Disentangling plant- and environment-mediated drivers of active rhizosphere bacterial community dynamics during short-term drought.

Mitigating the effects of climate stress on crops is important for global food security. The microbiome associated with plant roots, the rhizobiome, can harbor beneficial microbes that alleviate stress, but the factors influencing their recruitment are unclear. We conducted a greenhouse experiment using field soil with a legacy of growing switchgrass and common bean to investigate the impact of short-term drought severity on the recruitment of active bacterial rhizobiome members. We applied 16S rRNA and 16S rRNA gene sequencing for both crops and metabolite profiling for switchgrass. We included planted and unplanted conditions to distinguish environment- versus plant-mediated rhizobiome drivers. Differences in community structure were observed between crops and between drought and watered and planted and unplanted treatments within crops. Despite crop-specific communities, drought rhizobiome dynamics were similar across the two crops. The presence of a plant more strongly explained the rhizobiome variation in bean (17%) than in switchgrass (3%), with a small effect of plant mediation during drought observed only for the bean rhizobiome. The switchgrass rhizobiome was stable despite changes in rhizosphere metabolite profiles between planted and unplanted treatments. We conclude that rhizobiome responses to short-term drought are crop-specific, with possible decoupling of plant exudation from rhizobiome responses.

16S rRNA/rRNA Gene Ratios and Cell Activity Staining Reveal Consistent Patterns of Microbial Activity in Plant-Associated Soil.

At any given time, only a subset of microbial community members are active in their environment. The others are in a state of dormancy, with strongly reduced metabolic rates. It is of interest to distinguish active and inactive microbial cells and taxa to understand their functional contributions to ecosystem processes and to understand shifts in microbial activity in response to change. Of the methods used to assess microbial activity-dormancy dynamics, 16S rRNA/rRNA gene amplicons (16S ratios) and active cell staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) are two of the most common, yet each method has limitations. Given that in situ activity-dormancy dynamics are proxied only by laboratory methods, further study is needed to assess the level of agreement and potential complementarity of these methods. We conducted two experiments investigating microbial activity in plant-associated soils. First, we treated corn field soil with phytohormones to simulate plant soil stress signaling, and second, we used rhizosphere soil from common bean plants exposed to drought or nutrient enrichment. Overall, the 16S ratio and CTC methods exhibited similar patterns of relative activity across treatments when treatment effects were large, and the instances in which they differed could be attributed to changes in community size (e.g., cell death or growth). Therefore, regardless of the method used to assess activity, we recommend quantifying community size to inform ecological interpretation. Our results suggest that the 16S ratio and CTC methods report comparable patterns of activity that can be applied to observe ecological dynamics over time, space, or experimental treatment. IMPORTANCE Although the majority of microorganisms in natural ecosystems are dormant, relatively little is known about the dynamics of the active and dormant microbial pools through both space and time. The limited knowledge of microbial activity-dormancy dynamics is in part due to uncertainty in the methods currently used to quantify active taxa. Here, we directly compared two of the most common methods (16S ratios and active cell staining) for estimating microbial activity in plant-associated soil and found that they were largely in agreement in the overarching patterns. Our results suggest that 16S ratios and active cell staining provide complementary information for measuring and interpreting microbial activity-dormancy dynamics in soils. They also support the idea that 16S rRNA/rRNA gene ratios have comparative value and offer a high-throughput, sequencing-based option for understanding relative changes in microbiome activity, as long as this method is coupled with quantification of community size.

Fine root tradeoffs between nitrogen concentration and xylem vessel traits preclude unified whole-plant resource strategies in Helianthus.

Recent work suggests variation in plant growth strategies is governed by a tradeoff in resource acquisition and use, ranging from a rapid resource acquisition strategy to a resource-conservative strategy. While evidence for this tradeoff has been found in leaves, knowledge of root trait strategies, and whether they reflect adaptive differentiation across environments, is limited. In the greenhouse, we investigated variation in fine root morphology (specific root length and tissue density), chemistry (nitrogen concentration and carbon:nitrogen), and anatomy (root cross-sectional traits) in populations of 26 Helianthus species and sister Phoebanthus tenuifolius. We also compared root trait variation in this study with leaf trait variation previously reported in a parallel study of these populations. Root traits varied widely and exhibited little phylogenetic signal, suggesting high evolutionary lability. Specific root length and root tissue density were weakly negatively correlated, but neither was associated with root nitrogen, providing little support for a single axis of root trait covariation. Correlations between traits measured in the greenhouse and native site characteristics were generally weak, suggesting a variety of equally viable root trait combinations exist within and across environments. However, high root nitrogen was associated with lower xylem vessel number and cross-sectional area, suggesting a tradeoff between nutrient investment and water transport capacity. This led to correlations between root and leaf traits that were not always consistent with an acquisition-conservation tradeoff at the whole-plant level. Given that roots must balance acquisition of water and nutrients with functions like anchorage, exudation, and microbial symbioses, the varied evidence for root trait covariation likely reflects the complexity of interacting selection pressures belowground. Similarly, the lack of evidence for a single acquisition-conservation tradeoff at the whole-plant level likely reflects the vastly different selection pressures shaping roots and leaves, and the resources they are optimized to obtain.

Evolutionary Divergences in Root Exudate Composition among Ecologically-Contrasting Helianthus Species.

