Transcriptomic and metabolomic approaches elucidate the systemic response of wheat plants under waterlogging.

Extreme weather conditions lead to significant imbalances in crop productivity, which in turn affect food security. Flooding events cause serious problems for many crop species such as wheat. Although metabolic readjustments under flooding are important for plant regeneration, underlying processes remain poorly understood. Here, we investigated the systemic response of wheat to waterlogging using metabolomics and transcriptomics. A 12 d exposure to excess water triggered nutritional imbalances and disruption of metabolite synthesis and translocation, reflected by reductions in plant biomass and growth performance. Metabolic and transcriptomic profiling in roots, xylem sap, and leaves indicated anaerobic fermentation processes as a local response in roots. Differentially expressed genes and ontological categories revealed that carbohydrate metabolism plays an important role in the systemic response. Analysis of the composition of xylem exudates revealed decreased root-to-shoot translocation of nutrients, hormones, and amino acids. Interestingly, among all metabolites measured in xylem exudates, alanine was the most abundant. Immersion of excised leaves derived from waterlogged plants in alanine solution led to increased leaf glucose concentration. Our results suggest an important role of alanine not only as an amino-nitrogen donor but also as a vehicle for carbon skeletons to produce glucose de novo and meet the energy demand during waterlogging.

Beneficial microbial consortium improves winter rye performance by modulating bacterial communities in the rhizosphere and enhancing plant nutrient acquisition.

The beneficial effect of microbial consortium application on plants is strongly affected by soil conditions, which are influenced by farming practices. The establishment of microbial inoculants in the rhizosphere is a prerequisite for successful plant-microorganism interactions. This study investigated whether a consortium of beneficial microorganisms establishes in the rhizosphere of a winter crop during the vegetation period, including the winter growing season. In addition, we aimed for a better understanding of its effect on plant performance under different farming practices. Winter rye plants grown in a long-time field trial under conventional or organic farming practices were inoculated after plant emergence in autumn with a microbial consortium containing Pseudomonas sp. (RU47), Bacillus atrophaeus (ABi03) and Trichoderma harzianum (OMG16). The density of the microbial inoculants in the rhizosphere and root-associated soil was quantified in autumn and the following spring. Furthermore, the influence of the consortium on plant performance and on the rhizosphere bacterial community assembly was investigated using a multidisciplinary approach. Selective plating showed a high colonization density of individual microorganisms of the consortium in the rhizosphere and root-associated soil of winter rye throughout its early growth cycle. 16S rRNA gene amplicon sequencing showed that the farming practice affected mainly the rhizosphere bacterial communities in autumn and spring. However, the microbial consortium inoculated altered also the bacterial community composition at each sampling time point, especially at the beginning of the new growing season in spring. Inoculation of winter rye with the microbial consortium significantly improved the plant nutrient status and performance especially under organic farming. In summary, the microbial consortium showed sufficient efficacy throughout vegetation dormancy when inoculated in autumn and contributed to better plant performance, indicating the potential of microbe-based solutions in organic farming where nutrient availability is limited.

The structure of root-associated fungal communities is related to the long-term effects of plant diversity on productivity.

Root-associated fungi could play a role in determining both the positive relationship between plant diversity and productivity in experimental grasslands, and its strengthening over time. This hypothesis assumes that specialized pathogenic and mutualistic fungal communities gradually assemble over time, enhancing plant growth more in species-rich than in species-poor plots. To test this hypothesis, we used high-throughput amplicon sequencing to characterize root-associated fungal communities in experimental grasslands of 1 and 15 years of age with varying levels of plant species richness. Specifically, we tested whether the relationship between fungal communities and plant richness and productivity becomes stronger with the age of the experimental plots. Our results showed that fungal diversity increased with plant diversity, but this relationship weakened rather than strengthened over the two time points. Contrastingly, fungal community composition showed increasing associations with plant diversity over time, suggesting a gradual build-up of specific fungal assemblages. Analyses of different fungal guilds showed that these changes were particularly marked in pathogenic fungi, whose shifts in relative abundance are consistent with the pathogen dilution hypothesis in diverse plant communities. Our results suggest that root-associated fungal pathogens play more specific roles in determining the diversity-productivity relationship than other root-associated plant symbionts.

