Impact of Winter Cover Crops on Total and Microbial Carbon and Nitrogen in Black Soil

: Winter cover crops have been shown to promote the accumulation of microbial biomass carbon and nitrogen, enhance nutrient cycling, reduce erosion, improve ecosystem stability, etc. In the black soil area of Northeast China, Triticum aestivum L., Medicago sativa L., Vicia villosa Roth., Triticum aestivum L. and Medicago sativa L. mixed planting, Triticum aestivum L. and Vicia villosa Roth. mixed planting, and winter fallow ﬁ elds (CK) were selected to investigate the e ﬀ ects of winter cover crops on soil total carbon and nitrogen and microbial biomass carbon and nitrogen. The results showed that (1) after seasonal freeze–thaw, the rate of change in SOC ( − 2.49~6.50%), TN ( − 1.54~5.44%), and C/N ( − 1.18~1.16%) was less than that in SMBC ( − 80.91~ − 58.33%), SMBN ( − 65.03~332.22%), and SMBC/SMBN ( − 45.52~ − 90.03%); (2) winter cover crops not only alleviated the negative e ﬀ ects of seasonal freeze–thaw, which reduces SMBC and qMBC, but also increased SMBN and qMBN; (3) there was an extremely signi ﬁ cant ( p < 0.01) positive correlation between SOC and TN, a signi ﬁ cant ( p < 0.05) negative correlation between SMBC and SMBN, and there was no significant correlation between SOC and SMBC or between TN and SMBN; (4) alkali-hydrolysable nitrogen had the greatest impact on SOC and TN, while the soil’s saturation degree had the greatest impact on SMBC and SMBN; and (5) the Triticum aestivum L. monoculture was the most e ﬀ ective in conserving soil microbial carbon and nitrogen. In conclusion, winter cover crops can mitigate the reduction in soil microbial biomass carbon caused by seasonal freeze–thaw and also increase the soil microbial nitrogen content in the black soil region of Northeast China, of which Triticum aestivum L. monoculture showed the best performance.


Introduction
Black soils have high organic matter and fertility, which are very suitable for farming [1].With its high productivity, the black soil region in Northeast China accounts for about a quarter of China's grain production, so it is crucial for China's food security [2].However, these centuries-long large-scale agricultural reclamation activities, starting from the end of the Qing Dynasty, have resulted in the thinning of the soil layers of farmland soils in this region, a serious degradation of soil quality, and a massive loss of soil organic carbon and nitrogen [3][4][5][6].
Soil organic carbon and nitrogen cycling is the most basic ecological process in agriculture ecosystems and is closely related to soil quality and essential for soil productivity and even ecosystem stability [7,8].As a comparison with the turnover time of organic soil, which is 5 to 10 years, the turnover time of soil microbial biomass is 1 to 3 years or even less than 1 year.Thus, soil microbial biomass can respond rapidly to the minor environmental changes of soil, which is a current research hotspot of the soil research field [9][10][11].
Soil microbial biomass carbon and nitrogen are crucial components of soil microbial biomass and are not only commonly used as a measure of soil fertility but also serve as an inter-conversion link between inorganic and organic carbon or nitrogen [12,13].The soil quotient of microbial biomass carbon (or nitrogen) is the ratio of soil microbial biomass carbon (or soil microbial biomass nitrogen) to soil organic carbon (or soil total nitrogen), which reflects both their assimilation by microbes and the demands of soil microbial maintenance [14].Its value is directly proportional to the accumulation of soil nutrients affecting the input and loss of soil organic matter.It is the basis for characterizing the change rate of soil nutrients that are converted into microbial biomass [15][16][17], which is often used as a measure of soil nutrients and quality conditions.The carbon-to-nitrogen ratio is closely related to the carbon and nitrogen cycling in ecosystems, affecting the process of plants absorbing or releasing organic matter from the soil, and it is an important factor in measuring the efficiency of plant nutrient utilization.The microbial biomass's carbon-to-nitrogen ratio reflects the community structure characteristics of soil microorganisms [18,19].Soil carbon and nitrogen also interact with each other and with other environmental factors.In the context of global climate change, their relationship has become a current research hotspot.
Cover crops can effectively supplement the nutrient loss caused by exposed farmland, and the vegetation cover formed on the surface can effectively alleviate soil erosion and improve the physical and chemical quality of that soil [20,21].Cover crops can increase the relative abundance of the dominant bacterial phyla in soil, increase soil microbial biomass, moderate soil microbial characteristics, and increase soil microbial activity [22][23][24].Meanwhile, cover crops can change the rate of soil carbon input and the rate of organic matter loss, reducing nitrogen loss [25].A number of cover crops have been put into actual agricultural production practice, such as Triticum aestivum L., Medicago sativa L., and Vicia villosa Roth L. [26,27].Currently, there are relatively more studies in the southern region of China on the improvement of the soil physical and chemical environment, soil nutrient and biological properties, and the carbon and nitrogen cycling of cover crops, while there are relatively few studies on the effects of winter cover crops on the soil quality of black soil in the northeastern region of China.It is not clear to what extent cover crops affect soil carbon, nitrogen, and microbial biomass, or how the soil's physical and chemical factors affect soil carbon, nitrogen, and microbial biomass after seasonal freeze-thaw.
The objective of this study is to identify the changes in soil total carbon and nitrogen and soil microbial biomass carbon and nitrogen before and after seasonal freeze-thaw under different monocultures and mixed cover crops, and further explore their influence factors.The final goal is to provide a scientific reference for improving the soil functions of farmland ecosystems in black soil regions and achieving the sustainable use of black soil.

