The research site is selected in the Changwu Agricultural Ecology Experimental Station of the Chinese Academy of Sciences, Shaanxi Province, China. Geographic coordinates are 107°41′E, 35°14′N, with an altitude of 1,220 m. It belongs to a semihumid continental monsoon climate. The average annual precipitation is 580 mm, the average annual temperature is 9.1°C, and the frost-free period is 171 days. The loose and higher permeability soil is Heilu soil (Cumulic Haplustoll, USDA). The long-term field experiment in this research began in 1984. The initial soil nutrient status was as follows: organic matter = 10.50 g·kg⁻¹, total nitrogen = 0.80 g·kg⁻¹, total phosphorus = 1.26 g·kg⁻¹, and pH = 8.10.

This experimental design had 7 different fertilization and planting pattern treatments: 1) wheat continuous cropping without fertilization (W-CK); 2) wheat continuous cropping with NP fertilizer (W-NP); 3) wheat continuous cropping with NP and organic fertilizer (W-NPM); 4) wheat and corn rotation with NP fertilizer (WC-NP); 5) wheat and corn rotation with NP and organic fertilizer (WC-NPM); 6) wheat, millet and pea rotation with NP fertilizer (WMP-NP); and 7) wheat, millet and pea rotation with NP fertilizer and organic fertilizer (WMP-NPM). The fertilization amounts of the three fertilizers were as follows: N (120 kg·ha⁻¹·y⁻¹ CH₄N₂O), P (90 kg·ha⁻¹·y⁻¹ P₂O₅), and M (7.5 × 10⁴ kg·ha⁻¹·y⁻¹ manure with 10.6% organic matter content). Each treatment had 3 replicates with a 36 m² (4 m × 9 m) plot area. Field activities such as weeding and spraying were carried out according to local field management habits.

In June 2019, soil samples were collected after crop harvest. After clearing the dead branches and leaves on the soil surface, a soil sample of 0–20 cm was taken using the five point sampling method. The same treated soil samples in each plot were mixed and bagged, stored at 4°C and taken back to the laboratory. After removing the visible sand, gravel, animal and plant residues, the soil was passed through a 2 mm stainless sieve. The screened soil sample was divided into 3 parts. One part was placed into a 4°C refrigerator to determine the soil microbial biomass and soil respiration, one part was stored at −86°C to test PLFAs, and the third part was used to determine the soil physical and chemical properties after air drying with protection from sunlight.

Data Processing System (DPS) software was used for data analysis (Tang and Zhang, 2013). One-way ANOVA was performed with least significant difference (LSD) analysis. Structural equation modeling (SEM) was used to analyze the effect of different treatments on mineralized nitrogen and soil respiration by soil microbial community structure. The maximum likelihood estimation method was used with Amos V18.0 (IBM, Chicago, IL, United States). R software was used to draw figures. The data in this research are expressed as the average ± standard deviation.

Planting pattern and fertilization treatments had significant effects on soil physical and chemical properties (Table 1). Compared with the W-CK, SOC was significantly increased in the W-NPM and WC-NPM (p < 0.05), whereas TC was significantly decreased in the W-NP, WC-NP and WMP-NP (p < 0.05). All treatments significantly enhanced NO₃ ⁻-N, while only W-NPM, WC-NP and WMP-NP increased NH₄ ⁺-N and WC-NPM increased TN (p < 0.05). AP and TP were significantly higher than those in the W-CK in all other treatments, and OP was only significantly higher in the WMP-NP treatment (p < 0.05). The pH and SWC both significantly decreased in all the treatments, but the physical clay content only increased in the WC-NP treatment (p < 0.05).

In the wheat continuous treatment, SOC, TC, NH₄ ⁺-N, TN, AP and TP were significantly higher in the organic fertilizer used plot whereas SWC had a reverse trend (p < 0.05). The wheat and corn rotation plot with organic fertilizer had significantly higher SOC, TC, NO₃ ⁻-N, TN, AP and TP, and significantly lower NH₄ ⁺-N, pH and SWC than the chemical fertilizer plot (p < 0.05). The plot with wheat, millet and pea rotation and organic fertilizer obviously enhanced AP, TP and SWC but abated NH₄ ⁺-N, NO₃ ⁻-N and OP (p < 0.05).

