Presence of the microbiome consistently decreased growth rates and modified phenotypes of the host plants
The main aim of our experiment was to assess the fitness and phenotypic consequences of the microbiome for L. minor . Although considerable recent work has investigated the importance of certain microbes (mostly bacteria) for L. minor growth (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), the aim has largely been to isolate certain PGPB that increase plant fitness, with few studies characterising the impact of the entire intact natural microbiome on plant performance. Here we isolate plants from eight different genotypes with their full natural microbiomes and assess the impact of the microbiome on host fitness and phenotype.
The effect of the microbiome on plant growth rates was strong and consistent. Contrary to our expectation, the presence of the microbiome decreased plant fitness, on average by 12%. This was the case across all environmental conditions (Fig. 1), and for seven of the eight genotypes (Fig. 2a). Although several important plant-bacteria and plant-fungi mutualisms have been identified for L. minor that increase plant fitness (Acosta et al. 2020, O’Brien et al. 2020a, 2020b, Tan et al. 2021), our results suggest that the importance of pathogens, parasites, and competitors in the microbial assemblage far surpass that of any mutualistic microbes. This is not necessarily surprising given the rich literature documenting the importance of fungal and bacterial pathogens (Rejmankova et al. 1986, Underwood and Baker 1991, Zhang et al. 2010, Ishizawa et al. 2017a, 2017b) and algal competition (van Moorsel 2022), on L. minor growth. In land plants, assemblages of PGPB are often unstable in the field (Parnell et al. 2016), and inL. minor , the effects of fitness-enhancing strains can be lost with the inclusion of additional strains due to non-additive effects (Ishizawa et al. 2017b).
The effects of the microbiome on plant phenotype were equally clear. The presence of the microbiome resulted in plants with shorter roots and smaller fronds across all genotypes (Fig. 3). One explanation for smaller fronds would be the presence of many microbes, including photosynthetic algae, that decrease nutrient availability through direct competition with L. minor . However, if this were the main mechanism through which the microbiome modified L. minorphenotype, then it would result in increased root length, the ubiquitous plastic response to decreased nutrient availability. However, we found the opposite, i.e. shorter roots, perhaps a plant response to limit the available surface area for microbes to colonize. In addition, although decreased nutrient availability results in an increase in colony size, we find that the presence of the microbiome increases frond abscission resulting in smaller colonies (Fig. 4). This is consistent with other work that has found microbially-mediated shifts in average colony size in L. minor (O’Brien et al. 2020a). We therefore conclude that the mechanism by which the microbiome supressed plant fitness in our experiment goes beyond changes in resource levels. Frond abscission in response to heavy metals has been extensively studied in L. minorin the ecotoxicology literature and it is well known that toxic stress generally decreases colony size (Severi 2001, Li and Xiong 2004a, 2004b, Henke et al. 2011, Topp et al. 2011, O’Brien et al. 2020a). The decrease in colony size we observe when the microbiome was present could be due to a similar phenomenon, resulting from toxic microbial secondary metabolites.
One reason for the apparent inconsistency of our results with studies that report a fitness enhancing effect of many microbes (eg. O’Brien et al. 2020a, 2020b, Tan et al. 2021), is that the majority of these studies are explicitly looking to identify only these mutualistic associations. Due to its extremely rapid growth rate, among the fastest of all plants, L. minor research is often in the context of its many industrial applications which include waste water remediation (Landesman et al. 2011, Iqbal and Baig 2016), biomass production as biofuel (Verma and Suthar 2015), animal feed (Islam et al. 2004, Cheng and Stomp 2009), and human consumption (Sree et al. 2016, Appenroth et al. 2017). For all these applications, there is a keen incentive to further enhance growth rate. Much research has focussed on identifying and selecting the most productive genetic strains of L. minor(Bergmann et al. 2000), and much of the work on the microbiome has been done in the same vein, aiming to identify and isolate specific strains of PGPB (Yamaga et al. 2010, Tang et al. 2015, Appenroth et al. 2016). This bias in the literature could lead to a general impression that the microbiome is dominated by mutualistic fitness-enhancing associations, despite a general lack of evidence. Most studies intentionally isolate strains of bacteria that are good candidates to promote plant growth, which are then artificially inoculated to the axenic plants. Here we take the opposite approach, to estimate the overall effect on their host of the large and diverse assemblages of microbes that make up the microbiome. There are few studies that have tested the effect of entireL. minor microbiome on plant fitness instead of just a small subset of carefully chosen bacteria, and those that did found conflicting results. The study that most resembles ours in design, reinoculated the full microbial community to axenic L. minor , and concluded that the microbiome increased frond senescence (Underwood and Baker 1991).
A limitation of this study is the fact that we did not characterize the microbial community and thus, we can only speculate on the mechanisms responsible for our results. Variation in the phenotypic and fitness consequences of the microbiome was surprisingly consistent across all genotypes. This is notable since our genotype treatment included not just different plant clones, but also independent microbiomes from each genotype. Despite the possibility of strong differences in microbial community composition among genotypes, their overall effect on each plant genotype was overwhelmingly uniform. This is consistent with work that has shown the absence of plant-microbe specialisation among genotypes in L. minor by manipulating plant genotype and microbial community source independently (O’Brien et al. 2020a). It appears that in our experiment, the eight independent microbial communities were of similar composition, at least in terms of board functional groups and their interactions with the plant host.