Nitrogen Fertilizer Type and Genotype as Drivers of P Acquisition and Rhizosphere Microbiota Assembly in Juvenile Maize Plants.

Phosphorus (P) is an essential nutrient for plant growth and development, as well as an important factor limiting sustainable maize production. Targeted nitrogen (N) fertilization in the form of ammonium has been shown to positively affect Pi uptake under P-deficient conditions compared to nitrate. Nevertheless, its profound effects on root traits, P uptake, and soil microbial composition are still largely unknown. In this study, two maize genotypes F160 and F7 with different P sensitivity were used to investigate phosphorus-related root traits such as root hair length, root diameter, AMF association, and multiple P efficiencies under P limitation when fertilized either with ammonium or nitrate. Ammonium application improved phosphorous acquisition efficiency in the F7 genotype but not in F160, suggesting that the genotype plays an important role in how a particular N form affects P uptake in maize. Additionally, metabarcoding data showed that young maize roots were able to promote distinct microbial taxa, such as arbuscular mycorrhizal fungi, when fertilized with ammonium. Overall, the results suggest that the form of chemical nitrogen fertilizer can be instrumental in selecting beneficial microbial communities associated with phosphorus uptake and maize plant fitness.

Response of the wheat mycobiota to flooding revealed substantial shifts towards plant pathogens.

Rainfall extremes are intensifying as a result of climate change, leading to increased flood risk. Flooding affects above- and belowground ecosystem processes, representing a substantial threat to crop productivity under climate change. Plant-associated fungi play important roles in plant performance, but their response to abnormal rain events is unresolved. Here, we established a glasshouse experiment to determine the effects of flooding stress on the spring wheat-mycobiota complex. Since plant phenology could be an important factor in the response to hydrological stress, flooding was induced only once and at different plant growth stages, such as tillering, booting and flowering. We assessed the wheat mycobiota response to flooding in three soil-plant compartments (phyllosphere, roots and rhizosphere) using metabarcoding. Key soil and plant traits were measured to correlate physiological plant and edaphic changes with shifts in mycobiota structure and functional guilds. Flooding reduced plant fitness, and caused dramatic shifts in mycobiota assembly across the entire plant. Notably, we observed a functional transition consisting of a decline in mutualist abundance and richness with a concomitant increase in plant pathogens. Indeed, fungal pathogens associated with important cereal diseases, such as Gibberella intricans, Mycosphaerella graminicola, Typhula incarnata and Olpidium brassicae significantly increased their abundance under flooding. Overall, our study demonstrate the detrimental effect of flooding on the wheat mycobiota complex, highlighting the urgent need to understand how climate change-associated abiotic stressors alter plant-microbe interactions in cereal crops.

Ammonium fertilization increases the susceptibility to fungal leaf and root pathogens in winter wheat.

Nitrogen (N) fertilization is indispensable for high yields in agriculture due to its central role in plant growth and fitness. Different N forms affect plant defense against foliar pathogens and may alter soil-plant-microbe interactions. To date, however, the complex relationships between N forms and host defense are poorly understood. For this purpose, nitrate, ammonium, and cyanamide were compared in greenhouse pot trials with the aim to suppress two important fungal wheat pathogens Blumeria graminis f. sp. tritici (Bgt) and Gaeumannomyces graminis f. sp. tritici (Ggt). Wheat inoculated with the foliar pathogen Bgt was comparatively up to 80% less infested when fertilized with nitrate or cyanamide than with ammonium. Likewise, soil inoculation with the fungal pathogen Ggt revealed a 38% higher percentage of take-all infected roots in ammonium-fertilized plants. The bacterial rhizosphere microbiome was little affected by the N form, whereas the fungal community composition and structure were shaped by the different N fertilization, as revealed from metabarcoding data. Importantly, we observed a higher abundance of fungal pathogenic taxa in the ammonium-fertilized treatment compared to the other N treatments. Taken together, our findings demonstrated the critical role of fertilized N forms for host-pathogen interactions and wheat rhizosphere microbiome assemblage, which are relevant for plant fitness and performance.

