The effect of the drought treatments on Oryza longistaminata performance
To test the plant-associated microbial community response to drought stress, drought was imposed on Oryza longistaminata for two months. Two months after water withdrawal, the soil moisture content in the treated samples decreased from 34.6% to 0.072% (Fig. S1). Notably, the drought-stressed plants remained viable throughout the experiment, which indicated O. longistaminata’s strong drought tolerance traits (data not shown).
Drought significantly decreased the leaf photosynthetic rate (Pn) (P<0.01) and the leaf intercellular CO2 concentration (Ci) (P<0.01), while increasing the stomatal limitation value (Ls) (P<0.01) (Fig. 1A). The results suggest that drought decreased the leaf photosynthesis of O. longistaminata through stomatal closure. To test the degree of membrane lipid peroxidation caused by drought stress, the MDA content was measured. Drought increased the MDA content (P<0.01), suggesting that drought decreased photosynthesis through metabolic impairment (Fig. 1B). Drought significantly decreased the antioxidant enzymatic activity levels of SOD and POD and while increased the antioxidant enzymatic activity levels of CAT (P<0.05) (Fig. 1C). Collectively, these data demonstrate that drought treatments lead to a corresponding increase in plant stress.
Figure 1
Drought shapes the microbiome taxonomic community structure in the different compartments
Across plant compartments, the composition of the microbial communities varied significantly. The taxonomic α-diversity increased from the leaf towards the rhizosphere (Kruskal-Wallis test, P<0.01) (Fig. 2A and S2). We quantified microbiome community composition using weighted UniFrac distances with principal coordinate analysis and found clear differences in the compositions of the leaf, stem, root, and rhizosphere compartments (PERMANOVA, R2=0.18375, P<0.001) (Fig. 2B). The results indicated that the O. longistaminata microbiomes was spatially structured in each distinct compartments.
Figure 2
The microbial taxonomic alpha diversity within each sample was analyzed based on the Shannon diversity index. The mean α-diversity reduced under drought-stress in the leaves and stems, suggesting the decreased growth in a few bacterial groups under drought (Fig. 2C). However, the α-diversity of the root and rhizosphere microbiomes did not differ significantly in response to drought (Kruskal-Wallis test, P>0.05) (Fig. 2C). Unconstrained principal coordinate analyses (PCoAs) based on the weighted UniFrac (WUF) distance matrix were performed to investigate patterns of separation among microbial communities between the control and drought treatments within different compartments. The microbiome communities were separated by drought in the leaves, stems, roots, and rhizosphere (PERMANOVA, P<0.05) (Fig. 2D).
We analyze the drought-mediated alterations within the individual compartments. There were notable differences in the proportions of various phyla and families influenced by water deprivation. Drought dramatically affected the relative abundances of the microbiome in the endosphere compartments (Fig. S3AB). To more clearly show the patterns of the drought-responsive taxa, we display the first 50 different taxa at the phylum and family levels (Fig. S3CD). At the phylum level, drought-affected not only the major groups but also the minor groups, indicating a broad exclusionary effect of drought on the relative abundances of the microbiome (Fig. S3C). In response to drought, the patterns of control over their resident microbiota communities in the four compartments were divided into two patterns, i.e., aboveground and belowground patterns (Fig. S3CD). In the aboveground tissues, the phyla Euryarchaeota, Actinobacteria, Fusobacteria, and Firmicutes were significantly enriched, and the phyla beta-proteobacteria, Bacteroidetes, Spirochaetes, and Chloroflexi, the genus Pseudomonadaceae were significantly depleted under drought stress. In belowground tissues, the phyla Actinobacteria, Fusobacteria, TM7, Tenericutes, and FBP, the genus Streptomycetaceae were significantly enriched, and the phyla Spirochaetes, Epsilon-proteobacteria and OD1 were significantly depleted under drought stress (Fig. S3C). The results indicated that aboveground and belowground plant parts host microbiome assemblies with different taxonomical structures in response to drought.
The microbiome co-occurrence networks of ASVs changed with drought stress in compartment-specific patterns
The α-diversity data showed that microbial communities in aboveground tissues are more sensitive to drought than those of belowground compartments (Fig. 2C). We generated a microbial ASV (amplicon sequence variant) co-occurrence network using significant correlations to explore the more detailed changes in potential interactions among microbiota under drought stress.
The diameter, the number of edges, and vertices of the co-occurrence networks increased, while the clustering coefficient decreased, in the order of aboveground tissues, roots and rhizosphere soil (Fig. 3A, Table S1). The data indicated that the co-occurrence networks become larger and more connected and have less modularity from the aboveground plant parts to the rhizosphere soil. The wild rice microbiota formed highly compartmentalized coexistence networks of coexistence within the host.
Surprisingly, the responses of the aboveground and belowground microbial networks to drought was quite opposites. Drought strongly decreased the connectedness of nodes, the number of edges and vertices, and the number of clusters in the aboveground microbial networks, while it increased these properties in the below-ground microbial networks (Fig. 3ABC, Table S1). In particular, the microbial network’s negative correlation was strengthened in the rhizosphere under drought stress, which could be interpreted as an increase in competitive relationships within the rhizosphere under drought (Fig. 3A, Table S1). Specific microbes that are highly connected to other microbes within the co-occurrence networks are often defined as “hub” or “keystone” species and likely exert a stronger influence than other speces on the structure of microbial communities. We then focused on how the network hubs respond to drought stress in belowground microbial networks. In the roots, Proteobacteria and Firmicutes were the dominant network hubs. Proteobacteria and Firmicutes accounted for 25.0% and 37.5% of the community, respectively, while their relative abundance increased to 54.55% and 9.09% respectively in response to drought (Fig. 3D). In the rhizosphere, drought increased the number of network hubs (Fig. 3D). We also found that the network hub scores were not related to the relative abundance of the ASVs in all four compartments, and ASVs with either low or high relative abundance can be network hubs (Fig. S5). Together, this suggests that the aboveground microbial networks were unstable under drought, but not belowground co-occurrence networks. Drought promoted the restructuring and strengthening of the belowground networks to more strongly interconnect network properties.
