The role of genetic diversity and arbuscular mycorrhizal fungal diversity in population recovery of the semi-natural grassland plant species Succisa pratensis.

BACKGROUND: Ecosystem restoration is as a critical tool to counteract the decline of biodiversity and recover vital ecosystem services. Restoration efforts, however, often fall short of meeting their goals. Although functionally important levels of biodiversity can significantly contribute to the outcome of ecosystem restoration, they are often overlooked. One such important facet of biodiversity is within-species genetic diversity, which is fundamental to population fitness and adaptation to environmental change. Also the diversity of arbuscular mycorrhizal fungi (AMF), obligate root symbionts that regulate nutrient and carbon cycles, potentially plays a vital role in mediating ecosystem restoration outcome. In this study, we investigated the relative contribution of intraspecific population genetic diversity, AMF diversity, and their interaction, to population recovery of Succisa pratensis, a key species of nutrient poor semi natural grasslands. We genotyped 180 individuals from 12 populations of S. pratensis and characterized AMF composition in their roots, using microsatellite markers and next generation amplicon sequencing, respectively. We also investigated whether the genetic makeup of the host plant species can structure the composition of root-inhabiting AMF communities. RESULTS: Our analysis revealed that population allelic richness was strongly positively correlated to relative population growth, whereas AMF richness and its interaction with population genetic diversity did not significantly contribute. The variation partitioning analysis showed that, after accounting for soil and spatial variables, the plant genetic makeup explained a small but significant part of the unique variation in AMF communities. CONCLUSIONS: Our results confirm that population genetic diversity can contribute to population recovery, highlighting the importance of within-species genetic diversity for the success of restoration. We could not find evidence, however, that population recovery benefits from the presence of more diverse AMF communities. Our analysis also showed that the genetic makeup of the host plant structured root-inhabiting AMF communities, suggesting that the plant genetic makeup may be linked to genes that control symbiosis development.

Description of the temporal dynamics in microbial community composition and beer chemistry in sour beer production via barrel ageing of finished beers.

Currently, there is a strong interest in barrel ageing of finished, conventionally fermented beers, as a novel way to produce sour beers with a rich and complex flavour profile. The production process, however, remains largely a process of trial and error, often resulting in profit losses and inconsistency in quality. To improve product quality and consistency, a better understanding of the interactions between microorganisms, wood and maturing beer is needed. The aim of this study was to describe the temporal dynamics in microbial community composition, beer chemistry and sensory characteristics during barrel ageing of three conventionally fermented beers that differed in parameters like alcohol content and bitterness. Beers were matured for 38 weeks in new (two types of wood) and used (one type of wood) oak barrels. Beer samples were taken at the start of the maturation and after 2, 12 and 38 weeks. Microbial community composition, determined using amplicon sequencing of the V4 region of the bacterial 16S rRNA gene and the fungal ITS1 region, beer chemistry and sensory characteristics substantially changed throughout the maturation process. Likewise, total bacterial and fungal population densities generally increased during maturation. PerMANOVA revealed significant differences in the bacterial and fungal community composition of the three beers and across time points, but not between the different wood types. By contrast, significant differences in beer chemistry were found across the different beers, wood types and sampling points. Results also indicated that the outcome of the maturation process likely depends on the initial beer properties. Specifically, results suggested that beer bitterness may restrain the bacterial community composition, thereby having an impact on beer souring. While the bacterial community composition of moderately-hopped beers shifted to a dominance of lactic acid bacteria, the bacterial community of the high-bitterness beer remained fairly constant, with low population densities. Bacterial community composition of the moderate-bitterness beers also resembled those of traditional sours like lambic beers, hosting typical lambic brewing species like Pediococcus damnosus, Lactobacillus brevis and Acetobacter sp. Furthermore, results suggested that alcohol level may have affected the fungal community composition and extraction of wood compounds. More specifically, the concentration of wood compounds like cis-3-methyl-4-octanolide, trans-3-methyl-4-octanolide, eugenol and total polyphenols was higher in beers with a high alcohol content. Altogether, our results provide novel insights into the barrel ageing process of beer, and may pave the way for a new generation of sour beers.