Location of the Field
The research site is located at Keshan Farm, Keshan County, Qiqihar City, Heilongjiang Province (125°07′40″~125°37′30″ E, 48°11′15″~48°24′07″ N).The climate is temperate continental steppic, supratemperate, and lower subhumid, with an average annual precipitation of 501.7 mm and an average temperature over the last 10 years of 2.1 °C [28].The soil types in this region are mainly black soil (Phaeozems in the WRB; Mollisols in the USST), mostly loamy clay or clay loam.A long history of reclamation has led to serious soil erosion in the area, with the soil erosion area of sloping farmland accounting for 67% of the total farmland area.
The basic physical and chemical properties of the sampling soil are given in Table 1.

Experimental Design and Sample Collection
The experiment was conducted from September 2022 to May 2023 on the cover crop plots of Keshan Farm.Triticum aestivum L. (Ta), Medicago sativa L. (Ms), and Vicia villosa Roth.(Vr), which are suitable for growing on black soil in Northeast China, were selected as cover crops to be planted in this study for monoculture and mixed sowing.Six treatments were set up in this experiment: Ta (Poaceae Barnhart), Ms (Fabaceae Lindl.),Vr (Fabaceae Lindl.),Ta and Ms (Poaceae Barnhart mixed with Fabaceae Lindl.),Ta and Vr (Poaceae Barnhart mixed with Fabaceae Lindl.), and naked land without a cover crop (CK).Cover crops were seeded uniformly on September 10, 2022.Each cover crops' plot was 5 m × 10 m in size.The sowing amounts were Ta 3000 g, Ms 200 g, Vr 400 g, Ta 1500 g + Ms 100 g, and Ta 1500 g + Vr 200 g.Each experimental plot was sampled before freezethaw (1 November 2022) and after freeze-thaw (8 May 2023).There was no duplication of the plots, but five points were selected for the sampling of surface soil (0-10 cm) using the S-shaped random sampling method within each plot.Both undisturbed (100 cm 3 ) and disturbed soils (about 1 kg) were collected.The undisturbed soils were utilized for the determination of mass water content and bulk density.The disturbed soil samples were brought back to the laboratory and the five sample points' soil was mixed well.Part of the soil was naturally air dried; impurities such as plant residues and gravel were removed.This was used to determine the total soil organic carbon and total nitrogen after grinding and passing through a 100-mesh sieve (0.15 mm opening size).Another portion of the airdried soil was ground and sieved through a 2 mm mesh (2 mm opening size) for measuring soil alkali-hydrolysable nitrogen and pH.The remaining fresh soil, sieved through a 2 mm mesh, was promptly stored at 4 °C in a refrigerator for the subsequent analysis of its soil microbial biomass carbon and nitrogen content.Each indicator was repeated 3 times.

