Co-occurrence networks reveal more complexity than community composition in resistance and resilience of microbial communities
Testing H1 and H2 at community composition levelAs noted above, the simple fact that fungi grow more slowly than bacteria is the basis of the hypotheses that (H1) fungal communities should be more resistant than bacterial communities to drought stress, and (H2) that fungal communities should be less resilient than bacterial communities when the stress is relieved by rewetting18. In addition to growth rate, these two hypotheses may be related to differences in the form of growth between fungi and bacteria. For example, multicellular hyphal growth versus unicellular division or the greater thickness of fungal cell walls as compared to those of bacteria47,48. We tested H1 and H2 at the community composition level by blending the fungal and bacterial datasets generated from the same leaf, root, rhizosphere and soil samples collected from field-grown sorghum that had been either irrigated as a control, or subjected to preflowering drought followed by regular wetting beginning at flowering10,11.We followed the approach of Shade et al.17 to detect resistance and resilience, which had been developed for univariate variables, e.g., richness. For multivariate data, e.g., community composition, we modified it by calculating pairwise community dissimilarity for two groups: within-group (control-control pairs, drought-drought pairs, or rewetting-rewetting pairs), and between-group (control-drought pairs, or control-rewetting pairs). Ecological resistance to drought stress is detected by comparing compositional dissimilarity of between-group pairs (control-drought pairs) against within-group pairs (control-control pairs and drought-drought pairs) for each of the droughted weeks (weeks 3–8). Ecological resilience to rewetting is detected by assessing, from before to after rewetting, the change in the difference of compositional dissimilarity between within-group pairs and between-group pairs. Here, the point just before rewetting was week 8 and the points after rewetting were weeks 9–17. A t-test was used to assess the statistical significance of the differences in resistance or resilience between bacterial and fungal communities at each time point for each compartment.To account for the different resolutions of ITS and 16 S, we compared bacterial 16 S OTUs against both fungal ITS, species-level OTUs as well the fungal family level (Supplementary Fig. 1). The results of analyses using either fungal families or OTUs are consistent. Out of 36 comparisons (15 roots, 15 rhizospheres and 6 soils), different family and OTUs results were detected in four instances. In two of these, significances detected by OTUs were not detected by family (root, weeks 4 and 17) and, in the other two cases, significances detected by family were not detected by OTUs (rhizosphere, weeks 7 and 8). (Fig. 1). We report only results that are consistent at both the species and family levels (Fig. 1).In line with our first hypothesis, H1, we found that the resistance to drought stress for fungal mycobiomes was consistently stronger than that for bacterial microbiomes for weeks 5 in root, weeks 4–6 in rhizosphere, and weeks 4 and 6–8 in soil (Fig. 1, Supplementary Fig. 1 and Supplementary Table 2). In support of our second hypothesis, H2, when the stress of pre-flowering drought was relieved by rewetting, we found that the resilience of the bacterial communities was consistently higher than that for the fungi in weeks 9–16 in root, and weeks 11–17 in rhizosphere (Fig. 1, Supplementary Fig. 1 and Supplementary Table 2).Surprisingly, we found that resilience was stronger for fungal than bacterial communities in the first week (week 9) of rewetting in the rhizosphere (Fig. 1, Supplementary Fig. 1 and Supplementary Table 2). This high resilience of fungi may be associated with the quick growth of sorghum roots when rewetted. The rhizosphere zone around these newly formed roots may be quickly colonized by soil fungi, a community that was weakly affected by drought. This result suggests that re-assembly of the rhizosphere microbial community is more complex than previously expected.The finding that fungal community composition in the soil is not shaped by drought prevented us from further detecting resilience (Fig. 1). Note fungal community in early leaves was excluded from analysis due to the high proportion of non-fungal reads in sequencing11.Testing H1 and H2 at all-correlation levelNext, we moved from the comparison of whole communities to correlation among individual bacterial and fungal taxa to test the hypotheses about resistance, H1, and resilience, H2. As noted above, previous research provided the foundation for the stress gradient hypothesis, which predicts an increase in positive associations in stress32,33,34,35,36,37. Further, ecological modeling predicts that negative associations promote stability40. Concerning specific associations, studies of Arabidopsis and associated microbes reported that positive associations are favored within kingdoms, i.e., within bacteria or within fungi, while negative associations predominate between kingdoms38,39. Given these foundations, concerning H1, we expected an increase in the proportion of positive correlation by drought stress that would be strongest for B-B, followed by F-F, and lastly by B-F; for H2 we expected rewetting to cause a decrease in the proportion of positive correlation, again most strongly for B-B, followed by F-F, and lastly by B-F.Overall, at the all-correlation level, we found no consistent support for the differences postulated for bacterial and fungal responses in H1. For example, strong increases in the proportion of positive correlations