In the chemical fertilizer treatment, AP and TP significantly increased with the number of rotations whereas SWC decreased (p < 0.05). NH₄ ⁺-N and NO₃ ⁻-N were significantly influenced by rotational management with a reverse trend (p < 0.05). In the organic fertilizer plot, NH₄ ⁺-N significantly decreased with the number of rotations, pH decreased first and then increased, and NO₃ ⁻-N, TN and AP had a reverse trend (p < 0.05).

Planting pattern and fertilization had a significant effect on the soil microbial community structure (Figure 1). Compared with W-CK, the total PLFA content was significantly decreased in W-NP and significantly increased in W-NPM, WC-NP and WC-NPM (p < 0.05). There was no obvious difference among the W-CK, WMP-NP and WMP-NPM (Figure 1A). The trend of bacteria, fungi, actinomycete, AMF, G⁺ and G⁻ PLFAs all showed a similar pattern with the total PLFAs. The Fungal/Bacteria ratio was not significantly different.

The plot with organic fertilizer had higher microbial PLFAs than the chemical fertilizer treatment, and this effect was weaked in the wheat, millet and pea rotation scenario. As the rotation number increased, all the microbial PLFAs first increased then decreased. Overall, the application of organic fertilizers has a greater impact on soil microbial PLFAs than rotation management.

A heatmap was used to show the correlation between soil physical and chemical properties and soil microbial communities (Figure 2). The PLFAs of bacteria, gram positive bacteria and fungi were significantly correlated with SOC, TC, TN, SWC and TP (p < 0.05). The PLFAs of actinomycete and AM fungi were significantly correlated with SOC, TC and TN (p < 0.05). Gram negative bacteria were only correlated with SOC and TN (p < 0.05).

Fertilization and planting pattern have significant effects on soil microbial biomass. Compared with W-CK, W-NP and WMP-NP significantly increased MBC, whereas WC-NPM and WMP-NPM decreased MBC (p < 0.05). The organic fertilizer had a negative effect on MBC and the effect obviously increased as the rotation number increased (Figure 3A). The MBN was significantly decreased in the W-NP and WMP-NP treatments but increased in WC-NPM (p < 0.05). Contrary to MBC, MBN was positively influenced by organic fertilizer and the effect was lower in the wheat and corn rotation treatment (Figure 3B).

Based on structural equation modeling, the driving mechanisms of different treatments on mineralized nitrogen and soil respiration rate were analyzed (Figure 7, χ² = 17.175, df = 10.000, p = 0.071, CFI = 0.916, GFI = 0.839, RMSEA = 0.189, AIC = 69.175). The results of the SEM showed that both TN and SOC directly positively affected total PLFAs and explaining 58% of the total variance with TC and SWC. The fungi/bacteria PLFAs were significantly negatively influenced by TN with −0.65 standardized path coefficients, and significantly positively affected by SOC with 0.63 standardized path coefficients. The combination of TC, TN, SOC and SWC explained 84% of its total variance. Mineralized nitrogen was significantly positively affected by TN, and 55% of the total variance was explained by soil environmental factors and soil microbial community structure. The soil respiration rate was significantly positively affected by TC and negatively affected by SWC and was explained 63% by soil physical, chemical and microbial properties.