Diversity and community structure of ericoid mycorrhizal fungi in European bogs and heathlands across a gradient of nitrogen deposition.

Despite the ecological significance of ericoid mycorrhizal fungi, little is known about the abiotic and biotic factors driving their diversity and community composition. To determine the relative importance of abiotic and biotic filtering in structuring ericoid mycorrhizal fungal communities, we established 156 sampling plots in two highly contrasting environments but dominated by the same Ericaceae plant species: waterlogged bogs and dry heathlands. Plots were located across 25 bogs and 27 dry heathlands in seven European countries covering a gradient in nitrogen deposition and phosphorus availability. Putatively ericoid mycorrhizal fungal communities in the roots of 10 different Ericaceae species were characterized using high-throughput amplicon sequencing. Variation in ericoid mycorrhizal fungal communities was attributed to both habitat and soil variables on the one hand and host plant identity on the other. Communities differed significantly between bogs and heathlands and, in a given habitat, communities differed significantly among host plant species. Fungal richness was negatively related to nitrogen deposition in bogs and phosphorus availability in bogs and heathlands. Our results demonstrate that both abiotic and biotic filtering shapes ericoid mycorrhizal fungal communities and advocate an environmental policy minimizing excess nutrient input in these nutrient-poor ecosystems to avoid loss of ericoid mycorrhizal fungal taxa.

Effects of single and multiple species inocula of arbuscular mycorrhizal fungi on the salinity tolerance of a Bangladeshi rice (Oryza sativa L.) cultivar.

Soil salinization due to sea level rise and groundwater irrigation has become an important agronomic problem in many parts of the world. Symbiosis between crop species and arbuscular mycorrhizal fungi (AMF) may alleviate salt stress-induced detrimental effects on crop growth and yield, for example, through helping the host plant to selectively absorb potassium while avoiding uptake of excessive sodium. Here, we performed a greenhouse experiment to evaluate growth, grain yield, and salt tolerance of a Bangladeshi rice cultivar under three levels of salt stress (0, 75, and 120 mM) after inoculation with three different AMF species from three different genera (Funnelliformis mosseae (BEG12), Acaulospora laevis (BEG13), and Gigaspora margarita (BEG34)), singly and in combination. We found that under salt stress, AMF inoculation enhanced total chlorophyll concentration, shoot K+/Na+ ratio, and lowered shoot Na+/root Na+ ratio, accompanied by increased root biomass, spikelet fertility, and grain yield compared with the non-inoculated control plants. Specifically, we found that the combination of BEG13 and BEG34 increased rice yield by 125 and 143% as compared with the non-inoculated controls, at the 75 and 120mM salt levels, respectively. In general, the low AMF diversity treatments (one species or a combination of two AMF species) were found to be the most effective in mediating salt stress tolerance for the majority of the measured crop performance variables. Overall, our results indicate that specific AMF species can promote the salt tolerance and productivity of rice, likely by increasing photosynthetic efficiency and restricting Na+ uptake and transport from root to shoot in AMF-inoculated plants.

Soil organic matter rather than ectomycorrhizal diversity is related to urban tree health.

Urban trees provide many ecosystem services, including carbon sequestration, air quality improvement, storm water attenuation and energy conservation, to people living in cities. Provisioning of ecosystem services by urban trees, however, may be jeopardized by the typically poor quality of the soils in urban areas. Given their well-known multifunctional role in forest ecosystems, ectomycorrhizal fungi (EcM) may also contribute to urban tree health and thus ecosystem service provisioning. Yet, no studies so far have directly related in situ EcM community composition to urban tree health indicators. Here, two previously collected datasets were combined: i) tree health data of 175 Tilia tomentosa trees from three European cities (Leuven, Strasbourg and Porto) estimated using a range of reflectance, chlorophyll fluorescence and physical leaf indicators, and ii) ectomycorrhizal diversity of these trees as characterized by next-generation sequencing. Tree health indicators were related to soil characteristics and EcM diversity using canonical redundancy analysis. Soil organic matter significantly explained variation in tree health indicators whereas no significant relation between mycorrhizal diversity variables and the tree health indicators was found. We conclude that mainly soil organic matter, through promoting soil aggregate formation and porosity, and thus indirectly tree water availability, positively affects the health of trees in urban areas. Our results suggest that urban planners should not overlook the importance of soil quality and its water holding capacity for the health of urban trees and potentially also for the ecosystem services they deliver. Further research should also study other soil microbiota which may independently, or in interaction with ectomycorrhiza, mediate tree performance in urban settings.

