Changes in root-exudate-induced respiration reveal a novel mechanism through which drought affects ecosystem carbon cycling.

Root exudates play an important role in ecosystem response to climate change, but the functional consequences of drought-induced changes in the quality of root exudates are unknown. Here, we addressed this knowledge gap in a unique experimental approach. We subjected two common grassland species that differ widely in their growth strategies and root systems, the grass Holcus lanatus and the forb Rumex acetosa, to 2 wk of drought. We collected root exudates and soils at the end of the drought and after 2 wk of recovery and readded all root exudates to all soils in a fully reciprocal set-up to measure root-exudate-induced respiration. We found that soil treatment was unimportant for determining root-exudate-induced respiration. By contrast, root exudates collected from plants that had experienced drought clearly triggered more soil respiration than exudates from undroughted plants. Importantly, this increased respiration compensated for the lower rates of root exudation in droughted plants. Our findings reveal a novel mechanism through which drought can continue to affect ecosystem carbon cycling, and a potential plant strategy to facilitate regrowth through stimulating microbial activity. These findings have important implications for understanding plant and ecosystem response to drought.

Microbiome and ecotypic adaption of Holcus lanatus (L.) to extremes of its soil pH range, investigated through transcriptome sequencing.

BACKGROUND: Plants can adapt to edaphic stress, such as nutrient deficiency, toxicity and biotic challenges, by controlled transcriptomic responses, including microbiome interactions. Traditionally studied in model plant species with controlled microbiota inoculation treatments, molecular plant-microbiome interactions can be functionally investigated via RNA-Seq. Complex, natural plant-microbiome studies are limited, typically focusing on microbial rRNA and omitting functional microbiome investigations, presenting a fundamental knowledge gap. Here, root and shoot meta-transcriptome analyses, in tandem with shoot elemental content and root staining, were employed to investigate transcriptome responses in the wild grass Holcus lanatus and its associated natural multi-species eukaryotic microbiome. A full factorial reciprocal soil transplant experiment was employed, using plant ecotypes from two widely contrasting natural habitats, acid bog and limestone quarry soil, to investigate naturally occurring, and ecologically meaningful, edaphically driven molecular plant-microbiome interactions. RESULTS: Arbuscular mycorrhizal (AM) and non-AM fungal colonization was detected in roots in both soils. Staining showed greater levels of non-AM fungi, and transcriptomics indicated a predominance of Ascomycota-annotated genes. Roots in acid bog soil were dominated by Phialocephala-annotated transcripts, a putative growth-promoting endophyte, potentially involved in N nutrition and ion homeostasis. Limestone roots in acid bog soil had greater expression of other Ascomycete genera and Oomycetes and lower expression of Phialocephala-annotated transcripts compared to acid ecotype roots, which corresponded with reduced induction of pathogen defense processes, particularly lignin biosynthesis in limestone ecotypes. Ascomycota dominated in shoots and limestone soil roots, but Phialocephala-annotated transcripts were insignificant, and no single Ascomycete genus dominated. Fusarium-annotated transcripts were the most common genus in shoots, with Colletotrichum and Rhizophagus (AM fungi) most numerous in limestone soil roots. The latter coincided with upregulation of plant genes involved in AM symbiosis initiation and AM-based P acquisition in an environment where P availability is low. CONCLUSIONS: Meta-transcriptome analyses provided novel insights into H. lanatus transcriptome responses, associated eukaryotic microbiota functions and taxonomic community composition. Significant edaphic and plant ecotype effects were identified, demonstrating that meta-transcriptome-based functional analysis is a powerful tool for the study of natural plant-microbiome interactions.

Effects of extreme weather events and legume presence on mycorrhization of Plantago lanceolata and Holcus lanatus in the field.