Fertilization and planting pattern, as important field management practices, can affect soil nutrients and alter soil physicochemical characteristics (Wang W. et al., 2015; Cai et al., 2019). Soil nutrients are the main source for crop nutrition, and the level of soil nutrient content affects the survival and health of crops. Similar to other research, fertilization can significantly increase soil NH₄ ⁺-N, NO₃ ⁻-N, TN, AP, TP, and clay content, and reduce soil pH and SWC in the wheat continuous cropping treatment (Guo et al., 2010; Sun et al., 2014; Lusiba et al., 2017; Liu J. et al., 2020; Lin et al., 2022). The reason for this change in soil physicochemical properties may be that fertilizers contain a large amount of nitrogen and phosphorus. The application of fertilizers can quickly supplement the nutrient elements, thereby greatly increasing the available nitrogen and phosphorus. The increased available nutrients could enhance microbial activity and accelerate the transformation of nutrients (Zhang et al., 2013). The hydrolysis of urea causes a short-term and rapid decrease in soil pH, and lower pH and sufficient urea hydrolysates (NH₄ ⁺-N) significantly stimulate nitrification in soils (Zhao et al., 2014). This is consistent with the significant increase in nitrate nitrogen content and the opposite trend of nitrate and ammonia nitrogen changes after fertilization in this study. Soil pH is an important factor affecting soil quality. Nitrogen in fertilizer promotes the absorption of soil cations by crop roots, resulting in the production of more hydrogen ions and a decrease in the soil pH value with soil acidification (Cai et al., 2019; Dong et al., 2021b). In addition, the H⁺ produced by nitrification will also reduce the pH (Dong et al., 2021a).

Compared with the application of inorganic fertilizers, the application of chemical with organic fertilizers can increase SOC and reduce SWC and pH. After being applied to the soil, organic fertilizers increase the rate of soil nutrient accumulation, improve the soil physical structure, and create a rich microenvironment, which is conducive to microbial reproduction and ultimately drives the carbon nitrogen cycle and is more conducive to the accumulation of organic carbon components (Chen et al., 2019). The decrease in pH may be due to the combination of organic and inorganic fertilizers regulating the carbon nitrogen ratio of soil, promoting microbial growth, increasing soil microbial diversity, stimulating soil enzyme activity, promoting organic matter degradation, and generating more low molecular organic acids such as acetic acid (CH₃COOH) (Zhang et al., 2021). This research also found that under the same fertilizer conditions, the soil nutrient content was also affected by planting pattern. Under the same fertilization conditions, the SOC, TN, AP, and TP of the wheat corn rotation were higher than those of the wheat continuous cropping. This may be due to differences in nutrient absorption among different crops in rotation. Reasonable crop rotation can achieve balanced utilization of soil nutrients and achieve the effect of land nutrition. Meanwhile, compared to wheat continuous cropping, the SWC of wheat corn rotation decreases, possibly due to the more developed corn roots and high water demand, resulting in a decrease in SWC (Wu et al., 2021).

Soil microorganisms are an important component of soil ecosystems and play a notable role in maintaining soil ecosystem function and services. Fertilization significantly affects the abundance and diversity of soil microorganisms (Chinnadurai et al., 2014; Tamilselvi et al., 2015). In this research, compared with no fertilization, the application of chemical with organic fertilizer significantly increased the content of total PLFAs, bacteria, fungi, G⁺, G⁻, AMF and actinomycete PLFAs in the wheat continuous cropping treatment, whereas the NP fertilizer had the opposite effect. The application of organic fertilizer can provide a suitable survival environment for microorganisms (Wang et al., 2020). The application of organic fertilizers could increase the organic matter content of the soil, improve the soil physicochemical traits and the structure of the soil, improve the soil fertility, provide a suitable environment for microbial growth and promote microbial activity, and they also directly introduced microorganisms in organic fertilizers into the soil and played the roles of “inoculating” and “importing” (Zeng et al., 2007; Dong et al., 2014). Long-term application of inorganic fertilizers alters soil physicochemical traits and inhibits the activity of soil microorganisms; at the same time, exogenous organic carbon sources are insufficiently supplemented, and there is not enough energy to satisfy the growth and development of microorganisms, resulting in a decline in the number of microorganisms, while the input of organic fertilizers exactly complements this shortcoming, thereby increasing soil microbial PLFA contents (Li et al., 2020).

With the same fertilization, the PLFA content was different among the different rotation systems. In this research, the PLFA content in the wheat millet pea rotation treatment was lower than that in the wheat corn rotation treatment, indicating that rotation has a certain impact on the soil microbial community structure. Compared with the effect of fertilization, the planting pattern has a smaller impact on the soil microbial community structure, which may be because the application of organic fertilizer allows microorganisms to obtain a large amount of nutrients, or the presence of a large number of different microbial groups in the organic fertilizer. The differences in soil microbial community structure among different treatments are mainly related to whether organic fertilizers are applied, and although planting pattern have an impact on microbial community structure, the effect is relatively small.

