Introduction
Recent decades have seen considerable interest in the relationship between plants and their associated microorganisms, the plant microbiome (Bahram et al. 2018, Arias-Sánchez et al. 2019, Schmid et al. 2019, Tan et al. 2021). It is well known that a taxonomically rich assemblage of microbes colonises every accessible plant tissue and often have important effects on plant functioning and fitness. Plant-associated microbiomes may confer fitness advantages to the plant host, via increased nutrient uptake, stress tolerance (Smith et al. 2010, Zhu et al. 2010, Lau and Lennon 2012, Kivlin et al. 2013), resistance to pathogens (Pieterse et al. 2014, Compant et al. 2019), and reduced herbivory (Hubbard et al. 2019). Examples include nitrogen-fixing arbuscular mycorrhizal fungi, whose mutualistic association with terrestrial plants often releases the host from severe nitrogen or phosphorus limitation (Smith and Read 2008). In addition, there are other important mutualistic plant-microbe interactions, namely plant growth-promoting bacteria (PGPB), which have the potential to increase the yield of agricultural plant crops and other applications (Glick 2012). However, the majority of reported plant–microbe interactions are negative (Bever 2003, Kulmatiski et al. 2008, Van der Putten et al. 2013), which is thought to be play a role in promoting plant coexistence. (Bever et al. 1997, Bever 2003).
Although the vast majority of work on the plant microbiome focuses on terrestrial plants, there is a growing literature investigating the consequences of microbiota for floating aquatic plants (Crump and Koch 2008, Xie et al. 2015). Much of this work has focused on Lemna minor , a tiny floating aquatic plant in the family Lemnaceaewhich is increasingly used as a model system for host-microbe interactions (Zhang et al. 2010). Among the smallest of all angiosperms, L. minor consists of only a single floating leaf-like frond to which a single unbranched root is attached. Its reproduction is almost exclusively asexual and vegetative with daughter fronds budding out of the mother frond’s two meristematic pouches located on the frond’s lower surface. Daughter fronds remain attached to the mother for a certain period of time by a stipe, a stem-like bundle of vascular tissue, resulting in colonies of varying sizes, before splitting apart after abscission severs the stipe (Landolt 1986, Lemon et al. 2001). They are widespread and abundant, often found growing on the surface of eutrophic ponds, wetlands, and slow-moving rivers. In the wild, the fronds and roots are covered in a species rich assemblage of microbes (Gilbert et al. 2018, Acosta et al. 2020), which can be removed by sterilisation in the lab when used as an experimental model (Bowker et al. 1980). Interest in the L. minor microbiome dates back to the early 20th C, with the observation of an association with N-fixing bacteria (Bottomley 1920), and has accelerated in recent years (Ishizawa et al. 2017a, 2017b, 2019, Gilbert et al. 2018, Chen et al. 2019, Acosta et al. 2020, Iwashita et al. 2020, O’Brien et al. 2020a, 2020b, Tan et al. 2021) with a general consensus that plant-microbe interactions play an important role in mediating plant fitness and function. Although most of this research focuses on identifying specific PGPB strains, recent work has characterised the complete core bacterial assemblage associated with L. minor(Acosta et al. 2020), which consists of largely Proteobacteria (Pseudomonas and Actinobacteria) and bears a close resemblance to the leaf microbiome in terrestrial plants like Arabidopsis and rice. Although certain select strains of microbes are important in promoting L. minor growth, it remains unclear how the full natural assemblage, which also includes countless microbial pathogens, parasites and competitors, impacts plant fitness. The small size of this plant makes it well-suited to highly replicated experiments, and its fast generation time and vegetative asexual reproduction means that the life-time fitness can be measured across multiple generations within a single experiment simply as population growth rate.
The effect of the microbiome on host fitness and phenotype may both depend on the environment and the host genotype. Plant genotypes often differ in their responses to abiotic environmental conditions (Rehfeldt et al. 2002, Wilczek et al. 2014). These GxE (genotype by environment) interactions have been shown in some cases to depend on the microbiome, whose composition may vary among plant genotypes (Wagner et al. 2016, O’Brien et al. 2020a), or whose impact may mediate plant phenotypic responses to the environment. Just like the abiotic environment, the biotic environment can affect expression of phenotypically plastic traits and fitness in terrestrial plants (Friesen et al. 2011, Wagner et al. 2014), and these microbially-mediated shifts in plant phenotype have been shown to effect plant tolerance to environmental stress (Wagner et al. 2014, Hubbard et al. 2019, O’Brien et al. 2019). Furthermore, certain environmental conditions can lead to the decoupling of plant-microbe mutualisms (Shantz et al. 2016). Thus, the traits and fitness of the host plant depend on the host genotype, its microbiome, the abiotic environment, and the interactions between these three factors.
In this study we have three aims. First, we ask how the presence of the natural L. minor microbiome affects plant fitness. Second, we ask whether fitness effects of the microbiome depend on the specific environmental conditions and are associated with changes in plant phenotypic plasticity. Thirdly, we ask if different genotypes and their associated microbiomes differ in terms plant fitness and phenotypic plasticity (GxE).