Different from previous studies containing only a single N or water treatment in tropical/subtropical forests, our study involved both the nitrogen addition and precipitation seasonality change (Fig. S1), so the single and interactive effects of the two factors could be checked. The one-year manipulation of the nitrogen and water treatments did not significantly change the above-ground tree communities and biomass in comparison with the control (data unpublished). Yet, as the sensitive ecological groups, soil physicochemical properties and microbial communities showed significant responses to the N and water treatments, which showed great fluctuations between the dry and wet seasons.
The effects of season and treatment in affecting soil physicochemical properties, enzyme activities, and microbial communities
We observed that the seasonal changes were prominent for soil physicochemical properties, soil enzyme activities, and all the aspects of bacterial and fungal community traits. The effects of treatments were generally less than the season (Figs. 1–3, Fig. 5, and Fig. S2). Other studies have also found such strong seasonal effects on soil physicochemical properties or soil microbial communities [70–72]. In the subtropical forests of southern China, the climate is characterized by the divergence of the dry season in autumn-winter and the wet season in spring-summer . The disparate water and temperature situations between the dry and wet seasons could have acclimated soils to exhibit different adaptive traits in different seasons. Remarkably, the SWC differed significantly between the dry and wet seasons, which correlated significantly with almost all the traits of bacterial and fungal communities (Fig. 6). Another explanation for the season’s dominance in affecting soil microbial communities was that the treatments were only conducted for 1 year in our study. The long-term effects of nitrogen addition and precipitation change could cause a great magnitude of changes in soil respiration and microbial communities .
By introducing the dummy variables for the N and water treatments, we did the statistical tests of the two factors and their interaction in affecting soil physicochemical properties and soil microbial communities. For fungal alpha diversity, their interaction played a significant role in the dry season (Table 1). Interestingly, both the N and water treatments tended to reduce the alpha diversity of fungi, while their interaction acted antagonistically to alleviate the decrease of the fungal diversity (Fig. 2). The opposite effect of the interaction of N and water treatments to any of the sole treatments was also observed in one desert ecosystem . In a drought environment, the water and nitrogen could be both limited (as the diffusive capacity of N in soil is hindered), while the addition of exogenous N possibly alleviate the N limitation (Fig. 1) , thus an antagonistic interaction could be observed for N addition and precipitation reduce in the dry season .
Like the situation for the fungal diversity, the season dependence of the N and water treatments’ (and their interaction’s) effects were also observed for the soil physicochemical properties and network topological features (Tables S1 and S4). In the dry season, the water and N treatments and their interaction had more significant effects on soil properties (mainly the SWC and pH) and microbial co-occurrence networks (mainly the within-fungi network and the bacteria-fungi network) than in the wet season. This season-dependence of water and N treatments’ effects were not only in the subtropical forest, but also observed in a temperate desert . In the dry season, the PC treatment (rainfall reduction) caused drastic changes in the SWC (Fig. 1), which served as one primary property that affected other soil physicochemical properties and microbial community traits (Fig. 6). On the contrary, the PC treatment in the wet season (rainfall increase) did not cause significant changes in the SWC; and the TN and NO3− did not change significantly when both the N and water additions were applied (the NPC treatment) (Fig. 1), which implicated high NO3− leaching loss due to the enhanced surface runoff and interflow in the NPC plots (data unpublished). The enhanced hydrologic leaching may downsize the interaction strength of N and water treatments in the wet season .
Fungal communities were more sensitive to short-term nitrogen and water treatments
Though statistically no significant effects of the sole N or water treatment were detected for the alpha and beta diversity of either fungal or bacterial communities (Table 1), we found that in comparison with the control, the N and PC treatments caused significantly lower alpha diversity in fungal but not bacterial communities in the dry season (Fig. 2); and fungal community compositions changed more than bacterial community compositions (Fig. 3 and Fig. S3). There were also more significant changes between different treatments in both the node-level and network-level topological features in the within-fungal networks than in the within-bacterial networks (Fig. 5 and Fig. S4). So it was rational to address that fungi were more sensitive than bacteria to the short-term N and water treatments. Similar results showing that fungal communities were more sensitive to the N or water treatment had been observed in forest ecosystems in other studies [13, 20, 18, 77]. There were also some studies conducted in grassland or desert ecosystems, in which bacteria rather than fungi were more sensitive to the N or water treatment [78–79]. This inconsistency might be associated with the difference in ecosystem types, while the underlying mechanisms are still to be inspected.
The PC treatment in the dry season (water reduction) caused a low soil water content (with a mean of 18.5%), corresponding to a value of less than − 0.4 Mpa of soil water potential and nearly 30% water holding capacity in the subtropical forest soil [80–81], which might represent a water condition causing mild drought stress for soil microbes . Fungi are more complex, larger organisms, which mainly live in large soil pores. The moderate drought stress (less water in large soil pore) might more readily affect the fungal community than bacteria which lives in a finer scale and often develop biofilms in soil . In the dry season, the N treatment caused lower pH, SWC and AvaiP compared with the control. The contents of soil pH, SWC, and AvaiP were positively correlated (marginally significantly, P < 0.1) with fungal Shannon diversity, while having no significant relationships with bacterial Shannon diversity (Figs. S5-6). That fungal communities were more sensitive than bacteria to nitrogen deposition had been indicated by a meta-analysis study, which was generally consistent across global terrestrial ecosystems . Our results also indicated that the interaction of N and water treatments in the dry season could cause significant effects on the OTU richness of the taxa Agaricomycetes, unclassified Ascomycota, Eurotiomycetes, and Sordariomycetes, while had no significant effects on bacterial main taxa (Table S2). Though both the N and PC treatments tended to decrease the OTU richness of these fungal taxa, their interaction (in the NPC treatment) act antagonistically to alleviate the decreasing effects (Fig. S2).