Leaf bacterial microbiota response to flooding is controlled by plant phenology in wheat (Triticum aestivum L.).

Leaf microbiota mediates foliar functional traits, influences plant fitness, and contributes to various ecosystem functions, including nutrient and water cycling. Plant phenology and harsh environmental conditions have been described as the main determinants of leaf microbiota assembly. How climate change may modulate the leaf microbiota is unresolved and thus, we have a limited understanding on how environmental stresses associated with climate change driven weather events affect composition and functions of the microbes inhabiting the phyllosphere. Thus, we conducted a pot experiment to determine the effects of flooding stress on the wheat leaf microbiota. Since plant phenology might be an important factor in the response to hydrological stress, flooding was induced at different plant growth stages (tillering, booting and flowering). Using a metabarcoding approach, we monitored the response of leaf bacteria to flooding, while key soil and plant traits were measured to correlate physiological plant and edaphic factor changes with shifts in the bacterial leaf microbiota assembly. In our study, plant growth stage represented the main driver in leaf microbiota composition, as early and late plants showed distinct bacterial communities. Overall, flooding had a differential effect on leaf microbiota dynamics depending at which developmental stage it was induced, as a more pronounced disruption in community assembly was observed in younger plants.

Flooding Causes Dramatic Compositional Shifts and Depletion of Putative Beneficial Bacteria on the Spring Wheat Microbiota.

Flooding affects both above- and below-ground ecosystem processes, and it represents a substantial threat for crop and cereal productivity under climate change. Plant-associated microbiota play a crucial role in plant growth and fitness, but we still have a limited understanding of the response of the crop-microbiota complex under extreme weather events, such as flooding. Soil microbes are highly sensitive to abiotic disturbance, and shifts in microbial community composition, structure and functions are expected when soil conditions are altered due to flooding events (e.g., anoxia, pH alteration, changes in nutrient concentration). Here, we established a pot experiment to determine the effects of flooding stress on the spring wheat-microbiota complex. Since plant phenology could be an important factor in the response to hydrological stress, flooding was induced only once and at different plant growth stages (PGSs), such as tillering, booting and flowering. After each flooding event, we measured in the control and flooded pots several edaphic and plant properties and characterized the bacterial community associated to the rhizosphere and roots of wheat plant using a metabarcoding approach. In our study, flooding caused a significant reduction in plant development and we observed dramatic shifts in bacterial community composition at each PGS in which the hydrological stress was induced. However, a more pronounced disruption in community assembly was always shown in younger plants. Generally, flooding caused a (i) significant increase of bacterial taxa with anaerobic respiratory capabilities, such as members of Firmicutes and Desulfobacterota, (ii) a significant reduction in Actinobacteria and Proteobacteria, (iii) depletion of several putative plant-beneficial taxa, and (iv) increases of the abundance of potential detrimental bacteria. These significant differences in community composition between flooded and control samples were correlated with changes in soil conditions and plant properties caused by the hydrological stress, with pH and total N as the soil, and S, Na, Mn, and Ca concentrations as the root properties most influencing microbial assemblage in the wheat mircobiota under flooding stress. Collectively, our findings demonstrated the role of flooding on restructuring the spring wheat microbiota, and highlighted the detrimental effect of this hydrological stress on plant fitness and performance.

Crop host signatures reflected by co-association patterns of keystone Bacteria in the rhizosphere microbiota.

