Title: Switchgrass metabolomics reveals striking genotypic and developmental differences in 3 saponins 4 Abstract 38Switchgrass (Panicum virgatum L.) is a bioenergy crop that grows productively on low-fertility 39 lands not suitable for food production. We hypothesize that traits such as low soil nitrogen 40 demand, tolerance to water limitation and resistance to insect pests and microbial pathogens are 41 influenced by low molecular weight compounds known as specialized metabolites. We leveraged 42 untargeted liquid chromatography-mass spectrometry (LC-MS) and quantitative gas 43 chromatography-mass spectrometry (GC-MS) to identify differences in above-and below-44 ground metabolomes of three northern upland and three southern lowland switchgrass cultivars. 45This analysis documented abundant steroidal saponins and terpenoid glycosides as well as varied 46 phenolic compounds in switchgrass extracts. We identified many metabolite 'features' 47 set the stage for testing the impacts of differentially accumulating metabolites on biotic and 53 abiotic stress tolerance and inform development of low-input bioenergy crops. 54 55 56 57 58 59 60 61 62 63 64 4 65

Plant derived bioactive small molecules have attracted attention of scientists across fundamental and applied scientific disciplines. We seek to understand the influence of these phytochemicals on functional phytobiomes. Increased knowledge of specialized metabolite bioactivities could inform strategies for sustainable crop production. Our recent investigations of metabolomes of the upland and lowland ecotypes of the bioenergy crop switchgrass revealed large differences in types and abundances of terpenoid specialized metabolites. We hypothesized that - consistent with accumulating evidence that switchgrass genotype impacts microbiome assembly - differential terpenoid accumulation contributes to switchgrass ecotype-specific microbiome composition. An initial plate-based disc diffusion screen of 18 switchgrass root derived fungal isolates revealed differential responses to upland- and lowland-isolated metabolites. To identify specific fungal growth-modulating metabolites, we tested fractions from root extracts on three ecologically important fungal isolates - Linnemania elongata, Trichoderma sp. and Fusarium sp. Saponins and diterpenoids were identified as the most prominent antifungal metabolites. Finally, analysis of liquid chromatography-purified terpenoids revealed fungal inhibition structure - activity relationships (SAR). Saponin antifungal activity was primarily determined by the number of sugar moieties - saponins glycosylated at a single core position were inhibitory whereas saponins glycosylated at two core positions were inactive. Saponin core hydroxylation and acetylation were also associated with reduced activity. Diterpenoid activity required the presence of an intact furan ring for strong fungal growth inhibition. This study demonstrates that switchgrass genotype-specific metabolites differentially inhibit fungal isolates from the root and rhizosphere, supporting the hypothesis that these molecules contribute to microbiome assembly and function.

Background. Mitigating the effects of climate stress on crops is important for global food security. The microbiome associated with plant roots, henceforth, the rhizobiome, can harbor beneficial microbes that alleviate stress impacts. However, the factors influencing the recruitment of the rhizobiome during stress are unclear. We conducted an experiment to understand bacterial rhizobiome responses to short-term drought for two crop species: switchgrass and common bean. We used 16S rRNA and 16S rRNA gene sequencing to investigate the impact of drought severity on the recruitment of active bacterial rhizobiome members. We included planted and unplanted conditions to distinguish the environment- versus plant-mediated drivers of the active rhizobiome. Results. Though each crop had a distinct rhizobiome, there were differences in the active microbiome structure between drought and watered and between planted and unplanted treatments. Despite their different community structures, the drought rhizobiome dynamics were similar across the two crops. However, 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 only observed for the bean rhizobiome. The switchgrass rhizobiome was stable despite differences in the rhizosphere metabolite profiles between planted and unplanted treatments. Specifically, steroidal saponins and diterpennoids were enriched in drought, planted switchgrass soils. Conclusions. We conclude that rhizobiome benefits to resist short-term drought are crop-specific, with the possibility of decoupling of plant exudation and rhizobiome responses, as we observed in switchgrass. We propose bacterial taxa uniquely associated with common bean plants during the short-term drought, which could be further evaluated to determine any plant benefit during drought.