Mutualism outcome across plant populations, microbes, and environments in the duckweedLemna minor

Abstract

1The picture emerging from the rapidly growing literature on host-associated micro-2 biota is that host traits and fitness often depend on complex and interactive effects 3 of host genotype, microbial interactions, and abiotic environment. However, testing 4 these main and interactive effects typically requires large, multi-factorial experiments 5 and thus remains challenging in many systems. Furthermore, most studies of plant 6 microbiomes focus on terrestrial hosts and microbes. Aquatic habitats may confer 7 unique properties to plant micriobiomes. We grew different populations of duck-8 weed (Lemna minor ), a floating aquatic plant of increasing popularity in freshwater 9 phytoremediation, in three microbial treatments (adding no, "home", or "away" 10 microbes) at two levels of zinc, a common water contaminant in urban areas. Thus, 11 we simultaneously manipulated plant source population, microbial community, and 12 the abiotic environment, and measured both plant and microbial performance as 13 well as plant traits. Although we found little evidence of interactive effects, we found 14 strong main effects of plant source, microbial treatment, and zinc on both duckweed 15 and microbial growth, with significant variation among both duckweed and microbial 16 communities. Despite strong growth alignment between duckweed and microbes, zinc 17 consistently decreased plant growth, but increased microbial growth. Furthermore, 18 as in recent studies of terrestrial plants, microbial interactions altered a duckweed 19 phenotype (frond aggregation). Our results suggest that the duckweed source pop-20 ulation, its associated microbiome, and the contaminant environment may all need 21 to be considered in real-world phytoremediation efforts. Lastly, we propose that 22 duckweed microbes offer a robust experimental system for study of host-microbiota 23 interactions under a range of environmental stresses.24

Resilience to multiple stressors in an aquatic plant and its microbiome

“…It has a nearly cosmopolitan distribution (RDSC, ) and is widespread in the Greater Toronto Area, including in stormwater ponds and other water bodies that are commonly exposed to runoff. Duckweeds have microbial communities living on or in their roots and fronds, and our previous work has found that experimental microbial inoculation of sterilized L. minor increases plant growth (O'Brien et al., [Preprint]).…”

“…We prepared microbial inocula from each site by swabbing across the stored agar plate, adding to yeast mannitol broth, culturing in a shaking incubator (VWR, Radnor, PA, USA) for 3 d at 30°C and 200 rpm, and diluting to control cell density across inocula. We prepared dilutions by measuring optical density, converting to cell counts by applying a single calibration curve across inocula (correlated to colony forming units, O'Brien et al., [Preprint]), and standardizing to approximately 3000 cells/μL. The exact relationship of optical density to cell count depends on both cell size and composition (Volkmer and Heinemann, ), both of which likely vary across the taxa in our cultures, so cell concentrations may differ slightly across inocula.…”

“…If a well had no pixels passing ImageJ thresholds, we regarded all duckweed tissue in the well as dead. Because duckweeds primarily reproduce asexually, the pixel area of live fronds is very tightly correlated to the number of reproductive individuals (O'Brien et al., [Preprint]). From images taken on day 10, we also scored two plant traits: greenness and aggregation (the ratio of frond area to frond perimeter, see Appendix S3).…”

“…We took two 70 μL samples from each well and measured optical density at 600 nm wavelength on a plate reader (BioTek Synergy HT with Gen5 1.10 software, Winooski, VT, USA). Optical density at 600 nm is proportional to the density of all microbial cells in the sample (O'Brien et al., [Preprint]); i.e., it is a measure of total microbial abundance.…”

Resilience to multiple stressors in an aquatic plant and its microbiome

“…It has a nearly cosmopolitan distribution (RDSC, 2016), and is widespread in the Greater Toronto Area, including in stormwater ponds and other water bodies that are commonly exposed to runoff. Duckweeds have microbial communities living on or in their roots and fronds, and our previous work has found that experimental microbial inoculation of sterilized L. minor increases plant growth (O’Brien et al, 2018).…”