Soil origin and plant genotype structure distinct microbiome compartments in the model legume Medicago truncatula

Abstract

Background: Understanding the genetic and environmental factors that structure plant microbiomes is necessary for leveraging these interactions to address critical needs in agriculture, conservation, and sustainability. Legumes, which form root nodule symbioses with nitrogen-fixing rhizobia, have served as model plants for understanding the genetics and evolution of beneficial plant-microbe interactions for decades, and thus have added value as models of plant-microbiome interactions. Here we use a common garden experiment with 16S rRNA gene amplicon and shotgun metagenomic sequencing to study the drivers of microbiome diversity and composition in three genotypes of the model legume Medicago truncatula grown in two native soil communities.Results: Bacterial diversity decreased between external (rhizosphere) and internal plant compartments (root endosphere, nodule endosphere, and leaf endosphere). Community composition was shaped by strong compartment x soil origin and compartment x plant genotype interactions, driven by significant soil origin effects in the rhizosphere and significant plant genotype effects in the root endosphere. Nevertheless all compartments were dominated by Ensifer, the genus of rhizobia that forms root nodule symbiosis with M. truncatula , and additional shotgun metagenomic sequencing suggests that the nodulating Ensifer were not genetically distinguishable from those elsewhere in the plant. We also identify a handful of OTUs that are common in nodule tissues, which are likely colonized from the root endosphere.Conclusions: Our results demonstrate strong host filtering effects, with rhizospheres driven by soil origin and internal plant compartments driven by host genetics, and identify several key nodule-inhabiting taxa that coexist with rhizobia in the native range. Our results set the stage for future functional genetic experiments aimed at expanding our pairwise understanding of legume-rhizobium symbiosis towards a more mechanistic understanding of plant microbiomes.

Co-inoculation with novel nodule-inhabiting bacteria reduces the benefits of legume-rhizobium symbiosis

“…To further investigate the ecological relevance of our NREs, we asked whether our three ASVs of interest, representing the three inoculum strains, could also be detected among amplicon data from a separate study of the leaf, root, and nodule endophytes of M. truncatula endophytes grown in native field soil (Brown et al 2020). Aligning our ASVs of interest against the 50 most abundant sequences generated by Brown et al (2020) revealed imperfect but numerous alignments to taxa in their study (Table S10). The best alignment of ASV1 was OTU00001, an “ Ensifer ” bacterium (now reclassified as Sinorhizobium ) that was found to be a significant nodule endophyte in the Brown et al (2020) study, at 99% identity with 92% coverage of the OTU (Table S10).…”

“…The Pseudomonas and Bacillus NRE strains studied here were originally isolated from nodules of M. truncatula grown in soils collected from its natural range. By comparing our sequencing results to those of another study that grew M. truncatula in such soils (Brown et al 2020), we found OTUs representing taxa closely related to Bacillus sp.…”

“…Lastly, our ASV5 [Pseudomonas sp. 733B] aligned equally well to OTU00013 and OTU00003, which were labeled by Brown et al (2020) as "Streptomyces" and "Pseudomonas", respectively, at 99% identity and 92% coverage (Table S10). Because the OTU sequence lengths in Brown et al (2020) were over 100 bases shorter than the ASVs generated here, the alignments of ASV5 to both Streptomyces and Pseudomonas OTUs are likely due to the lower sequence resolution in their study.…”

“…To further investigate the ecological relevance of our NREs, we asked whether our three ASVs of interest, representing the three inoculum strains, could also be detected among amplicon data from a separate study of the leaf, root, and nodule endophytes of M. truncatula endophytes grown in native field soil (Brown et al 2020). Aligning our ASVs of interest against the 50 most abundant sequences generated by Brown et al (2020) revealed imperfect but numerous alignments to taxa in their study (Table S10). The best alignment of ASV1 was OTU00001, an "Ensifer" bacterium (now reclassified as Sinorhizobium) that was found to be a significant nodule endophyte in the Brown et al…”

“…Table S10. Nucleotide BLAST (Camacho et al 2009) of ASV sequences in Table S7 against the top 50 OTU sequences from Brown et al (Brown et al 2020). Minimum percent identity of 80% and maximum e-value of 0.01.…”