BACKGROUND: The native crop bacterial microbiota of the rhizosphere is envisioned to be engineered for sustainable agriculture. This requires the identification of keystone rhizosphere Bacteria and an understanding on how these govern crop-specific microbiome assembly from soils. We identified the metabolically active bacterial microbiota (SSU RNA) inhabiting two compartments of the rhizosphere of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), rye (Secale cereale), and oilseed rape (Brassica napus L.) at different growth stages. RESULTS: Based on metabarcoding analysis the bacterial microbiota was shaped by the two rhizosphere compartments, i.e. close and distant. Thereby implying a different spatial extent of bacterial microbiota acquirement by the cereals species versus oilseed rape. We derived core microbiota of each crop species. Massilia (barley and wheat) and unclassified Chloroflexi of group 'KD4-96' (oilseed rape) were identified as keystone Bacteria by combining LEfSe biomarker and network analyses. Subsequently, differential associations between networks of each crop species' core microbiota revealed host plant-specific interconnections for specific genera, such as the unclassified Tepidisphaeraceae 'WD2101 soil group'. CONCLUSIONS: Our results provide keystone rhizosphere Bacteria derived from for crop hosts and revealed that cohort subnetworks and differential associations elucidated host species effect that was not evident from differential abundance of single bacterial genera enriched or unique to a specific plant host. Thus, we underline the importance of co-occurrence patterns within the rhizosphere microbiota that emerge in crop-specific microbiomes, which will be essential to modify native crop microbiomes for future agriculture and to develop effective bio-fertilizers.

DNA Metabarcoding for the Characterization of Terrestrial Microbiota-Pitfalls and Solutions.

Soil-borne microbes are major ecological players in terrestrial environments since they cycle organic matter, channel nutrients across trophic levels and influence plant growth and health. Therefore, the identification, taxonomic characterization and determination of the ecological role of members of soil microbial communities have become major topics of interest. The development and continuous improvement of high-throughput sequencing platforms have further stimulated the study of complex microbiota in soils and plants. The most frequently used approach to study microbiota composition, diversity and dynamics is polymerase chain reaction (PCR), amplifying specific taxonomically informative gene markers with the subsequent sequencing of the amplicons. This methodological approach is called DNA metabarcoding. Over the last decade, DNA metabarcoding has rapidly emerged as a powerful and cost-effective method for the description of microbiota in environmental samples. However, this approach involves several processing steps, each of which might introduce significant biases that can considerably compromise the reliability of the metabarcoding output. The aim of this review is to provide state-of-the-art background knowledge needed to make appropriate decisions at each step of a DNA metabarcoding workflow, highlighting crucial steps that, if considered, ensures an accurate and standardized characterization of microbiota in environmental studies.

Dynamics of Soil Bacterial Communities Over a Vegetation Season Relate to Both Soil Nutrient Status and Plant Growth Phenology.

Soil microorganisms regulate element cycling and plant nutrition, mediate co-existence of neighbors, and stabilize plant communities. Many of these effects are dependent upon environmental conditions and, in particular, on nutrient quality and availability in soils. In this context, we set up a pot experiment in order to examine the combined effects of soil nutrient availability and microbial communities on plant-soil interactions and to investigate assemblage rules for soil bacterial communities under changed nutrient conditions. Four gamma-sterilized soils, strongly differing in their nutrient contents, were obtained from different fertilization treatments of a centenary field experiment and used to grow communities of grassland plants. The sterilized soils were either self- or cross-inoculated with microbial consortia from the same four soils. Molecular fingerprinting analyses were carried out at several time points in order to identify drivers and underlying processes of microbial community assemblage. We observed that the bacterial communities that developed in the inoculated sterilized soils differed from those in the original soils, displaying dynamic shifts over time. These shifts were illustrated by the appearance of numerous OTUs that had not been detected in the original soils. The community patterns observed in the inoculated treatments suggested that bacterial community assembly was determined by both niche-mediated and stochastic-neutral processes, whereby the relative impacts of these processes changed over the course of the vegetation season. Moreover, our experimental approach allowed us not only to evaluate the effects of soil nutrients on plant performance but also to recognize a negative effect of the microbial community present in the soil that had not been fertilized for more than 100 years on plant biomass. Our findings demonstrate that soil inoculation-based approaches are valid for investigating plant-soil-microbe interactions and for examining rules that shape soil microbial community assemblages under variable ecological conditions.