Temporal and spatial variation in bacterial communities of "Jonagold" apple (Malus x domestica Borkh.) and "Conference" pear (Pyrus communis L.) floral nectar.

Production of many agricultural crops and fruits strongly depends on pollinators. For instance, pome fruits such as apple and pear are highly dependent on pollination for fruit set, fruit quality, and yield. Nectar is often inhabited by microbes, most often yeasts and bacteria, which may change nectar quality and therefore also affect plant-pollinator interactions. Here, we used high-throughput 16S ribosomal RNA gene amplicon sequencing to investigate the temporal and spatial variation in bacterial communities in floral nectar of apple and pear. We sampled 15 apple (Malus x domestica Borkh.) and 15 pear (Pyrus communis L.) orchards distributed over the eastern part of Belgium over a timespan of seven days. Nectar bacterial community composition differed strongly among fruit species. Nectar of pear was dominated by Actinobacteria, followed by Proteobacteria and Firmicutes. Apple nectar was strongly enriched in Bacteroidetes, a phylum which until now has been found to be rarely associated with floral nectar. Nectar was dominated by only a few bacterial species, with Brevibacterium (Actinobacteria) and Undibacterium (Proteobacteria) as the most abundant bacteria in pear and apple nectar, respectively. Bacterial richness and diversity were found to fluctuate during flowering, likely due to changing environmental conditions. Additionally, spatial structure in nectar bacterial community composition was found in apple orchards, while this was not the case for pear. Differences in nectar bacterial communities between apple and pear nectar may differently affect the chemical and nutritional composition of the nectar, influencing pollinator attraction and visitation, and thus pollination efficacy in general.

Variation in arbuscular mycorrhizal fungal communities associated with lowland rice (Oryza sativa) along a gradient of soil salinity and arsenic contamination in Bangladesh.

Rice is an essential food crop that nourishes >50% of the world population. In many regions of Bangladesh rice production is constrained by high soil salinity and heavy metal contamination due to irrigation practices. Plants may naturally overcome such stress through mutualistic interactions with arbuscular mycorrhizal fungi (AMF). Yet, little is known regarding the diversity and composition of AMF communities in rice fields with high saline and arsenic concentration. Here we used high throughput Illumina sequencing to characterize AMF communities in rice roots from 45 Bangladeshi rice fields, along a large geographical gradient of soil salinity and arsenic contamination. We obtained 77 operational taxonomic units (OTUs, based on a sequence similarity threshold of 97%) from eight AMF families, and showed that high soil salinity and arsenic concentration are significantly associated with low AMF diversity in rice roots. Soil salinity and arsenic concentration also explained a large part of the variation in AMF community composition, but also soil pH, moisture, organic matter content and plant available soil phosphorus played an important role. Overall, our study showed that even at very high salinity and arsenic levels, some AMF OTUs are present in rice roots. Their potential role in mediating a reduction of rice stress and arsenic uptake remains to be investigated.

Vegetation reflectance spectroscopy for biomonitoring of heavy metal pollution in urban soils.

