa roots, we treated a soil with 7,4′-dihydroxyflavone and naringenin in two separate experiments using three different rates: medium (equivalent to the exudation rate of 7,4′-dihydroxyflavone from M. sativa seedlings), high (10× the medium rate), and low (0.1× the medium rate). Controls received no flavonoid. Soil samples were subjected to ATP assays and 16S rRNA gene amplicon sequencing. The flavonoid treatments caused no significant change in the soil ATP content. With the high 7,4′-dihydroxyflavone treatment rate, operational taxonomic units (OTUs) classified as Acidobacteria subdivision 4 increased in relative abundance compared with the control samples, whereas OTUs classified as Gaiellales, Nocardioidaceae, and Thermomonosporaceae were more prevalent in the control. The naringenin treatments did not cause significant changes in the soil bacterial community structure. Our results suggest that the root exudate flavonoid 7,4′-dihydroxyflavone can interact with a diverse range of soil bacteria and may have other functions in the rhizosphere in addition to nod gene induction in legume—rhizobia symbiosis

Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions

peer-reviewedThe importance of geographic location and annual variation on the detection of differences in the rhizomicrobiome caused by the genetic modification of maize (Bt-maize, event MON810) was evaluated at experimental field sites across Europe including Sweden, Denmark, Slovakia and Spain. DNA of the rhizomicrobiome was collected at the maize flowering stage in three consecutive years and analyzed for the abundance and diversity of PCR-amplified structural genes of Bacteria, Archaea and Fungi, and functional genes for bacterial nitrite reductases (nirS, nirK). The nirK genes were always more abundant than nirS. Maize MON810 did not significantly alter the abundance of any microbial genetic marker, except for sporadically detected differences at individual sites and years. In contrast, annual variation between sites was often significant and variable depending on the targeted markers. Distinct, site-specific microbial communities were detected but the sites in Denmark and Sweden were similar to each other. A significant effect of the genetic modification of the plant on the community structure in the rhizosphere was detected among the nirK denitrifiers at the Slovakian site in only one year. However, most nirK sequences with opposite response were from the same or related source organisms suggesting that the transient differences in community structure did not translate to the functional level. Our results show a lack of effect of the genetic modification of maize on the rhizosphere microbiome that would be stable and consistent over multiple years. This demonstrates the importance of considering annual variability in assessing environmental effects of genetically modified crops

Glyphosate and glyphosate-based herbicides (GBHs) induce phenotypic imipenem resistance in Pseudomonas aeruginosa

GBHs are the most widely used herbicides for weed control worldwide that potentially affect microorganisms, but the role of their sublethal exposure in the development of antibiotic resistance of Pseudomonas aeruginosa is still not fully investigated. Here, the effects of glyphosate acid (GLY), five glyphosate-based herbicides (GBHs), and POE(15), a formerly used co-formulant, on susceptibility to imipenem, a potent carbapenem-type antibiotic, in one clinical and four non-clinical environmental P. aeruginosa isolates were studied. Both pre-exposure in broth culture and co-exposure in solid media of the examined P. aeruginosa strains with 0.5% GBHs resulted in a decreased susceptibility to imipenem, while other carbapenems (doripenem and meropenem) retained their effectiveness. Additionally, the microdilution chequerboard method was used to examine additive/antagonistic/synergistic effects between GLY/POE(15)/GBHs and imipenem by determining the fractional inhibitory concentration (FIC) indexes. Based on the FIC index values, glyphosate acid and Total demonstrated a potent antagonistic effect in all P. aeruginosa strains. Dominator Extra 608 SL and Fozat 480 reduced the activity of imipenem in only one strain (ATCC10145), while POE(15) and three other GBHs did not have any effect on susceptibility to imipenem. Considering the simultaneous presence of GBHs and imipenem in various environmental niches, the detected interactions between these chemicals may affect microbial communities. The mechanisms of the glyphosate and GBH-induced imipenem resistance in P. aeruginosa are yet to be investigated

Hidden heterogeneity and co‐occurrence networks of soil prokaryotic communities revealed at the scale of individual soil aggregates

