4.1. Long-term crop rotation and nitrogen application-induced shifts in soil nitrogen forms
Within the context of soybean-corn rotation, corn often gains a competitive edge in soil nitrogen acquisition, as its nitrogen consumption exceeds that of soybeans. This, in turn, stimulates soybean's nitrogen fixation capacity. Wang et al. (2022), in their study concerning the impact of rotation on soil physicochemical properties in black calcareous soil regions, corroborated a notable decrease of 15.5% in soil total nitrogen content following crop rotation. In the current work, the rotational practice significantly lowered the soil's total nitrogen content during the maturation phase. This phenomenon primarily arose from the mutualistic interaction between corn and soybeans. During periods of low nitrogen availability, leguminous plants' nitrogen fixation capacity adapts to enhance fixation rather than diminish it. Consequently, soybeans transfer nitrogen to corn, strengthening the nutrient translocation that augments corn's nitrogen nourishment and stimulates its growth (Batista et al., 2020).
Within this study, the soil's post-rotation levels of AHN and NN were noticeably greater than those of AN. This divergence can be attributed to the transformation of AN nitrogen into AHN and NN during the nitrification process. A greater disparity between AHN and NN leads to higher availability of nitrate nitrogen for translocation (Ding et al., 2022). Hohman et al. (2020) reported enhanced NN content in soybean-corn rotation systems. Unlike continuous cultivation, the extended rotation of corn and soybean substantially elevates soil TON levels, with LON content exerting the greatest influence. As compared to the MS0 and SS0 treatments, long-term fertilization had a considerable influence on TON content. Within the components of TON, rotation, and fertilization primarily elevated soil LON, whose concentration increased with the duration of the rotation. The order of RON content across treatments was: rotation > continuous cultivation, fertilized > non-fertilized, signifying that the combination of fertilization and crop rotation can enhance RON. This outcome primarily stems from the differing cultivation practices. In conventionally tilled soils, nitrogen predominantly transforms into amide nitrogen, while amino sugar nitrogen and amino acid nitrogen prevail in rotated soils (Alison et al., 2020).
4.2. The prolonged practice of crop rotation and nitrogen applications alters the structure of soil microbial communities
The dynamics of soil nitrogen reflect shifts in the overall microbial population within the soil. In the context of long-term rotation between rice and soybean, the bacterial composition at the phylum and genus levels remains quite similar. The relative abundance of these has been consistently higher in rotational soil compared to continuous cropping, indicating that cultivation practices have a major influence on the distribution of bacterial phyla and genera. This could be attributed to the influence of alternating crops on bacterial composition (Fan et al., 2022). Microbial community analysis via PCA and functional prediction revealed that after rotation, Proteobacteria and Acidobacteriota significantly contributed to soil nitrogen content. This contribution was intertwined with soil urease and protease activities, consequently promoting nitrification and ammonification processes within the soil. However, there was a negative correlation with the key enzyme for denitrification, nitrate reductase, thereby reducing nitrogen loss. This could be explained by the aerobic nature of both Proteobacteria and Acidobacteriota.
Rotational cultivation of leguminous and graminaceous crops enhances soil aeration, and these two bacterial phyla can fix atmospheric nitrogen under low oxygen pressure (Liu, 2020; Zhang et al., 2019). Construction of a co-occurrence network model revealed that rotation increased the edges among soil bacteria, leading to a more complex interplay among phyla. As planting years grow, soil microbial communities display increased species richness and reduced inter-species competition. This shift is due to improved survival conditions for dominant bacterial phyla post-rotation, resulting in strengthened mutualistic relationships and reduced competitive interactions (Rong et al., 2019). Rotation effectively enhances the relative abundance of Ascomycota while decreasing that of Basidiomycota. Basidiomycota, being a large and complex fungal group, includes several plant pathogens like Chaetomium and Fusarium. These pathogens, which are major drivers of soybean root rot, often increase in abundance during continuous soybean cropping, potentially elevating the incidence of crop diseases (Zhou et al., 2018).
In this study, Ascomycota effectively increased soil nitrogen content while simultaneously boosting the activities of soil urease and protease enzymes. This effect can be attributed to yeast symbionts in the soil, which proliferate around plant roots. Their gelatinous secretions enhance soil structure by increasing looseness, aeration, water retention, and nutrient preservation. This, in turn, decomposes nitrogen, phosphorus, potassium, and other immobilized elements in the soil, transforming them into nutrients that plants can directly absorb and utilize. As a result, the utilization efficiency of fertilizers is enhanced (Naumova et al., 2017; Danka et al., 2022).
4.3. Impact of long-term crop rotation on nitrogen form transformation: Insights from microbial community dynamics and enzyme activities
In this study, the pivotal species and enzymatic activities associated with variations in nitrogen forms were identified using PCA analysis and a random forest predictive model. Within the TN group, the proportions of RON and AAN components were notably predominant in TON and TIN, respectively. This phenomenon is most likely due to the modulation of key microbial communities and enzymatic activities regulating the formation of RON and AAN. AAN contains elements like AN and NN that are plausible nitrogen sources for plant uptake and constitute one of the most dynamic nitrogen reservoirs for crop growth (Brown et al., 2022). The fluctuation of AAN in the soil could be linked to variations in AHN, given its significant presence within AAN and its role as a swiftly releasable fraction. Proteobacteria and Acidobacteriota as well as Ascomycota have previously been identified as members of the Dissimilatory Nitrate Reduction to Ammonium (DNRA) community (Sakuntala et al., 2016).
Crop rotation and nitrogen fertilization might induce ammonium reduction, curbing nitrogen loss via ammonia volatilization and thereby promoting AN and NN accumulation. The production of chitinase by Ascomycota has been reported (Hu et al., 2022) and thus, chitinase decomposition of organic nitrogen products could add to the pool of AN and NN. Furthermore, recent reports indicate Ascomycota's participation in crop pathogen suppression, assistance in crop growth, and the accumulation of AN and NN components through enhanced root exudation (Challacombe et al., 2019). The significant influence of SU, SP, and SNR on the changes in AN components was documented in the present work. SP is engaged in the breakdown of chitin and lignin, key constituents of bacterial and fungal cell walls (Zhang et al., 2020). Following microbial cell wall shedding, decomposition generates low-molecular-weight organic compounds like free amino acids and amino sugars, contributing to the TIN pool. On the other hand, UR's end product is NH4+, a precursor of AN. However, when nitrogen saturation occurs, SNR may lead to a reduction in AN and NN content (Alizadeh et al., 2017).
In the SM1 treatment, AN and NN content significantly surpassed those of MS0 and SS0 treatments (Fig. 1), suggesting that Proteobacteria, Acidobacteriota, and Ascomycota excel in modulating AAN content compared to SNR. Certain fungal species have been reported to possess genes associated with amino acids (Frey et al., 2000), participating in nitrogen cycling. Their ability to produce an amino acid oxidase has also been observed (Isobe et al., 2014). The lower ammonia-producing capability following crop rotation is explained by decreased fungal richness relative to continuous cropping. Notably, in this study, Fungi demonstrate greater resilience to lower pH levels than bacteria in microbial communities (Zhang et al., 2020). Thus, the fungal-to-bacterial richness ratio rose with nitrogen fertilization due to the anticipated pH decrease caused by nitrogen input. This possibly elucidates the critical relevance of fungi in AAN morphological changes, as AAN is intricately tied to microbial metabolism (Durani et al., 2016).