Rhizosphere microbial community composition shifts diurnally and in response to natural variation in host clock phenotype.

Plant-associated microbial assemblages are known to shift at time scales aligned with plant phenology, as influenced by the changes in plant-derived nutrient concentrations and abiotic conditions observed over a growing season. But these same factors can change dramatically in a sub-24-hour period, and it is poorly understood how such diel cycling may influence plant-associated microbiomes. Plants respond to the change from day to night via mechanisms collectively referred to as the internal "clock," and clock phenotypes are associated with shifts in rhizosphere exudates and other changes that we hypothesize could affect rhizosphere microbes. The mustard Boechera stricta has wild populations that contain multiple clock phenotypes of either a 21- or a 24-hour cycle. We grew plants of both phenotypes (two genotypes per phenotype) in incubators that simulated natural diel cycling or that maintained constant light and temperature. Under both cycling and constant conditions, the extracted DNA concentration and the composition of rhizosphere microbial assemblages differed between time points, with daytime DNA concentrations often triple what were observed at night and microbial community composition differing by, for instance, up to 17%. While we found that plants of different genotypes were associated with variation in rhizosphere assemblages, we did not see an effect on soil conditioned by a particular host plant circadian phenotype on subsequent generations of plants. Our results suggest that rhizosphere microbiomes are dynamic at sub-24-hour periods, and those dynamics are shaped by diel cycling in host plant phenotype. IMPORTANCE We find that the rhizosphere microbiome shifts in composition and extractable DNA concentration in sub-24-hour periods as influenced by the plant host's internal clock. These results suggest that host plant clock phenotypes could be an important determinant of variation in rhizosphere microbiomes.

Whole-Genome Duplication and Host Genotype Affect Rhizosphere Microbial Communities.

The composition of microbial communities found in association with plants is influenced by host phenotype and genotype. However, the ways in which specific genetic architectures of host plants shape microbiomes are unknown. Genome duplication events are common in the evolutionary history of plants and influence many important plant traits, and thus, they may affect associated microbial communities. Using experimentally induced whole-genome duplication (WGD), we tested the effect of WGD on rhizosphere bacterial communities in Arabidopsis thaliana. We performed 16S rRNA amplicon sequencing to characterize differences between microbiomes associated with specific host genetic backgrounds (Columbia versus Landsberg) and ploidy levels (diploid versus tetraploid). We modeled relative abundances of bacterial taxa using a hierarchical Bayesian approach. We found that host genetic background and ploidy level affected rhizosphere community composition. We then tested to what extent microbiomes derived from a specific genetic background or ploidy level affected plant performance by inoculating sterile seedlings with microbial communities harvested from a prior generation. We found a negative effect of the tetraploid Columbia microbiome on growth of all four plant genetic backgrounds. These findings suggest an interplay between host genetic background and ploidy level and bacterial community assembly with potential ramifications for host fitness. Given the prevalence of ploidy-level variation in both wild and managed plant populations, the effects on microbiomes of this aspect of host genetic architecture could be a widespread driver of differences in plant microbiomes. IMPORTANCE Plants influence the composition of their associated microbial communities, yet the underlying host-associated genetic determinants are typically unknown. Genome duplication events are common in the evolutionary history of plants and affect many plant traits. Using Arabidopsis thaliana, we characterized how whole-genome duplication affected the composition of rhizosphere bacterial communities and how bacterial communities associated with two host plant genetic backgrounds and ploidy levels affected subsequent plant growth. We observed an interaction between ploidy level and genetic background that affected both bacterial community composition and function. This research reveals how genome duplication, a widespread genetic feature of both wild and crop plant species, influences bacterial assemblages and affects plant growth.

Accuracy of de novo assembly of DNA sequences from double-digest libraries varies substantially among software.