Soil microbial biomass serves as both a reservoir of available nutrients and a driving force for soil nutrient cycling and organic matter transformation (Yang et al., 2017). MBN is a key interconversion of inorganic and organic N in soils and often serves as a sensitive indicator for the evolution of soil fertility status and soil quality. Previous studies have shown that the application of chemical fertilizers can enhance MBC and MBN (Daniel and Kate, 2014). The results of this study differed in that, compared with no fertilization, the application of NP and organic manure had no significant effect on MBC and MBN, but the application of NP significantly increased the MBC content and decreased the MBN content. This may be due to the difference in the amount of NP applied compared to other studies, or it may be related to soil cultivation methods or background physicochemical indicators, resulting in the application of NP affecting microorganisms and causing a decrease in MBN. In addition, research has found that compared to applying NP, applying NP and organic fertilizer significantly reduces MBC content and increases MBN content. This may be due to unreasonable chemical fertilizer application, resulting in insufficient carbon sources. The increase in MBN may be due to the long-term input of exogenous organic materials, which can regulate the soil nitrogen supply (Liu et al., 2012).

Soil respiration is often used to measure the total activity of soil microorganisms and to evaluate soil fertility and is affected by fertilization and planting pattern. Compared with no fertilization, fertilization can significantly improve soil respiration, except when chemical fertilizer is applied in wheat continuous and wheat and corn rotation systems. This may be because fertilizer improves the availability of nitrogen in the soil, promotes the growth of crop roots and their secretions, and increases microbial respiration (Ding et al., 2006; Li et al., 2010). Compared with applying NP, applying NP and organic fertilizer can significantly improve soil respiration in wheat continuous and wheat and corn rotation systems. In addition, by comparing W-NP, W-NPM, WC-NP and WC-NPM, although rotation had an impact on soil respiration, the impact was far less than that of the combined application of organic fertilizer. The main reason may be that the combination of organic and inorganic fertilizers significantly increases the soil organic matter content and thus increases the number of soil microorganisms, thereby increasing CO₂ emissions from microbial respiration (Han et al., 2008). Different soil microenvironments lead to different soil respiration (Raich and Tufekciogul, 2000), and changes in fertilization and planting pattern can change soil physical and chemical properties, which indirectly affect soil respiration by affecting the number and distribution of microorganisms (Zhou et al., 2011).

The nitrogen required by crops comes not only from the input of exogenous fertilizer but also from the inorganic nitrogen released by soil organic nitrogen mineralization. Therefore, studying the changes in soil nitrogen mineralization under different fertilization and planting pattern is crucial for the nitrogen supply to crops (Zhang et al., 2019). In this study, the trends of changes in soil mineralization nitrogen content, soil net nitrification rate, and soil net mineralization rate were the same among the different treatments. When the fertilization was the same, the wheat and corn rotation treatment had the highest soil mineralization nitrogen content, soil net mineralization rate, and soil net nitrification rate. The combination of organic fertilizer and wheat and corn rotation has a promoting effect on soil N transformation. According to the heatmap analysis in Figure 5, there is a highly significant positive correlation between soil mineralization nitrogen content and SOC, nitrate nitrogen, TN, and MBN, indicating that the richer the soil organic carbon, nitrate nitrogen, TN, and MBN content, the more substrates and more conducive to mineralization (He et al., 2005; Wang S. C. et al., 2015). Soil urease and protease are key enzymes for soil nitrogen conversion, and their activity is closely related to the intensity of soil nitrogen conversion and soil nitrogen supply capacity (Wang et al., 2010). It is speculated that the impact mechanism of fertilization and planting pattern on N mineralization is, on the one hand, by changing the mineralization substrate and the number of related microorganisms, and on the other hand, it may affect enzyme activity by changing soil physical and chemical properties, thereby affecting nitrogen mineralization.