Abstract Sequencing PCR‐amplified gene fragments from metagenomic DNA is a widely applied method for studying the diversity and dynamics of soil microbial communities. Typically, DNA is extracted from 0.25 to 1 g of soil. These amounts, however, neglect the heterogeneity of soil present at the scale of soil aggregates and thus ignore a crucial scale for understanding the structure and functionality of soil microbial communities. Here, we show with a nitrogen‐depleted agricultural soil the impact of reducing the amount of soil used for DNA extraction from 250 mg to approx. 1 mg to access spatial information on the prokaryotic community structure, as indicated by 16S rRNA gene amplicon analyses. Furthermore, we demonstrate that individual aggregates from the same soil differ in their prokaryotic community compositions. The analysis of 16S rRNA gene amplicon sequences from individual soil aggregates allowed us, in contrast to 250 mg soil samples, to construct a co‐occurrence network that provides insight into the structure of microbial associations in the studied soil. Two dense clusters were apparent in the network, one dominated by Thaumarchaeota, known to be capable of ammonium oxidation at low N concentrations, and the other by Acidobacteria subgroup 6, representing an oligotrophic lifestyle to obtain energy from SOC. Overall this study demonstrates that DNA obtained from individual soil aggregates provides new insights into how microbial communities are assembled

The Effect of Root Exudate 7,4'-Dihydroxyflavone and Naringenin on Soil Bacterial Community Structure.

Our goal was to investigate how root exudate flavonoids influence the soil bacterial community structure and to identify members of the community that change their relative abundance in response to flavonoid exudation. Using a model system that approximates flavonoid exudation of Medicago sativa roots, we treated a soil with 7,4'-dihydroxyflavone and naringenin in two separate experiments using three different rates: medium (equivalent to the exudation rate of 7,4'-dihydroxyflavone from M. sativa seedlings), high (10× the medium rate), and low (0.1× the medium rate). Controls received no flavonoid. Soil samples were subjected to ATP assays and 16S rRNA gene amplicon sequencing. The flavonoid treatments caused no significant change in the soil ATP content. With the high 7,4'-dihydroxyflavone treatment rate, operational taxonomic units (OTUs) classified as Acidobacteria subdivision 4 increased in relative abundance compared with the control samples, whereas OTUs classified as Gaiellales, Nocardioidaceae, and Thermomonosporaceae were more prevalent in the control. The naringenin treatments did not cause significant changes in the soil bacterial community structure. Our results suggest that the root exudate flavonoid 7,4'-dihydroxyflavone can interact with a diverse range of soil bacteria and may have other functions in the rhizosphere in addition to nod gene induction in legume-rhizobia symbiosis

Non-metric multidimensional scaling (NMS) ordination plots.

<p>(A) 7,4′-Dihydroxyflavone experiment, (B) naringenin experiment. Points represent samples, crosses are group centroids. Samples of the same treatment are enclosed in convex hulls. Stress is calculated on a scale of 0 to 100. (Treatments: ● high, ♦ medium, ■ low, and ▲ control).</p

Increased microbial anabolism contributes to soil carbon sequestration by mineral fertilization in temperate grasslands

Ecosystem responses to nitrogen (N) additions are manifold and complex, and also affect the carbon (C) cycle. It has been suggested that increased microbial carbon use efficiency (CUE), i.e. growth per C uptake, due to higher N availability potentially increases the stabilization rates of organic inputs to the soil. However, evidence for a direct link between altered microbial anabolism and soil organic C (SOC) stocks is lacking. In this study, unfertilized (control) and NPK-fertilized (NPK) treatments of seven temperate grassland experiments were used to test the hypothesis that fertilizer-induced differences in SOC stocks (ΔSOC) cannot be explained by differences in C input alone, but that microbial anabolism plays an important role in C sequestration. At two experimental sites, microbial CUE and related metabolic parameters was determined using an 18O labeling approach at two different incubation temperatures (10 °C and 20 °C). Fertilization effects on the abundance of Bacteria, Archaea and Fungi were also determined using quantitative PCR targeting the respective rRNA genes. Due to the availability of yield and belowground biomass data, the introductory carbon balance model (ICBM) could be used for all seven sites to estimate the contribution of C input to ΔSOC. A significantly higher microbial growth (+102 ± 6%), lower specific respiration (−16 ± 7%) and thus significantly higher CUE (+53 ± 21%) was found for the NPK treatments, which was consistent across experiments and incubation temperatures and correlated with measured root C:N ratios. Growth (+49 ± 5%) and respiration (+70 ± 9%) were increased by a higher incubation temperature, but this was not the case for CUE. The fungi to bacteria ratio changed significantly from 0.18 ± 0.02 (control) to 0.09 ± 0.02 (NPK). On average, only 77% (51% when excluding one extreme site) of observed ΔSOC was explained by C inputs. The optimized humification coefficient h of the model used to fit the observed ΔSOC was strongly correlated to differences in the root C:N ratio between the control and NPK treatments (R2 = 0.71), thus confirming a link between microbial anabolism and substrate C:N ratio. Furthermore, varying h directly by observed differences in CUE improved the model fit at the two sites investigated. This study provides direct evidence that CUE of soil microbial communities is relevant for SOC sequestration, and its dependency on soil N availability or substrate C:N ratio might allow for its inclusion in models without explicit microbial C pools.</p