Advances in DNA sequencing have made it feasible to gather genomic data for non-model organisms and large sets of individuals, often using methods for sequencing subsets of the genome. Several of these methods sequence DNA associated with endonuclease restriction sites (various RAD and GBS methods). For use in taxa without a reference genome, these methods rely on de novo assembly of fragments in the sequencing library. Many of the software options available for this application were originally developed for other assembly types and we do not know their accuracy for reduced representation libraries. To address this important knowledge gap, we simulated data from the Arabidopsis thaliana and Homo sapiens genomes and compared de novo assemblies by six software programs that are commonly used or promising for this purpose (ABySS, CD-HIT, Stacks, Stacks2, Velvet and VSEARCH). We simulated different mutation rates and types of mutations, and then applied the six assemblers to the simulated data sets, varying assembly parameters. We found substantial variation in software performance across simulations and parameter settings. ABySS failed to recover any true genome fragments, and Velvet and VSEARCH performed poorly for most simulations. Stacks and Stacks2 produced accurate assemblies of simulations containing SNPs, but the addition of insertion and deletion mutations decreased their performance. CD-HIT was the only assembler that consistently recovered a high proportion of true genome fragments. Here, we demonstrate the substantial difference in the accuracy of assemblies from different software programs and the importance of comparing assemblies that result from different parameter settings.

The effect of rhizosphere microbes outweighs host plant genetics in reducing insect herbivory.

Rhizosphere microbes affect plant performance, including plant resistance against insect herbivores; yet, a direct comparison of the relative influence of rhizosphere microbes versus plant genetics on herbivory levels and on metabolites related to defence is lacking. In the crucifer Boechera stricta, we tested the effects of rhizosphere microbes and plant population on herbivore resistance, the primary metabolome, and select secondary metabolites. Plant populations differed significantly in the concentrations of six glucosinolates (GLS), secondary metabolites known to provide herbivore resistance in the Brassicaceae. The population with lower GLS levels experienced ~60% higher levels of aphid (Myzus persicae) attack; no association was observed between GLS and damage by a second herbivore, flea beetles (Phyllotreta cruciferae). Rhizosphere microbiome (disrupted vs. intact native microbiome) had no effect on plant GLS concentrations. However, aphid number and flea beetle damage were respectively about three- and seven-fold higher among plants grown in the disrupted versus intact native microbiome treatment. These differences may be attributable to shifts in primary metabolic pathways previously implicated in host defence against herbivores, including increases in pentose and glucoronate interconversion among plants grown with an intact microbiome. Furthermore, native microbiomes with distinct community composition (as estimated from 16s rRNA amplicon sequencing) differed two-fold in their effect on host plant susceptibility to aphids. The findings suggest that rhizosphere microbes, including distinct native microbiomes, can play a greater role than population in defence against insect herbivores, and act through metabolic mechanisms independent of population.

The plant circadian clock influences rhizosphere community structure and function.

Plants alter chemical and physical properties of soil, and thereby influence rhizosphere microbial community structure. The structure of microbial communities may in turn affect plant performance. Yet, outside of simple systems with pairwise interacting partners, the plant genetic pathways that influence microbial community structure remain largely unknown, as are the performance feedbacks of microbial communities selected by the host plant genotype. We investigated the role of the plant circadian clock in shaping rhizosphere community structure and function. We performed 16S ribosomal RNA gene sequencing to characterize rhizosphere bacterial communities of Arabidopsis thaliana between day and night time points, and tested for differences in community structure between wild-type (Ws) vs clock mutant (toc1-21, ztl-30) genotypes. We then characterized microbial community function, by growing wild-type plants in soils with an overstory history of Ws, toc1-21 or ztl-30 and measuring plant performance. We observed that rhizosphere community structure varied between day and night time points, and clock misfunction significantly altered rhizosphere communities. Finally, wild-type plants germinated earlier and were larger when inoculated with soils having an overstory history of wild-type in comparison with clock mutant genotypes. Our findings suggest the circadian clock of the plant host influences rhizosphere community structure and function.