Figure 3
Phylogenetic conservation of microbial drought responses
We quantified the strength of the relationship between phylogeny and drought to determine whether phylogenetic information could be predictive of the response of microbial taxa to the global drought stress. The drought responses were strongly phylogenetically conserved in all compartments. The mean genetic depth (τD) ranged from 0.07282 to 0.2099. The D-test of Fritz and Purvis also confirmed that drought responses were dispersed in a mode between a Brownian motion and a random model (0<D<1) in every compartment, suggesting that closely related species exhibited more similar ecological preferences for drought stress (Fig. 4).
Figure 4
Microbial community assembly processes in drought-stressed Oryza longistaminata
To understand the assembly processes governing the composition of microbial communities of Oryza longistaminata in response to drought stress, we first analyzed changes in the microbial community under drought using Sorensen beta diversity based on the ASV level. The Sorensen beta diversity (βSOR) was the lowest in the rhizosphere, followed by the roots, leaves, and stems (Fig. 5A). To explain those changes in the microbial community, we partitioned the Sorensen beta diversity into turnover (species replacement through dispersal) and nestedness-resultant dissimilarity (species loss through death or emigration) components. We found that species replacement (βSIM) was a much greater contributor to the observed beta diversity than species loss (βSNE) in all four compartments in response to drought (Fig. 5B).
We considered which deterministic and/or stochastic processes give structure to microbial communities. To evaluate the ecological process controlling the composition of each microbial community, the microbial phylogenetic community composition within each community was determined. The phylogenetic alpha diversity (PD) of the rhizosphere microbiome decreased significantly in response to drought (Kruskal-Wallis test, P<0.001) (Fig. 5C). In the leaves and stems, we found that the values of the standardized effect sizes of MNTD (SESMNTD, equivalent to -NTI) calculated using the null model were in the range of -2 to +2 (Fig 5D, Table S2). The values of SESMNTD were < -2 in the roots and rhizospheres, suggesting that microbial communities within these samples were more significantly phylogenetic clustering among co-occurring species than expected by chance (mntd.obs.z < 0, mntd.obs.p < 0.05), and drought significantly increased microbial community clustering in the rhizospheres (p < 0.001) (Fig. 5D, Table S2). In addition, based on the AIC values, the fitness of the null, log-normal, pre-emption, Mandelbrot, and Zipf models was compared to investigate which processes were important in shaping the microbial community structure. The results showed that a large amount of data for the leaf and stem communities fit to the null models (Fig S5), while the nonrandom models best fit the data for the roots and rhizospheres (Fig S5), these results are consistent with the MNTD analysis.
Figure 5
To evaluate the relative influences of stochastic and deterministic processes in controlling community dynamics in response to drought stress, turnover in the phylogenetic composition turnover (phylogenetic β-diversity) between control and drought conditions was calculated using the β nearest taxon index (βNTI) to explore the mechanisms underlying community assembly. The βNTI score for microbial communities was in the range of -2 to +2, indicating that stochastic (neutral) processes dominated microbial community dynamics in response to drought across all four compartments (Fig. 5E). To evaluate the influence of spatial position on phylogenetic turnover, we observed that phylogenetic turnover increased from the leaves to the rhizosphere (Fig. 5F). Density plots showed clear separations among these distributions in different compartments (Fig. 5G). Subsequently, the taxonomic β-diversity metric (Bray-Curtis-based Raup-Crick, RCBray) was used to partition the pairwise comparisons with an absolute βNTI values of < 2. The majority of the RCBray scores were in the range of -0.95 and +0.95 (94.5% in leaves, 92.7% in stems, 66.7% in roots, 86.2% in the rhizosphere) (Fig. 5H), which indicated that weak selection, weak dispersal, diversification, and/or drift dominated the microbial community dynamics (treated as an “undominated” fraction). The RCBray scores that were > 0.95 (5.45% in leaves, 7.27% in stems, 33.3% in roots, 13.8% in rhizospheres) indicated that dispersal limitation dominated the microbial community dynamics (Fig. 5H).
Drought affected the functional composition of the microbial community in belowground compartments
Given the evidence for links between microbial communities and biogeochemical cycles, we expected drought-induced changes in microbial communities and networks to influence microbial functions. This expectation was tested using AFPPOTAX. Our data showed that rhizosphere microbiota performed a complete set of biogeochemical processes involving methanogenesis, sulfur-cycling, nitrogen-cycling, carbohydrate metabolism, metal metabolism, and photoautotrophy (Fig. 6A). Drought strongly affected the biogeochemical processes involving a one-carbon cycling, sulfur cycling, iron respiration, and phototrophy. In response to drought stress, the functional composition of the microbial community in the rhizospheres shown more robust than that in the roots (Fig. 6AB). In detail, methylotrophy, sulfur, and iron respiration were significantly decreased under drought in the roots (Wilcoxon test, P<0.01) (Fig 6AB). In the rhizosphere, iron respiration decreased significantly in response to drought (Wilcoxon test, P<0.01), while phototrophy increased significantly (P<0.05) (Fig. 6AB).
Figure 6