Heavy metals in urban soils may impose a threat to public health and may negatively affect urban tree viability. Vegetation spectroscopy techniques applied to bio-indicators bring new opportunities to characterize heavy metal contamination, without being constrained by laborious soil sampling and lab-based sample processing. Here we used Tilia tomentosa trees, sampled across three European cities, as bio-indicators i) to investigate the impacts of elevated concentrations of cadmium (Cd) and lead (Pb) on leaf mass per area (LMA), total chlorophyll content (Chl), chlorophyll a to b ratio (Chla:Chlb) and the maximal PSII photochemical efficiency (Fv/Fm); and ii) to evaluate the feasibility of detecting Cd and Pb contamination using leaf reflectance spectra. For the latter, we used a partial-least-squares discriminant analysis (PLS-DA) to train spectral-based models for the classification of Cd and/or Pb contamination. We show that elevated soil Pb concentrations induced a significant decrease in the LMA and Chla:Chlb, with no decrease in Chl. We did not observe pronounced reductions of Fv/Fm due to Cd and Pb contamination. Elevated Cd and Pb concentrations induced contrasting spectral changes in the red-edge (690-740 nm) region, which might be associated with the proportional changes in leaf pigments. PLS-DA models allowed for the classifications of Cd and Pb contamination, with a classification accuracy of 86% (Kappa = 0.48) and 83% (Kappa = 0.66), respectively. PLS-DA models also allowed for the detection of a collective elevation of soil Cd and Pb, with an accuracy of 66% (Kappa = 0.49). This study demonstrates the potential of using reflectance spectroscopy for biomonitoring of heavy metal contamination in urban soils.

Variation in ectomycorrhizal fungal communities associated with Silver linden (Tilia tomentosa) within and across urban areas.

Trees in urban areas face harsh environmental conditions. Ectomycorrhizal fungi (EcM) form a symbiosis with many tree species and provide a range of benefits to their host through their extraradical hyphal network. Although our understanding of the environmental drivers and large scale geographical variation of EcM communities in natural ecosystems is growing, our knowledge of EcM communities within and across urban areas is still limited. Here, we characterized EcM communities using Illumina miseq sequencing on 175 root samples of the urban tree Tilia tomentosa from three European cities, namely Leuven (Belgium), Strasbourg (France) and Porto (Portugal). We found strong differences in EcM richness and community composition between cities. Soil acidity, organic matter and moisture content were significantly associated with EcM community composition. In agreement, the explained variability in EcM communities was mostly attributed to general soil characteristics, whereas very little variation was explained by city and heavy metal pollution. Overall, our results suggest that EcM communities in urban areas are significantly associated with soil characteristics, while heavy metal pollution and biogeography had little or no impact. These findings deliver new insights into EcM distribution patterns in urban areas and contribute to specific inoculation strategies to improve urban tree vitality.

Mycorrhizal fungi show regular community compositions in natural ecosystems.

Dissimilarity overlap curve analysis has shown that 'universality' is a common feature in many complex microbial communities, suggesting that the same taxa interact in a similar manner when shared between communities. We present evidence that arbuscular mycorrhizal fungi, common plant root symbionts, show universal community compositions in natural ecosystems and that this pattern is conserved even at larger spatial scales. However, universality was not detected in agricultural ecosystems potentially implying that agricultural symbiont communities are formed in a different manner.

Abiotic rather than biotic filtering shapes the arbuscular mycorrhizal fungal communities of European seminatural grasslands.

Although it is well known that arbuscular mycorrhizal fungi (AMF) play a key role in the functioning of natural ecosystems, the underlying drivers determining the composition of AMF communities remain unclear. In this study, we established 138 sampling plots at 46 grassland sites, consisting of 26 acidic grasslands and 20 calcareous grasslands spread across eight European countries, to assess the relative importance of abiotic and biotic filtering in driving AMF community composition and structure in both the grassland soils and in the roots of 13 grassland plant species. Soil AMF communities differed significantly between acidic and calcareous grasslands. In root AMF communities, most variance was attributable to soil variables while very little variation was explained by host plant identity. Root AMF communities in host plant species occurring in only one grassland type closely resembled the soil AMF communities of that grassland type and the root AMF communities of other host plant species occurring in the same grassland type. The observed AMF-host plants networks were not modular but nested. Our results indicate that abiotic conditions, rather than biotic filtering through host plant specificity, are the most important drivers in shaping AMF communities in European seminatural grasslands.

Assessing the composition of microbial communities in textile wastewater treatment plants in comparison with municipal wastewater treatment plants.

