Rapid differentiation of soil and root microbiomes in response to plant composition and biodiversity in the field

The ongoing loss of biodiversity necessitates a deeper understanding of the ecological processes maintaining biodiversity. Emerging evidence points to the role of microbes in mediating plant species coexistence through host-specific microbiome differentiation. These plant-microbiome feedbacks can contribute to native plant diversity via negative feedbacks (accumulation of host-specific pathogens at low diversity) or positive feedbacks (changes in mutualist density). Litter decomposition rates also vary among plant species, influencing plant-soil feedback (PSF) dynamics and affecting community composition and productivity. Key questions remain regarding the patterns and rates of microbiome component differentiation, particularly in field settings. Plant microbiome feedback is driven by various components (pathogens, mutualists, saprotrophs), with their relative importance depending on their differential impacts on hosts and differentiation rates. Greenhouse studies have shown rapid pathogen and AMF differentiation, but field studies are limited, often spanning multiple years and focusing on subsets of microbiome components. The strength of host-specific differentiation is likely influenced by host plant phylogenetic similarity; closely related species share functional traits, including defenses. Phylogenetic signals exist in pathogen specialization, AMF impacts on host growth, and saprotrophic community composition, but their relative strength in differentiation remains unexplored. Plant community diversity also impacts microbiome composition, with reduced host-specific pathogen density at high diversity (dilution effect) being a likely mechanism for productivity gains. Mycorrhizal and saprotroph composition also respond to plant diversity, potentially mediating neighbor benefits and productivity. However, the relative strength of change in these components with plant richness remains largely unknown. Plant-driven microbiome changes are expected to be more pronounced in roots than soil due to host selection during root colonization and differences in root traits. This study aimed to investigate soil microbiome differentiation across plant species with varying phylogenetic distances, manipulating plant biodiversity, phylogenetic dispersion, and composition four months after planting. The research focused on dissecting the relative strength of microbiome differentiation across microbial functional and taxonomic groups and soil/root compartments, hypothesizing stronger differentiation of pathogens and AMF in roots and stronger responses to plant diversity in more specialized groups.

Previous research has established the significant role of plant-soil feedbacks in shaping plant communities and ecosystem processes. Studies have demonstrated the development of host-specific plant-microbe interactions, including both positive and negative feedbacks impacting plant growth and diversity. Greenhouse studies have shown that pathogen differentiation can occur rapidly within a single growing season, while other studies have shown similar patterns with AMF. However, most research has been conducted under controlled greenhouse conditions, which may not fully reflect the complex dynamics of natural field settings. In the field, several studies have explored the spatial patterns of microbiome composition, noting variation in pathogen, AMF, and saprophyte composition in close proximity to mature plants. The phylogenetic relationships among plant species have also been shown to influence the strength of plant-soil feedback, suggesting the potential for co-evolutionary interactions and specificity. The effects of plant diversity on microbiome composition have also been investigated, with evidence indicating that increasing plant species richness can lead to reduced densities of host-specific pathogens. This phenomenon, known as the "dilution effect," has been linked to increased plant productivity in diverse communities.

This field experiment was conducted in a tallgrass prairie region of North America. 240 plots (1.5m x 1.5m) were established in June 2018, with soil tilled and augmented with soil from a native prairie remnant. The experimental design included 18 prairie plant species (6 from each of three families: Poaceae, Fabaceae, and Asteraceae) in monocultures and mixtures (2, 3, or 6 species) with varying phylogenetic dispersion (under- or over-dispersed) and precipitation treatments (50% or 150% ambient). 18 seedlings pre-inoculated with native soil were planted in each plot. Soil samples were collected in September 2018 (four months post-planting), and paired plots were pooled before analysis, resulting in 120 samples. DNA was extracted from soil and roots separately, and bacterial, fungal, oomycete, and AMF communities were sequenced using a two-step PCR process. Bioinformatics analysis used the QIIME2 pipeline, including quality filtering, denoising, merging, chimera removal, and taxonomic assignment using SILVA and UNITE databases. FUNGuild was used to identify fungal guilds (pathogens, saprotrophs). AMF sequences were filtered to exclude non-AMF sequences. Oomycete OTUs were identified against NCBI databases or by phylogenetic placement. Statistical analysis included co-occurrence network analysis (Spearman's rank correlations), GLMs for microbial diversity (Shannon-Wiener index), PERMANOVAs to assess variance explained by experimental factors (block, species richness, phylogenetic dispersion, plant species proportions), and PCAs to visualize community divergence. Relative abundance of putative pathogen OTUs within genera was analyzed using usearch10 to determine responses to plant family composition.