Little is known about direct and indirect effects of extreme weather events on arbuscular mycorrhizal fungi (AMF) under field conditions. In a field experiment, we investigated the response of mycorrhization to drought and heavy rain in grassland communities. We quantified AMF biomass in soil, mycorrhization of roots of the grass Holcus lanatus and the forb Plantago lanceolata, as well as plant performance. Plants were grown in four-species communities with or without a legume. We hypothesised that drought increases and heavy rain decreases mycorrhization, and that higher mycorrhization will be linked to improved stress resistance and higher biomass production. Soil AMF biomass increased under both weather extremes. Heavy rain generally benefitted plants and increased arbuscules in P. lanceolata. Drought neither reduced plant performance nor root mycorrhization. Arbuscules increased in H. lanatus several weeks after drought, and in P. lanceolata several weeks after heavy rain spells. These long-lasting effects of weather events on mycorrhization highlight the indirect influence of climate on AMF via their host plant. Legume presence increased plant community biomass, but had only minor effects on mycorrhization. Arbuscule colonisation was negatively correlated with senescence during the dry summer. Mycorrhization and biomass production in P. lanceolata were positively related. However, increased mycorrhization was related to less biomass in the grass. AMF mycelium in soil might generally increase under extreme events, root colonisation, however, is host species specific. This might amplify community shifts in grassland under climate change by further increasing stress resistance of species that already benefit from changed precipitation.

Genetic evidence for reproductive isolation among sympatric Epichloë endophytes as inferred from newly developed microsatellite markers.

Reproductive isolation is central to the maintenance of species, and especially in sympatry, effective barriers to prevent interspecific crosses are expected. Host specificity is thought to constitute an effective mechanism for the formation of barriers in different genera of Fungi, but evidence for endophytes is so far lacking. Sexual Epichloë species (Ascomycota, Clavicipitaceae) represent an ideal study system to investigate the mechanisms underlying speciation as mediated by host specificity because they include species complexes with several host-specific taxa. Here, we studied genetic differentiation of three host-specific Epichloë species using microsatellite markers that were newly in silico identified on the genome of Epichloë poae. Among these, 15 were experimentally tested and applied to study an extensive sampling of isolates representing Epichloë typhina infecting Dactylis glomerata and Epichloë clarkii infecting Holcus lanatus from a site with sympatric populations in Switzerland, as well as a reduced sampling of E. poae infecting Poa nemoralis to create a three-taxon dataset. Both principal coordinate analysis and Bayesian clustering algorithm showed three genetically distinct groups representing the three host-specific species. High pairwise F ST values among the three species, as well as sequencing data of the tefA gene revealing diagnostic single nucleotide polymorphisms (SNPs), further support the hypothesis of genetic discontinuities among the taxa. These results provide genotypic evidence of the maintenance of reproductive isolation of the species in a context of sympatry. In silico testing of 885 discovered microsatellites on the genome of Epichloë festucae extend their applicability to a wider taxonomic range of Epichloë.

Potential contribution of natural enemies to patterns of local adaptation in plants.

Genetic differentiation among plant populations and adaptation to local environmental conditions are well documented. However, few studies have examined the potential contribution of plant antagonists, such as insect herbivores and pathogens, to the pattern of local adaptation. Here, a reciprocal transplant experiment was set up at three sites across Europe using two common plant species, Holcus lanatus and Plantago lanceolata. The amount of damage by the main above-ground plant antagonists was measured: a rust fungus infecting Holcus and a specialist beetle feeding on Plantago, both in low-density monoculture plots and in competition with interspecific neighbours. Strong genetic differentiation among provenances in the amount of damage by antagonists in both species was found. Local provenances of Holcus had significantly higher amounts of rust infection than foreign provenances, whereas local provenances of Plantago were significantly less damaged by the specialist beetle than the foreign provenances. The presence of surrounding vegetation affected the amount of damage but had little influence on the ranking of plant provenances. The opposite pattern of population differentiation in resistance to local antagonists in the two species suggests that it will be difficult to predict the consequences of plant translocations for interactions with organisms of higher trophic levels.

Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing, denitrifying and nitrifying bacterial communities in grassland soils.

