The characteristics of ecoenzymatic stoichiometry and microbial nutrient limitation in proso millet/mung bean intercropping system
Soil extracellular activities focusing on C, N, and P metabolism played an important role in soil organic matter mineralization and nutrient cycling, and thus can be used to evaluate microbial metabolic activities in the terrestrial ecosystem (Xiao et al., 2020; Cui et al., 2023). In current study, intercropping significantly decreased the BG and AP activities, increased the NAG + LAP activities, regardless of proso millet or mung bean (Fig. S4), suggesting that soil microorganisms may enhance their investments in N acquisition enzymes in response to soil nutrient availability due to biological N2 fixation, such as TN and MBN contents that contributed to provide more substrates for microorganisms (Figs. S2 and S3). The addition of leguminous crops in the intercropping system increased the rhizosphere soil N level, and affected the C and P contents of soil and microorganisms to some extent based on the relationship of nutrient balance. However, the responses of proso millet and mung bean to intercropping were different. This phenomenon might be associated with the growth state of the crop in the intercropping system, that is, to positive or negative growth (Wang et al., 2017; Gong et al., 2021). Moreover, ecoenzymatic C:N:P stoichiometry can reflect a biogeochemical equilibrium between the environmental substrates availability and microbial resource allocation on account of soil microorganisms configure their biochemical characteristics to obtain relatively limited nutrient resources (Zhang et al., 2019). Intercropping significantly decreased MBC:MBN, MBC:MBP, BG:(NAG + LAP), BG:AP and increased (NAG + LAP):AP in rhizosphere soil of two crops (Table 1), which were mainly due to increased MBN and NAG + LAP. Cereal/legume intercropping may regulate the ratio of soil bacteria to fungi, because bacteria correspond to soil high N availability and fungi need more C than bacteria (Gong et al., 2019; Zhang et al., 2023). Another explanation is that changes in substrate concentration can upset the underlying equilibrium relationship due to the synergistic coupling effect between microbial nutrients (Peng et al., 2022; Ye et al., 2022). Consequently, proso millet/mung bean intercropping might indirectly stimulate microbial C and P metabolism because soil N improvement can support the synthesis of critical phosphate–containing compounds.
Microbial nutrient limitation has strong impact on microbial growth and metabolism, as decomposing cells must maintain a balanced composition of C, N, and P to ensure microbial biomass homeostasis (Shen et al., 2021). We found that microbial C limitation was significantly decreased by intercropping (Fig. 1), regardless of proso millet or mung bean. This result indicated that cereal/legume intercropping can provide more sufficient labile C than nutrients for microbes due to a higher diversity of rhizodeposits in the rhizosphere of intercropped species (Cong et al., 2014). Interestingly, compared to the sole cropping, intercropping significantly caused microbial N limitation (from 44.9° to 39.7° for proso millet and from 44.2° to 40.8° for mung bean) in rhizosphere soil of two crops (Fig. 1), indicating that C:N and N:P imbalances were responsible for the aggravated microbial N limitation. The prediction of microbial N limitation could be supported by the observed TERC:N (Fig. 2). Although mung bean rhizobium has the ability to fix nitrogen N2 and microbial metabolic activities can provide nutrients for plants, plants would compete with microbes for nutrients, especially in oligotrophic ecosystems (Inselsbacher et al., 2010). Intercropping with high cropping index enhanced competition between plants and microbes for nitrogen in DNA and protein synthesis. (Christian et al., 2009). Furthermore, in terms of nutrient requirements, microbial growth requires ten to twenty times more N than P, indirectly leading to this phenomenon (Li et al., 2022). Relevant studies reported that intercropping promoted plant root growth to produce a mass of died roots, root exudates and microbial necromass (Gong et al., 2020; Jiang et al., 2022). These increased C sources improved microbial N demand for stoichiometric balance. Variations in microbial nutrient limitation depend on complex taxonomic and functional microbial characterization strategies and stepwise responses to soil environments and plant physiological activities (Shi et al., 2022). However, soil microbial communities in proso millet/mung bean intercropping system had no strong homeostasis (Fig. 2). This phenomenon further emphasized that microbes are sensitive to the external environment, and their physiological metabolism would correspond with the changes of the environment to acquire low N resources. Consistent with other results (Cui et al., 2020; Li et al., 2022), microbial nutrient limitation was governed by multiple stoichiometric imbalances between microbial communities and available nutrient supplies.
Responses of soil microbial communities to nutrient limitation in proso millet/mung bean intercropping system
The composition of a microbial community determines its nutrient demands, which are influenced by the fundamental differences in element content and life strategy. For example, bacteria with a low C:N ratio (r–strategists) and fungi with a high C:N ratio (k–strategists) (Chen et al., 2023). In the present study, we found that intercropping rhizosphere had greater Shannon index (9.96 for IP and 9.37 for IM) than sole cropping (9.58 for SP and 9.04 for SM) (Fig. 3). This suggests that intercropping system had the more complex structure of the bacterial network, which provides vital information for soil sustainability and the ecological function of microorganisms (Zhang et al., 2023). The Venn diagram showed that IM (25) had higher bacterial specific OTUs than SM (21), whereas the response of the number of specific OTUs in the rhizosphere of proso millet and mung bean to intercropping was completely opposite (SP, 74; IP, 102; SM, 100; IM, 89). This response may be attributed to the physiological characteristics, biology and habitats of microorganisms (Jiao et al., 2019; Xu et al., 2020). Different ecological environment of two crops in intercropping system resulted in variations in ecological strategy and oxygen supply for aerobic microbes. Furthermore, the Shannon index of bacteria decreased with vector angle (Table S8), illustrating that the distinct microbial balance and community differentiation in intercropping system contribute to regulating soil N turnover and thereby weakening microbial N limitation (Bi et al., 2022). Moreover, analysis of the community structure response to intercropping indicated that soil microbial beta diversity (PCoA) changed significantly with the planting systems, as well as the species (Fig. 4), which was supported by the results that differences in microbial community characterizing can explain the soil micro–ecology characteristics to a certain extent.
