According to the plant biomass (Fig. 1) in this study, 180 kg N ha− 1 was confirmed as the reasonable N fertilization for maize grown in the test soil [31], and excessive N fertilizer application (300 kg ha− 1) resulted in a significant decrease in plant biomass and in the diazotroph diversity (Fig. 3). This result was consistent with previous studies that plant biomass was not directly proportional to N fertilizer application rates [32, 33]. However, the decreased biomass of maize under the excessive N fertilizer application is an interesting phenomenon, which was also reported in other plants [34, 35]. According to the Leibig Law of minimum, the plant growth (biomass accumulation) is determined by the nutrient with the lowest concentration, so the addition of excessive N in the present study could not help the plant growth. The negative effects of excessive N on maize growth may be due to that the excessive N fertilizer is toxic for maize that fit the Shelford Law of tolerance. The toxic effect of excessive N fertilizer application might be from the modification of plant metabolism, or from modification of soil environments, such as the degradation of soil properties [34, 35] and the alternation of soil microbial community that in turn could decrease the growth of maize. The present study offered evidence of alternation in soil properties and soil microbial communities caused by excessive N application, as well as the correlation among them.
Firstly, the results in the present study clearly evidenced the alternation in soil properties under the excessive N fertilization, which is similar to those in some other studies. Previous studies have shown that excessive N fertilizer input can lead to soil acidification and increase of soluble salt concentration [20]. Soil pH has been considered to be the main factor affecting microbial abundance and diversity [13, 36–39], and high EC values may increase the incidence of some crop diseases (such as maize root rot) [40–42]. Similar to the previous studies, excessive N fertilizer input led to a decrease in soil pH and an increase in EC values in our present study. No doubt that the low pH (4.60 in rhizosphere and 4.46 in bulk soil) and high salinity (EC 19.37 mS m− 1 in rhizosphere and 22.77 mS m− 1 in bulk soil) (Table 1) could affect the metabolism and nutrient absorption of maize, which presented optimal growth at pH 5.5-7.0 or pH 4.9–7.3 [43], and then decrease the growth of plant [34, 35]. In addition, the acidic condition could enhance the solubility of Fe, Mn, Bo, Cu and Zn and decreased the solubility of Mo, Mg, Ca, K and S, which may cause the toxic effects of the heavy metal and reduce the availability of some mineral nutrients [44].
Secondly, the application of excessive N fertilizer decreased the α-diversity (Fig. 2) and changed the community composition (Figs. 4 and S2b) of diazotrophs in both rhizosphere and bulk soils. These results were similar to those obtained in several previous studies. A long-term addition of NPK fertilizer in a Calcic Kastanozems soil with wheat-soybean rotation lead to decreased abundance and diversity of diazotrophs [8]; while N fertilizer application at dose of 300 kg ha− 1 in Red Soil of wheat-maize rotation also lead to decreased abundance and diversity of diazotrophic [10]. And our study was performed with different levels of N fertilizer (N0, N60, N180, N300) in black soil with mono-cropping maize. Therefore, the decrease of abundance and alternation of the structure composition of diazotrophs in response to the long-term excessive N fertilization is common in different soil types (including rhizosphere soil) and in different cropping systems. The shifting of diazotrophs community composition might be explained by their great genomic and biochemical diversity, which is determinant for their adaptation to the environmental factors.
The application of different levels of N fertilizer in the present study made it possible to horizontally compare the influence of different N fertilizer application rates on the diazotrophs, which was not reported up to date. Interestingly, no significant difference was found in diversity of diazotrophs among the low N (N0, N60) and normal fertilization (N180), and only the long-term excessive N fertilization significantly decreased the abundance and diversity of diazotrophs (Figs. 2 and 3). These phenomena were consistent to the biomass increase and decrease, implying the possible relationships between the diazotrophs and the plant growth.
The different compositions of diazotrophic community were clearly demonstrated by the clustering analysis, in which the diazotrophic communities were separated according to the N fertilizer application rates; moreover, the communities from excessive N fertilization formed a cluster far distant with those from the other treatments (Figs. 4 and 5). So, the structure of diazotrophic community would be seriously changed by the N fertilizer application dose that exceeded a certain threshold. Among the ten most abundant genera (Fig. 5, Table S7), the stability of relative abundance in both rhizosphere and bulk soils against the N levels for Azospirillum, Herbaspirillum, Klebsiella, Methylobacterium, Paraburkholderia and Pseudomonas demonstrated that they might live as saprophytes by using the combined N as their N source. This estimation is consistent to the TN contents in the tested soils, in which the background N content is very high (1.38–1.68 g kg− 1, Table 1) [45] and is sufficient to inhibit the biological N fixation, e.g. the diazotrophs can not fix N2 in these soils.