under drought could be found in all microbial pairings for some compartments (B-B in leaf and root, F-F in rhizosphere and soil, and B-F in root and rhizosphere) (Fig. 2a, Supplementary Figs. 2, 3). Neither did we find consistent support for the differences ascribed to bacteria and fungi in H2 as the strongest decreases in the proportion of positive correlations during rewetting occurred at F-F in rhizosphere and soil, and B-B in leaf and root (Fig. 2b, Supplementary Figs. 2, 3).Fig. 2: Correlations of microbes in drought stress and drought relief.Estimates of combined correlations (row a) show an increase in positive correlations under drought stress across the four compartments (root, black; rhizosphere, blue; soil, red; leaf, green). Data points underlying the lines in the figure are provided in the alternative version in Supplementary Fig. 2. This result is in line with the stress gradient hypothesis which posits that stressful environments favor positive associations because competition will be less intense than in benign environments32,33,36,37. Note that positive trends in combined correlations can arise in two ways. First, from an increase of positive correlations (row b) that exceeds the rise in negative correlations (row c), e.g., Leaf bacterial-bacterial (Bac-Bac) correlations or rhizosphere fungal-fungal (Fun-Fun) correlations in the drought period (Negative correlations in row C values are multiplied by −1 to facilitate comparison). Second, from a decrease in negative correlations that exceeds a decrease in positive correlations, e.g., root bacterial-bacterial correlations or root bacterial-fungal (Bac-Fun) correlations in drought. Combined (a), positive (b) and negative (c) estimates of correlation (Spearman’s rho, ρ) are given for four compartments (root, rhizosphere, soil and leaf), and three types of correlations (Bacterium-Bacterium, Fungus-Fungus, Bacterium-Fungus). T-tests (two sided) were carried out for linear mixed effect modelling that incorporates link type and compartments as random factors. Detailed distribution densities of correlations are presented in Supplementary Fig. 3. Source data are provided as a Source Data file.Full size imageWe found support for the stress gradient hypothesis because drought increased the relative frequency of positive correlations among microbial taxa (Fig. 2a, Supplementary Figs. 2, 3). The increases were due, largely, to B-B correlations in leaf and F-F correlations in the rhizosphere during drought, when the relative frequency of positive correlations was increased (Fig. 2b, Supplementary Figs. 2, 3) and the frequencies of negative correlations were decreased or weakly increased (Fig. 2c, Supplementary Figs. 2, 3). Less obvious increases in the relative frequency of positive correlations (such as B-B in root, F-F in soil, and B-F in root and rhizosphere) occurred where drought reduced both positive and negative correlations, but the losses of negative correlations exceeded those of positive correlations (Fig. 2, Supplementary Figs. 2, 3).In support of the expectation that correlations would be more negative between taxonomic groups than within taxonomic groups, we found that the relative frequency of positive correlations was generally lower for B-F than B-B and F-F correlations (Fig. 2, Supplementary Figs. 2, 3). Moreover, as ecological modeling has indicated that negative associations should promote stability of communities40, we hypothesize that B-F correlations would be more stable than B-B and F-F networks in response to drought stress. However, we found no support for this hypothesis, as B-F correlations (for example in root) did not always show the least response to drought stress (Fig. 2, Supplementary Figs. 2, 3).Testing H1 and H2 at species co-occurrence levelFor our final test of H1 (resistance) and H2 (resilience) we focused on co-occurrence networks based on significant, positive correlations. These networks have been reported to be destabilized for bacteria but not for fungi in mesocosms subject to drought stress19, and shown to be disrupted for bacteria in natural vegetation studied over gradients of increasing aridity41,42. Using these results as guides, for H1 we expected that drought stress should disrupt co-occurrence networks most strongly for B-B, followed by F-F, and lastly by B-F. For H2 we expected that relief of stress by rewetting should strengthen microbial co-occurrence networks most strongly for B-B, followed by F-F, and lastly by B-F.For this test we constructed microbial co-occurrence networks using significant positive pairwise correlations between microbial taxa, B-B, F-F and B-F, and compared the network complexity between fully irrigated control and drought, and between control and rewetting following drought. In general, we found no consistent support for the difference between bacteria and fungi inherent in H1. Rhizosphere was the one compartment where B-B vertices dropped and F-F vertices rose in response to drought, as expected, but this result was offset in root and soil, where vertices dropped in all networks, B-B, F-F and B-F (Figs. 3, 4; Supplementary Figs. 4, 5). Analysis by co-occurrence networks highlighted the differences between plant compartments. In root drought strongly disrupted networks of B-B, B-F and F-F, but in the other three compartments, network disruption was weaker, and networks were even enhanced by drought for F-F in rhizosphere and B-B in leaf (Figs. 3, 4).Fig. 3: Networks of significant positive cross-taxonomic group correlations (bacteria and fungi).a Fungal operational taxonomic units (OTUs) (blue) and bacterial OTUs (black) are graphed as nodes. Significant positive Spearman correlations are graphed as edges (ρ > 0.6, false discovery rate adjusted P More