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).