Management by combined grazing and mowing events is commonly used in grasslands, which influences the activity and composition of soil bacterial communities. Whether observed effects are mediated by management-induced disturbances, or indirectly by changes in the identity of major plant species, is still unknown. To address this issue, we quantified substrate-induced respiration (SIR), and the nitrification, denitrification and free-living N(2)-fixation enzyme activities below grass tufts of three major plant species (Holcus lanatus, Arrhenatherum elatius and Dactylis glomerata) in extensively or intensively managed grasslands. The genetic structures of eubacterial, ammonia oxidizing, nitrate reducing, and free-living N(2)-fixing communities were also characterized by ribosomal intergenic spacer analysis, and denaturing gradient gel electrophoresis (DGGE) or restriction fragment length polymorphism (RFLP) targeting group-specific genes. SIR was not influenced by management and plant species, whereas denitrification enzyme activity was influenced only by plant species, and management-plant species interactions were observed for fixation and nitrification enzyme activities. Changes in nitrification enzyme activity were likely largely explained by the observed changes in ammonium concentration, whereas N availability was not a major factor explaining changes in denitrification and fixation enzyme activities. The structures of eubacterial and free-living N(2)-fixing communities were essentially controlled by management, whereas the diversity of nitrate reducers and ammonia oxidizers depended on both management and plant species. For each functional group, changes in enzyme activity were not correlated or were weakly correlated to overall changes in genetic structure, but around 60% of activity variance was correlated to changes in five RFLP or DGGE bands. Although our conclusions should be tested for other ecosystems and seasons, these results show that predicting microbial changes induced by management in grasslands requires consideration of management-plant species interactions.

Effects of gamma irradiation on Holcus lanatus (Yorkshire fog grass) and associated soil microorganisms.

An investigation was conducted to determine the impact of acute doses of gamma radiation on the microbial community structure of a Holcus lanatus dominated grassland soil. Mesocosms containing soil and established grass were irradiated using a sealed (137)Cs source (7.0 Gy min(-1)). Doses ranged from 5 to 160 Gy, analyses were conducted on the day of irradiation, then 7 and 30 days later. Plant growth and arbuscular mycorrhizal fungal colonisation of roots were reduced by irradiation. Gram-negative bacteria, and microbial metabolic capacity were also negatively affected by treatment. Microbial biomass measured by phospholipid fatty acid (PLFA) analysis, showed an increase at doses above 20 Gy, 7 and 30 days after treatment. Proportions of Gram-positive bacterial and fungal PLFAs fluctuated inversely to each other, in response to both sampling time and radiation dose. We hypothesise that many of the observed soil microbial responses are indirect effects mediated by the influence of ionising radiation on the plants in this system.

Impact of temperature on the arbuscular mycorrhizal (AM) symbiosis: growth responses of the host plant and its AM fungal partner.

The growth response of the hyphae of mycorrhizal fungi has been determined, both when plant and fungus together and when only the fungus was exposed to a temperature change. Two host plant species, Plantago lanceolata and Holcus lanatus, were grown separately in pots inoculated with the mycorrhizal fungus Glomus mosseae at 20/18 degrees C (day/night); half of the pots were then transferred to 12/10 degrees C. Plant and fungal growth were determined at six sequential destructive harvests. A second experiment investigated the direct effect of temperature on the length of the extra-radical mycelium (ERM) of three mycorrhizal fungal species. Growth boxes were divided in two equal compartments by a 20 micro m mesh, allowing only the ERM and not roots to grow into a fungal compartment, which was either heated (+8 degrees C) or kept at ambient temperature. ERM length (LERM) was determined on five sampling dates. Growth of H. lanatus was little affected by temperature, whereas growth of P. lanceolata increased with temperature, and both specific leaf area (SLA) and specific root length (SRL) increased independently of plant size. Percentage of colonized root (LRC) and LERM were positively correlated with temperature when in symbiosis with P. lanceolata, but differences in LRC were a function of plant biomass. Colonization was very low in H. lanatus roots and there was no significant temperature effect. In the fungal compartment LERM increased over time and was greatest for Glomus mosseae. Heating the fungal compartment significantly increased LERM in two of the three species but did not affect LRC. However, it significantly increased SRL of roots in the plant compartment, suggesting that the fungus plays a regulatory role in the growth dynamics of the symbiosis. These temperature responses have implications for modelling carbon dynamics under global climate change.