Co-occurrence networks reveal more complexity than community composition in resistance and resilience of microbial communities

Testing H₁ and H₂ at community composition level

As 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 bacteria^47,48. 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 flowering^10,11.

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 level

Next, 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 stress^{32,33,34,35,36,37}. 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 kingdoms^38,39. 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).

We 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 level

For 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 aridity^41,42. 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).

Our second hypothesis H₂ was supported by comparison of rewetted and control microbial networks in the rhizosphere and soil where F-F networks became less complex and B-B networks became more complex, again likely due to the slower growth rate of fungi than bacteria. Behavior of the B-F network largely followed the patterns of B-B in root, rhizosphere and soil (Figs. 3, 4, Supplementary Figs. 4, 5). However, we found no support for the H₂ in leaf and root where the F-F did not lose complexity, and where both the B-B and B-F networks gained complexity (Figs. 3, 4, Supplementary Figs. 4, 5). The results in root and leaf indicate that, upon rewetting, the co-occurrence network disrupted by drought quickly strengthens to become even more complex than the undisturbed control. Our results suggest that resilience does not necessarily stop when approaching the control values, but that resilience of biotic association can exceed the control. Our data highlight a phenomenon that has rarely been reported¹⁷.

We then turned our attention to detecting network modules, finding that network modularity is generally increased by drought stress, and decreased by rewetting (Supplementary Figs. 6–9). The exceptions were the F-F network in rhizosphere and the B-B network in leaf, both of which showed lower modularity under drought (network modularity, F-F: 0.483; B-B: 0.600) than control (network modularity, F-F: 0.698; B-B: 0.835), and higher modularity in rewetting (network modularity F-F: 0.529; B-B: 0.314) than control (network modularity F-F: 0.390; B-B: 0.247) (Supplementary Figs. 6–9).

We then detected the hub taxa of the networks based on their links to other microbes within modules (modular hubs, Zi) and connector taxa based on their links to other microbes in other modules (connectors, Pi). Interestingly, recalling the observed decrease during drought of modularity of the networks for rhizosphere F-F and leaf B-B, we found that the numbers of connectors of both networks were higher for drought than control (Supplementary Figs. 10–13), indicating that higher modularity can result in fewer, larger modules and fewer opportunities for connectors. Specifically, in the rhizosphere F-F network, four connectors [arbuscular mycorrhizal OTU70_Claroideoglomus (Pi = 0.643); saprotroph OTU93_Mortierella (Pi = 0.625); plant pathogens OTU87_Spizellomyces (Pi = 0.64) and OTU624_Cylindrocarpon (Pi = 0.667)] and one modular hub (saprotroph OTU59_Chaetomium, Zi = 2.54) were detected under drought, whereas no network hub, module hub or connector was detected in controls (Supplementary Fig. 11; Supplementary Data 1). In the leaf B-B network, five connectors [four Actinobacteria (Pi = 0.625–0.678) and one Chloroflexi (Pi = 0.667)] and three module hubs [two Actinobacteria (Zi = 3.254) and one Proteobacteria (Zi = 2.852)] were detected under drought, whereas no network hub, module hub or connector was detected in controls (Supplementary Fig. 12; Supplementary Data 1). The decrease of modularity and increase of hub numbers indicate that rhizosphere fungi and leaf bacteria are more interconnected under drought (Fig. 3b).

Guilds

The strong response to drought stress demonstrated by rhizosphere fungi and leaf bacteria encouraged us to sort fungi and bacteria into functional groups, guilds for fungi and phyla for bacteria. When fungi or bacteria, alone, were displayed in networks, the gain in complexity in stress was more apparent (Fig. 3b) and it became possible, for the gain, to calculate the proportion of new correlations that were within or between guilds or phyla (Fig. 3c). For rhizosphere fungi, the inter-guild correlations formed during rewetting were higher than those of constantly watered controls, as was the case with leaf bacteria where the distribution of inter-phyla correlations formed during rewetting was higher than control (Fig. 3c).

Fungi belonging to one of the guilds, arbuscular mycorrhizal fungi (AMF), form key mutualistic symbiosis with plants, and interact with soil microbiome to contribute to the host plant’s adaption to various biotic and abiotic stresses⁴⁹. Here we explored the resistance and resilience of significant, positive, co-occurrences between AMFs and other fungi, and between AMFs and bacteria. We found that the network of AMF and other fungi was disrupted in root and soil but was strengthened in rhizosphere, and that the network of AMF and bacteria was disrupted in root, rhizosphere, and soil. Networks in roots and soil of both AMF and other fungi and AMF and bacteria, when re-wetted, largely recovered their pre-drought complexity. In rhizosphere, however, the network of AMF and other fungi was less complex in rewetting than the control (Fig. 5a), and the network of AMF and bacteria was not different from the control (Fig. 5b).

Source: Ecology - nature.com