In addition to the alpha diversity and community compositions, the network topological features of fungi were also more sensitive to the N and water treatments than those of bacteria (Fig. 5, Fig. S4 and Table S4). In the dry season, the fungal members might have sparser relationships (lower closeness) and less interaction influence (lower betweenness) in the N treatment; while in the wet season, fungal members might develop sparser relationships and have higher interaction influence in the N treatment in comparisons with the control (Fig. 5) . N addition might down-regulate the potential cooperation between different fungal species in acquiring N, while in the wet season, the significantly higher N content might favor the growth efficiency and biomass of some fungal species , which might exert a higher influence capacity on other members in the community.
The contributions of different soil factors to the variations of enzyme activities
Soil functions, which are often represented by the enzyme activities in soils, are closely linked with microbial activities . In this study, four enzymes related to carbon, nitrogen, and phosphorous cycling were included to represent the basic yet sensitive soil functions to environmental change. Similar to soil physicochemical properties, the seasonal dynamics of soil enzyme activities were more apparent than the differences between different treatments (Fig. 1). In the wet season, all 4 enzyme activities were higher than those in the dry season. The 4 enzymes were highly correlated with each other in their activities and were all significantly correlated with soil water content (Fig. 6). Such positive relationships between soil moisture with the activities of BG and NAG were also observed in other studies [87–88]. For a certain gradient, higher water availability may result in more active microbial populations and higher enzymatic activities [89–90].
For the short-term simulated environmental changes, soil physicochemical properties explained a greater part of the variations of enzyme activities than the community traits (Table 2 and Fig. 6). Soil physicochemical properties, such as soil water content, pH, NH4+, and AvaiP were significantly correlated with enzyme activities (Fig. 6), and may readily affect enzyme activities through the regulation of reaction conditions or substrate concentrations. Soil physicochemical properties had been suggested as the primary regulators of soil enzyme activities which overwhelmed plant diversity or agronomic management [91–92]. Due to the widespread functional redundancy among different microbial taxa, the changes in microbial community traits (e.g., compositional change) may have less influential capacity on soil enzyme activities  (Table 2). What’s more, different community traits of bacteria and fungi may have differential influential effects on soil functions (Table 2 and Fig. 6). The network structure of the meta-community explained a higher proportion of variations of enzyme activities than the alpha or beta diversity of bacteria and fungal communities. This implicated the importance of microbial connections or interactions in affecting soil enzyme activities and ecosystem functions .
Our results also suggested that the inter-kingdom microbial associations possibly had great effects in affecting soil enzyme activities (even larger than the effects of within-kingdom associations) (Table 2). Bacteria and fungi may interact far more often than previously thought . They can establish close physical associations ranging from seemingly disordered polymicrobial communities to highly specific symbiotic relationships, such as fungal hyphae and bacterial cells; Their interactions were suggested to be important in gut health, rumen ecosystem functions, and also in biogeochemical processes . The cooperations between bacteria and fungi in degrading litters were also well known . Our results revealed that the links between Ascomycota with a variety of bacteria (such as those from Gammaproteobacteria, Alphaproteobacteria and Verrucomicrobiota) might be important in mediating the interactions between fungi and bacteria (Table S5). Ascomycota fungi could interact with bacteria through the hyphae or inter-kingdom gene transfer, which promoted nutrient transportation and enzyme activities [98–100]. Take the most important edge in the between bacteria and fungi network (Table S5) for example; The Archaeorhizomyces are global distributed fungi, which live in soil or around hardwoods roots. It may play great roles in nutrient turnover and can establish links with other fungi or bacteria [101–102]. The uncultured KF-JG30-C25 was also found to have many links with other fungi (such as the Ascomycota), and their potential interactions may contribute to the assimilation of acidobacterial extracellular polymeric substances . Individually, bacterial diversity (alpha and beta) and network features were more important than those of fungi in explaining the variations of enzyme activities. This may be due in part to the fact that bacteria may be more effective (higher biomass-specific activities) than fungi in regulating enzyme activities . For a specific season, the relative abundances of 4 bacterial taxa (Acidobacteriota, Gammaproteobacteria, Planctomycetota, Verrucomicrobiota), but only 1 fungal taxa (Eurotiomycetes) were significantly correlated with enzyme activities (Figs. S7-11). Besides, the 4 determined enzymes were mainly corresponding to the degradation of labile organics, which were preferentially linked with bacteria’s functions . It is possible that when including the enzymes specific for the recalcitrant carbon such as lignin, the importance of fungal community traits might arise in explaining the variation of enzyme activities.