Co-occurrence network analysis revealed a significant correlation between root and soil bacterial communities, but other groups showed weak correlations. Fungal pathogen diversity in soil increased with plant species richness, while oomycete diversity decreased. In roots, fungal pathogen and saprobe diversity increased with richness, while bacterial diversity decreased. PERMANOVA analysis showed that all microbial communities responded to the planting design. Soil fungal pathogens responded significantly to plant species richness and the proportion of Fabaceae species *C. fasciculata*. Soil saprotrophs significantly responded to *C. fasciculata* and marginally to other Fabaceae and Poaceae species. Soil oomycetes showed a significant response to *E. pallida*. Root bacteria showed a significant response to plant family composition and the interaction of plant family and species richness. Root fungal saprobes differentiated based on the proportion of specific plant species and the interaction of plant family and richness. Root bacterial composition differed significantly based on plant family, proportion of specific species from the three plant families and marginally on plant species richness. Root AMF composition differed significantly according to the proportion of specific species. Beta dispersion analyses revealed significant differences in root fungal saprotrophs with plant species richness, and in root bacteria with plant family composition. PCA showed that soil fungal pathogen differentiation was detected in PC1 and PC5 with plant family, while root non-pathogenic fungi and oomycetes responded in multiple PC axes. Analysis of fungal genera revealed that soil fungal pathogens in genera *Monographella*, *Cercospora*, and *Erysiphe* were more abundant in legume-only plots. *Stagonospora* showed a marginal response. In roots, *Papiliotrema* and *Lophiostoma* showed weak responses.

The study demonstrated rapid differentiation of the soil microbiome in response to plant community composition and diversity, with significant responses observed in root bacteria and soil fungal pathogens four months after planting. The largely independent responses of microbial groups suggest their relative potential importance in plant community dynamics. Root bacteria and soil fungal pathogens showed the strongest differentiation with plant composition, indicating their potential role in generating plant-soil feedbacks. AMF showed the weakest differentiation, suggesting less immediate impact in the short-term. The differentiation of fungal pathogens with plant family supports the idea of pathogen specificity, which could drive stronger negative feedbacks between phylogenetically distant plant pairs. Shifts in pathogen composition with plant density are consistent with the dilution effect mediating productivity benefits of increased plant species richness. The weaker response of AMF could indicate delayed responses compared to pathogens and bacteria, suggesting the relative rates of pathogen and mutualist community dynamics could impact plant productivity responses to plant diversity over time. The stronger impacts of plant composition on root compared to soil compartments support the strong filtering of root colonization, driving microbe specialization. The strong response of fungal pathogens in soil compared to roots may reflect the rapid turnover of root-colonized pathogens, potentially magnifying their growth rates in low diversity plots. Strong responses of fungal saprotrophs to plant composition suggest potential host-specificity, potentially contributing to the Home-Field Advantage effect, reflecting the differential decomposition of plant litter.

This study demonstrated rapid microbiome differentiation in response to plant composition and diversity just four months after planting. Root bacteria and soil fungal pathogens showed the strongest responses. Pathogen and mutualist communities differentiated according to plant family, suggesting host specificity. The findings suggest that pathogens and bacteria may be important drivers of short-term plant-soil feedbacks, while AMF responses might be delayed. Future studies should investigate the long-term effects of microbiome dynamics on plant coexistence and ecosystem functions. The rapid responses observed highlight the importance of considering the microbiome in ecological restoration.

The study was limited to a four-month timeframe, which might not capture the full extent of long-term microbiome dynamics. The pooling of replicate plots before analysis could have masked some variation. The focus on three plant families may limit the generalizability of findings to other plant communities. Further research is needed to investigate the functional roles of bacteria and their relationship to plant-soil feedbacks.