Different from the six genera mentioned above, the relative abundances of Bradyrhizobium, Burkholderia, Rhodobacter and Sphingobium were significantly changed sequentially (Fig. 5, Table S7): addition of N fertilizer decreased the relative abundance of Burkholderia, so it was most abundant in N0 treatment; while the abundances of Rhodobacter, Bradyrhizobium and Sphingobium were significantly enhanced in the treatments of N60, N180 and N300, respectively (Fig. 5, Table S7). These changes demonstrated that different levels of N fertilizer application enriched distinct diazotrophic bacteria, which might be related to their adaption to the decreased pH values and increased EC values in soils caused by the fertilization (Table 1).
It is well known that the roots of plant can enrich the some microbes via secretion of secondary metabolites, casts of roots and modification the rhizosphere microenvironments [46–48]. Previous studies had found that the diazotrophs in the rhizosphere soil of sorghum, soybean, maize was more abundant than that in the bulk soil [10, 27, 49, 50]. Our study also showed that maize rhizosphere had a significant effect on the copy numbers of nifH gene, and the diazotrophs in rhizosphere soil were also more abundant than that that in bulk soil (Fig. 2, Table S2). In addition, NO3−-N in rhizosphere soil was lower than that in bulk soil (Table 1), which was due to the selective absorption of soil nutrients by plants [51]. The selectivity may be one of the reasons for the enrichment of diazotrophic in rhizosphere. The proliferation of more diazotrophic in the rhizosphere should help provide more nitrogen nutrients or other beneficial effects to the plant. However, there was no significant difference in the compositions of diazotrophic communities in rhizosphere and bulk soil, indicating that rhizosphere effect had little influence on the diazotrophic community in the tested soils, which was consistent with previous studies [10, 17].
Based on our research, through the construction of structural equation model (SEM), it seems that the N fertilizer application rates and rhizosphere effect could directly lead to the variation of soil characteristics and diazotrophic abundance. Then, the variation of α-diversity of diazotrophic mediated by soil characteristics. Finally, α-diversity and N fertilizer application rates mediated the separation of β-diversity of diazotrophs in the tested soils (Fig. 6). The results revealed the influencing factors of diazotrophic community structure variation and the causal relationship among each of the variables, which provided a good idea for the study of subsequent variation mechanisms of microbial community structure.
In our study, excessive N fertilizer input led to a decrease in soil pH and an increase in EC values, which were significantly related to the abundance and diversity of diazotrophs (Tables 2 and S5). Among them, pH was negatively correlated with Sphingobium, while positively correlated with Burkholderia (Fig. S5), indicating that the low soil pH under excessive N application led to the accumulation of Sphingobium, while reducing the relative abundance of Burkholderia. Sphingobium were reported can degrade many kinds of polycyclic aromatic hydrocarbons (PAHs) and their derives [52], and Burkholderia was famous for its bioremediation and antifungal properties [53], suggested that excess N fertilizer despite increased the degradation ability of soil pollutants, but its resistance to pathogen was reduced, thus increasing the probability of occurrence of soil spread diseases.
Long-term excessive N fertilizer input also had a profound impact on soil nutrient content (Table 1). The variation of soil properties also had a significant impact on the compositions of diazotrophic community (Figs. S3 and S4, Table 3). In particular, the NO3−-N and EC values in soil were positively correlated with Sphingobium and negatively correlated with Burkholderia, which were agreed with the relationship between soil pH and these two diazotrophic bacteria, indicating the synergistic effect on the structure of diazotrophic community of high nitrate content, EC values and low pH caused by excessive N fertilizer input. On the contrary, the soil AP and AK negative correlation with Sphingobium and positive correlation with Burkholderia imply that the proportion of nitrogen, phosphorus and potassium in soil was also crucial for maintaining a normal diazotrophic community, which proved the importance of balanced application of NPK from the perspective of soil microbes [54].
The variation of soil microbial community structure has a feedback regulation effect on plant [1]. However, little is known about the feedback of plant from the change of diazotrophic community. Based on our study, it was found that plant biomass was positively correlated with the relative abundances of Bradyrhizobium and Methylobacterium (P < 0.05, Fig. S5). Bradyrhizobium, which is widely distributed in soil, has important biological functions, including photosynthesis, symbiotic and free-living nitrogen fixation, denitrification and aromatic compound degradation, and plays an important role in the global nitrogen cycle. The multiple functions make it important for agricultural production [55–57]. In addition, Methylobacterium is also widely existed in the environment. It is a kind of diazotrophic bacteria that can produce plant growth hormone and can be classified as plant growth-promoting bacteria [59, 60]. This indicated that the increase of biomass under normal N fertilization (N180) was more likely related to the enrichment of these two diazotroph. Based on the above analysis, Bradyrhizobium and Methylobacterium may be utilized to develop efficient microbial fertilizers.