Generally, crop roots recruit specific microbial communities by secreting various substances. As a result, there are significant differences in the rhizosphere microbial structure among different planting systems (Xie et al., 2022). However, we found that intercropping had relatively weak effects on soil bacterial community composition, especially in proso millet rhizosphere soil such as Proteobacteria, Actinobacteria, and Chloroflexi, even though these were the dominant phyla (Fig. 5 and Table S3). A possible explanation is that microorganisms of the positive growth crop with competitive advantage had relatively stronger soil heterogeneity and altered the variability among rhizosphere bacterial communities (Ablimit et al., 2022). Compared to SM, IM significantly altered the abundances of certain bacterial phyla, including increased Proteobacteria and Bacteroidetes and decreased Acidobacteria, Actinobacteria, and Gemmatimonadetes (p < 0.05, Fig. 5 and Table S3). Proteobacteria are eutrophic whose growth is promoted in N–rich environments (Kim et al., 2021) and was negatively associated with the vector angle (p < 0.001), illustrating the important role of microbial community composition in optimizing soil microbial N limitation. Acidobacteria thrive in barren soil (Ablimit et al., 2022) and the decreased abundance observed in the intercropping system suggests that it has an impact on the functional characteristics of bacterial communities, leading to an enrichment of species with distinct functional traits. At the taxonomic level, Betaproteobacteria and Deltaproteobacteria, branches of the Proteobacteria phyla; Subgroup_6 and Blastocatellia, branches of the Acidobacteria phyla, these compositions corresponded to the microbial N limitation in intercropping system. Furthermore, the abundance of the main fungal phyla was affected by intercropping to a certain extent (Fig. 5 and Table S3) because fungi are crucial in enhancing farmland productivity and soil nutrient storage in crop diversity systems (Pan et al., 2023). Compared with sole cropping, the relative abundances of Ascomycota and Mortierellomycota under intercropping system decreased and the relative abundance of Basidiomycota increased in proso millet rhizosphere soil (SP vs IP), while for mung bean, only the relative abundance of Basidiomycota was significantly affected and the trend was decreased (SM vs IM). Ascomycota and Mortierellomycota are known to be able to live in barren soil, which are often associated with soil nutrients (Zhao et al., 2023). It is likely that soil environments and biogeography are likely to play a crucial role in driving community nutrient metabolism. (Tian et al., 2023). Our findings indicate that specific biomarkers are associated with the variation of microbial community structures, potentially playing a crucial role in microbial N limitations.
Microbial co–occurrence network analyses provide a novel perspective for the potential microbial interactions, thereby determining microbial ecological functions of various soil microbial taxa (Barberan et al., 2012). As shown in Fig. 6, the soil bacterial community co–occurrence network was tighter than the fungal network, illustrating that bacteria play an important ecological role in the regulation of microecology on the Loess Plateau of China. Proteobacteria are essential and exist in all planting systems, regardless of proso millet or mung bean, because they provide energy and nutrients for other microorganisms by degrading cellulose and polysaccharides (Kim et al., 2021). We found that the number of bacterial nodes and edges, average path length, average degree, average weighted degree in IP treatment was lower than those in SP treatment (Table S5). Intercropping practice improved the microenvironmental homogeneity and increased the nutrient levels of the soil by mitigating the competition among soil bacterial communities (Xiao et al., 2023). In contrast, for fungi, intercropping had greater numbers of nodes and edges, average degree, and average weighted degree than the sole cropping system, indicating that intercropping exhibited greater ecological stability and might better adapt to environmental changes. Cooperation among the communities might contribute to the improving microbial resilience in a changing environment (Oliveira et al., 2014). These results demonstrated that intercropping has the potential to fundamentally alter the co–occurrence network’s properties and structure. Furthermore, the PLS–PM results suggested that the change in soil stoichiometry caused by intercropping was the most fundamental cause of microbial nutrient limitation (Fig. 7). Shifty soil nutrient ratios and microbial biomass ratios had a direct impact on the growth of the microbial community and microbial metabolism. This, in turn, drived microorganisms to degrade more soil organic matter in order to acquire the necessary nutrients to maintain stoichiometric homeostasis (Cui et al., 2018; Song et al., 2020). This conclusion was also supported by the relationship between microbial communities and soil properties, as shown that TC:TN and BG:(NAG + LAP) had strong influence on bacterial and fungal community structures, respectively (Tables S6 and S7). Taken together, the microbial metabolism in the intercropping ecosystems have potentially nutrient preferences, and thus adapt the resource availability and environmental stress.