It is assumed that microbial communities involved in the biological treatment of different wastewaters having a different chemical composition harbor different microbial populations which are specifically adapted to the environmental stresses encountered in these systems. Yet, little is known about the composition of these microbial communities. Therefore, the aim of this study was to assess the microbial community composition over two seasons (winter and summer) in activated sludge from well-operating textile wastewater treatment plants (WWTPs) in comparison with municipal WWTPs, and to explain observed differences by environmental variables. 454-pyrosequencing generated 160 archaeal and 1645 bacterial species-level Operational Taxonomic Units (OTUs), with lower observed richness in activated sludge from textile WWTPs compared to municipal WWTPs. The bacterial phyla Planctomycetes, Chloroflexi, Chlorobi, and Acidobacteria were more abundant in activated sludge samples from textile WWTPs, together with archaeal members of Thaumarchaeota. Nonmetric multidimensional scaling analysis of the microbial communities showed that microbial communities from textile and municipal WWTPs were significantly different, with a seasonal effect on archaea. Nitrifying and denitrifying bacteria as well as phosphate-accumulation bacteria were more abundant in municipal WWTPs, while sulfate-reducing bacteria were almost only detected in textile WWTPs. Additionally, microbial communities from textile WWTPs were more dissimilar than those of municipal WWTPs, possibly due to a wider diversity in environmental stresses to which microbial communities in textile WWTPs are subjected to. High salinity, high organic loads, and a higher water temperature were important potential variables driving the microbial community composition in textile WWTPs. This study provides a general view on the composition of microbial communities in activated sludge of textile WWTPs, and may provide novel insights for identifying key players performing important functions in the purification of textile wastewaters.

Microbial community dynamics linked to enhanced substrate availability and biogas production of electrokinetically pre-treated waste activated sludge.

The restricted hydrolytic degradation rate of complex organic matter presents a considerable challenge in anaerobic digestion of waste activated sludge (WAS). Within this context, application of pre-treatment of digester substrate has potential for improved waste management and enhanced biogas production. Anaerobic degradation of untreated or electrokinetically pre-treated WAS was performed in two pilot-scale digesters for 132days. WAS electrokinetically pre-treated with energy input 0.066kJ/kg sludge was used in a first phase of operation and WAS pre-treated with energy input 0.091kJ/kg sludge was used in a second phase (each phase lasted at least three hydraulic retention times). Substrate characteristics before and after pre-treatment and effects on biogas digester performance were comprehensively analysed. To gain insights into influences of altered substrate characteristics on microbial communities, the dynamics within the bacterial and archaeal communities in the two digesters were investigated using 16S rRNA gene sequencing (pyrosequencing) and quantitative PCR (qPCR). Specific primers targeting dominant operation taxonomic units (OTUs) and members of the candidate phylum Cloacimonetes were designed to further evaluate their abundance and dynamics in the digesters. Electrokinetic pre-treatment significantly improved chemical oxygen demand (COD) and carbohydrate solubility and increased biogas production by 10-11% compared with untreated sludge. Compositional similarity of the bacterial community during initial operation and diversification during later operation indicated gradual adaptation of the community to the higher solubility of organic material in the pre-treated substrate. Further analyses revealed positive correlations between gene abundance of dominant OTUs related to Clostridia and Cloacimonetes and increased substrate availability and biogas production. Among the methanogens, the genus Methanosaeta dominated in both digesters. Overall, the results showed that electrokinetic pre-treatment of WAS increases substrate solubility and biogas production. Changes in bacterial community composition and abundances of dominant bacterial OTUs were observed during anaerobic degradation of pre-treated WAS, whereas the relative abundance of methanogenic community members remained stable.

Microwave and ultrasound pre-treatments influence microbial community structure and digester performance in anaerobic digestion of waste activated sludge.

Comparative analyses of bacterial and archaeal community structures and dynamics in three biogas digesters during start-up and subsequent operation using microwaved, ultrasonicated or untreated waste activated sludge were performed based on 454 pyrosequencing datasets of part of 16S ribosomal RNA sequences and quantitative PCR. The pre-treatment increased the solubility, and thus the availability of the substrate for microbial degradation and significantly affected the succession of the anaerobic community structure over the course of the digestion. Bacteroidetes, Proteobacteria and Firmicutes were the dominant phyla in all digesters throughout operation. Proteobacteria decreased in relative abundance from 23-26 % to 11-13 % in association with enhanced substrate availability. Negative correlations between relative abundance of Alpha-, Beta- and Gammaproteobacteria and the substrate availability and/or biogas production were disclosed in statistical analyses. Clostridiales was the dominant order in Firmicutes, and Clostridiales, Clostridia and Firmicutes relative abundance and richness were shown to positively correlate with substrate availability and biogas generation. Methanogenic communities had a fairly restricted structure, highly dominated by Methanosaeta and Methanobrevibacter phylotypes. A gradual decline in Methanobrevibacter and increased representation of Methanosaeta concilii over time were particularly apparent in the digester receiving untreated waste activated sludge, whereas more diversified archaeal communities were maintained in the pre-treatment digesters. The quantitative PCR analyses revealed a methanogenic community distribution that coincided with the 454 pyrosequencing data.