Plant roots exude numerous metabolites into the soil that influence nutrient availability. Although root exudate composition is hypothesized to be under selection in low fertility soils, few studies have tested this hypothesis in a phylogenetic framework. In this study, we examined root exudates of three pairs of Helianthus species chosen as phylogenetically-independent contrasts with respect to native soil nutrient availability. Under controlled environmental conditions, seedlings were grown to the three-leaf-pair stage, then transferred to either high or low nutrient treatments. After five days of nutrient treatments, we used gas chromatography-mass spectrometry for analysis of root exudates, and detected 37 metabolites across species. When compared in the high nutrient treatment, species native to low nutrient soils exhibited overall higher exudation than their sister species native to high nutrient soils in all three species pairs, providing support for repeated evolutionary shifts in response to native soil fertility. Species native to low nutrient soils and those native to high nutrient soils responded similarly to low nutrient treatments with increased exudation of organic acids (fumaric, citric, malic acids) and glucose, potentially as a mechanism to enhance nutrition acquisition. However, species native to low nutrient soils also responded to low nutrient treatments with a larger decrease in exudation of amino acids than species native to high nutrient soils in all three species pairs. This indicates that species native to low nutrient soils have evolved a unique sensitivity to changes in nutrient availability for some, but not all, root exudates. Overall, these repeated evolutionary divergences between species native to low nutrient soils and those native to high nutrient soils provide evidence for the adaptive value of root exudation, and its plasticity, in contrasting soil environments.

Evolutionary divergences in root system morphology, allocation, and nitrogen uptake in species from high- versus low-fertility soils.

Root morphology and nutrient uptake processes are essential for acquisition of mineral resources from soil. However, our understanding of how root form and function have diverged across environments is limited. In this study, we addressed hypotheses of adaptive differentiation using three pairs of Helianthus species chosen as phylogenetically-independent contrasts with respect to native soil nutrients. Under controlled environmental conditions, root morphology, allocation, and nitrogen (N) uptake (using a 15N tracer) were assessed for seedlings under both high and low N treatments. Species native to low nutrient soils (LNS) had lower total root length than those native to high nutrient soils (HNS), reflecting the slower growth rates of species from less fertile environments. Contrary to expectations, species did not consistently differ in specific root length, root tissue density, or root system plasticity, and species native to LNS had lower root:total mass ratio and higher 15N uptake rates than species native to HNS. Overall, these evolutionary divergences provide support for adaptive differentiation among species, with repeated evolution of slow-growing root systems suited for low resource availability in LNS. However, species native to LNS maintain a high capacity for N uptake, potentially as a means of maximising nutrient acquisition from transient pulses.

Macroevolution of leaf defenses and secondary metabolites across the genus Helianthus.

Leaf defenses are widely recognized as key adaptations and drivers of plant evolution. Across environmentally diverse habitats, the macroevolution of leaf defenses can be predicted by the univariate trade-off model, which predicts that defenses are functionally redundant and thus trade off, and the resource availability hypothesis, which predicts that defense investment is determined by inherent growth rate and that higher defense will evolve in lower resource environments. Here, we examined the evolution of leaf physical and chemical defenses and secondary metabolites in relation to environmental characteristics and leaf economic strategy across 28 species of Helianthus (the sunflowers). Using a phylogenetic comparative approach, we found few evolutionary trade-offs among defenses and no evidence for defense syndromes. We also found that leaf defenses are strongly related to leaf economic strategy, with higher defense in more resource-conservative species, although there is little support for the evolution of higher defense in low-resource habitats. A wide variety of physical and chemical defenses predict resistance to different insect herbivores, fungal pathogens, and a parasitic plant, suggesting that most sunflower defenses are not redundant in function and that wild Helianthus represents a rich source of variation for the improvement of crop sunflower.

Heterogeneity of clonal patterns among patches of kudzu, Pueraria montana var. lobata, an invasive plant.

BACKGROUND AND AIMS: Viny species are among the most serious invasive plants, and better knowledge of how vines grow to dominate landscapes is needed. Patches may contain a single genotype (i.e. genet), a competitively dominant genet or many independent but interacting genets, yet the clonal structure of vining species is often not apparent. Molecular markers can discriminate among the genetic identities of entwined vines to reveal the number and spatial distribution of genets. This study investigated how genets are spatially distributed within and among discrete patches of the invasive vine kudzu, Pueraria montana var. lobata, in the United States. It was expected that ramets of genets would be spatially clustered within patches, and that an increase in the number of genets within a patch would be associated with a decrease in the average size of each genet. METHODS: Six discrete kudzu patches were sampled across 2 years, and 1257 samples were genotyped at 21 polymorphic allozyme loci. Variation in genotypic and genetic diversity among patches was quantified and patterns of genet interdigitation were analysed. KEY RESULTS: Substantial genotypic and genetic variation occurred within and among patches. As few as ten overlapping genets spanned up to 68 m(2) in one patch, while >90 % of samples were genetically unique in another patch. Genotypic diversity within patches increased as mean clone size decreased, although spatially widespread genets did not preclude interdigitation. Eight genets were shared across ≥2 patches, suggesting that vegetative dispersal can occur among patches. CONCLUSIONS: Genetically unique kudzu vines are highly interdigitated. Multiple vegetative propagules have become established in spatially discrete patches, probably through the movement of highway construction or maintenance machinery. The results suggest that common methods for controlling invasive vines (e.g. mowing) may inadvertently increase genotypic diversity. Thus, understanding vine architecture and growth has practical implications.