Effects of slope exposure on soil physico-chemical and microbiological properties along an altitudinal climosequence in the Italian Alps.

Due to their sensitivity to changing environmental conditions sub- and alpine soils are often monitored in the context of climate change, usually, however, neglecting slope exposure. Therefore, we set up a climosequence-approach to study the effect of exposure and, in general, climate, on the microbial biomass and microbial diversity and activity, comprising five pairs of north (N)- and south (S)-facing sites along an altitudinal gradient ranging from 1200 to 2400m a.s.l. in the Italian Alps (Trentino Alto Adige, Italy). Soil physico-chemical properties were related to microbiological properties (microbial biomass: double strand DNA yield vs. substrate-induced respiration; diversity of bacterial, fungal and archaeal communities: genetic fingerprinting DGGE vs. real-time PCR; microbial activity: basal respiration vs. multiple hydrolytic enzyme assays) to monitor shifts in the diversity and activity of microbial communities as a function of slope exposure and to evaluate the most determinant chemical parameters shaping the soil microbiota. The exposure-effect on several hydrolytic key-enzymes was enzyme-specific: e.g. acid phosphomonoesterase potential activity was more pronounced at the N-facing slope while the activities of alkaline phosphomonoesterase, pyrophosphate-phosphodiesterase and arylsulfatase were higher at the S-facing slope. Furthermore, this exposure-effect was domain-specific: bacteria (S>N, altitude-independent); fungi (N~S); and archaea (N>S; altitude-dependent). Additionally, the abiotic parameters shaping the community composition were in general depending on soil depth. Our multidisciplinary approach allowed us to survey the exposure and altitudinal effects on soil physico-chemical and microbiological properties and thus unravel the complex multiple edaphic factor-effects on soil microbiota in mountain ecosystems.

Mineral vs. Organic Amendments: Microbial Community Structure, Activity and Abundance of Agriculturally Relevant Microbes Are Driven by Long-Term Fertilization Strategies.

Soil management is fundamental to all agricultural systems and fertilization practices have contributed substantially to the impressive increases in food production. Despite the pivotal role of soil microorganisms in agro-ecosystems, we still have a limited understanding of the complex response of the soil microbiota to organic and mineral fertilization in the very long-term. Here, we report the effects of different fertilization regimes (mineral, organic and combined mineral and organic fertilization), carried out for more than a century, on the structure and activity of the soil microbiome. Organic matter content, nutrient concentrations, and microbial biomass carbon were significantly increased by mineral, and even more strongly by organic fertilization. Pyrosequencing revealed significant differences between the structures of bacterial and fungal soil communities associated to each fertilization regime. Organic fertilization increased bacterial diversity, and stimulated microbial groups (Firmicutes, Proteobacteria, and Zygomycota) that are known to prefer nutrient-rich environments, and that are involved in the degradation of complex organic compounds. In contrast, soils not receiving manure harbored distinct microbial communities enriched in oligotrophic organisms adapted to nutrient-limited environments, as Acidobacteria. The fertilization regime also affected the relative abundances of plant beneficial and detrimental microbial taxa, which may influence productivity and stability of the agroecosystem. As expected, the activity of microbial exoenzymes involved in carbon, nitrogen, and phosphorous mineralization were enhanced by both types of fertilization. However, in contrast to comparable studies, the highest chitinase and phosphatase activities were observed in the solely mineral fertilized soil. Interestingly, these two enzymes showed also a particular high biomass-specific activities and a strong negative relation with soil pH. As many soil parameters are known to change slowly, the particularity of unchanged fertilization treatments since 1902 allows a profound assessment of linkages between management and abiotic as well as biotic soil parameters. Our study revealed that pH and TOC were the majors, while nitrogen and phosphorous pools were minors, drivers for structure and activity of the soil microbial community. Due to the long-term treatments studied, our findings likely represent permanent and stable, rather than transient, responses of soil microbial communities to fertilization.

Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples.

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.