Decrease in diversity and changes in community composition of arbuscular mycorrhizal fungi in roots of apple trees with increasing orchard management intensity across a regional scale.

Understanding which factors drive the diversity and community composition of arbuscular mycorrhizal fungi (AMF) is important due to the role of these soil micro-organisms in ecosystem functioning and current environmental threats to AMF biodiversity. Additionally, in agro-ecosystems, this knowledge may help to evaluate their use in making agriculture more sustainable. Here, we used 454-pyrosequencing of small subunit rRNA gene amplicons to quantify AMF diversity and community composition in the roots of cultivated apple trees across 24 orchards in central Belgium. We aimed at identifying the factors (soil chemical variables, organic vs. conventional farming, and geographical location) that affect AMF diversity and community composition. In total, 110 AMF OTUs were detected, of which the majority belonged to the Glomeraceae (73%) and the Claroideoglomeraceae (19%). We show that soil characteristics and farming system, rather than the geographical location of the orchards, shape AMF communities on apple trees. Particularly, plant-available P content of the soil was associated with lower AMF diversity. In orchards with a lower plant-available P content of the soil (P < 100 mg/kg soil), we also found a significantly higher AMF diversity in organically managed orchards as compared to conventionally managed orchards. Finally, the degree of nestedness of the AMF communities was related to plant-available P and N content of the soil, pointing at a progressive loss of AMF taxa with increasing fertilization. Overall, we conclude that a combination of organic orchard management and moderate fertilization may preserve diverse AMF communities on apple trees and that AMF in the roots of apple trees appear not to be dispersal limited at the scale of central Belgium.

Evaluation of six primer pairs targeting the nuclear rRNA operon for characterization of arbuscular mycorrhizal fungal (AMF) communities using 454 pyrosequencing.

In the last few years, 454 pyrosequencing-based analysis of arbuscular mycorrhizal fungal (AMF; Glomeromycota) communities has tremendously increased our knowledge of the distribution and diversity of AMF. Nonetheless, comparing results between different studies is difficult, as different target genes (or regions thereof) and primer combinations, with potentially dissimilar specificities and efficacies, are being utilized. In this study we evaluated six primer pairs that have previously been used in AMF studies (NS31-AM1, AMV4.5NF-AMDGR, AML1-AML2, NS31-AML2, FLR3-LSUmBr and Glo454-NDL22) for their use in 454 pyrosequencing based on both an in silico approach and 454 pyrosequencing of AMF communities from apple tree roots. Primers were evaluated in terms of (i) in silico coverage of Glomeromycota fungi, (ii) the number of high-quality sequences obtained, (iii) selectivity for AMF species, (iv) reproducibility and (v) ability to accurately describe AMF communities. We show that primer pairs AMV4.5NF-AMDGR, AML1-AML2 and NS31-AML2 outperformed the other tested primer pairs in terms of number of Glomeromycota reads (AMF specificity and coverage). Additionally, these primer pairs were found to have no or only few mismatches to AMF sequences and were able to consistently describe AMF communities from apple roots. However, whereas most high-quality AMF sequences were obtained for AMV4.5NF-AMDGR, our results also suggest that this primer pair favored amplification of Glomeraceae sequences at the expense of Ambisporaceae, Claroideoglomeraceae and Paraglomeraceae sequences. Furthermore, we demonstrate the complementary specificity of AMV4.5NF-AMDGR with AML1-AML2, and of AMV4.5NF-AMDGR with NS31-AML2, making these primer combinations highly suitable for tandem use in covering the diversity of AMF communities.