Indicator Measurements and Data Processing
Soil total organic carbon (SOC) and total nitrogen (TN) were quantified using the elemental analyser method [29].Soil microbial biomass carbon (SMBC) and soil microbial biomass nitrogen (SMBN) were quantified using the chloroform fumigation extraction method [30].pH was measured using the potentiometric method with an aqueous solution.[31].Alkali-hydrolysable nitrogen was determined using the alkaline hydrolysis diffusion method [32].Mass water content was determined by the drying method [33].The soil bulk density was determined by the ring knife method [32].Soil capillary porosity was measured using the water immersion method [32].Soil total porosity, volume water content, soil saturation degree, quotient of microbial biomass carbon (qMBC), quotient of microbial biomass nitrogen (qMBN), carbon-to-nitrogen ratio (SOC/TN), and microbial biomass carbon-to-nitrogen ratio (SMNC/SMBN) were obtained by calculation methods.
Comparing the changes in various indicators of the cover crops' plots before and after freeze-thaw with the changes in fallowed land can be better reflect the sensitivity of the soil to the impact of seasonal freeze-thaw on various indicators with different winter cover crops than only focusing on the content of various indicators before or after seasonal freeze-thaw.Thus, we proposed the freeze-thaw change rate, which is calculated as follows: freeze-thaw change rate (△Ftcr) = (index contentafter freeze-thaw − index contentbefore freeze-thaw)/index contentbefore freeze-thaw × 100%".
A statistical analysis was performed with a t-test and one-way ANOVA using SPSS software (version 26.0,IBM SPSS, Chicago, IL, USA), for which the p values of <0.05 and <0.01 were considered to be significant (*) and extremely significant (**), respectively.Origin 2021 (OriginLab, Northampton, PA, USA) was used to plot all data.Redundancy analysis (RDA) was performed using Canoco 5 software (Cabit Information Technology Co., Ltd., Shanghai, China).

The Impact of Cover Crops on SOC and SMBC
As seen in Figure 1, before seasonal freeze-thaw, the SOC content of all plots was Ta + Ms (26.07 g/kg) > Ta (25.88 g/kg) > CK (25.79 g/kg) > Ms (24.88 g/kg) > Ta + Vr (24.66 g/kg) > Vr (19.88 g/kg), with Ta + Ms being significantly higher than CK, and Ms, Vr, and Ta + Vr being significantly lower than CK (p < 0.05).After seasonal freeze-thaw, the SOC content of all plots was Ta (27.57g/kg) > CK (25.63 g/kg) > Ms (25.46 g/kg) > Ta + Ms (25.42 g/kg) > Ta + Vr (24.75 g/kg) > Vr (20.35 g/kg).Compared with the CK, the SOC content of Ta significantly increased, and the SOC content of Vr and Ta + Vr significantly decreased (p < 0.05).Compared with before freeze-thaw, the SOC content of Ta, Ms, and Vr significantly increased after freeze-thaw (p < 0.05).Seasonal freeze-thaw caused a significant decrease in the SOC content of Ta + Ms (p < 0.05).The SOC content of CK decreased after the plot experienced freeze-thaw, but not significantly (p > 0.05).

The Impact of Cover Crops on TN and SMBN
As seen in Figure 3, before seasonal freeze-thaw, the TN content of all plots was CK (2.10 g/kg) > Ta + Ms (2.08 g/kg) > Ta (2.02 g/kg) > Ms (1.97 g/kg) > Ta + Vr (1.96 g/kg) > Vr (1.57g/kg).TN content of all cover crops plots lower than that of CK.All cover crops plots showed significant differences from CK (p < 0.05), except for Ta + Ms.After seasonal freeze-thaw, the TN content of all plots was Ta (2.13 g/kg) > CK (2.08 g/kg) > Ta + Ms (2.05 g/kg) > Ms (2.01 g/kg) > Ta + Vr (1.94 g/kg) > Vr (1.63 g/kg).Compared with CK, the TN content of Ms, Ta + Vr and Vr showed a significant decrease compared to CK (p < 0.05).Compared with before seasonal freeze-thaw, the TN content of Ta and Vr significantly increased after seasonal freeze-thaw (p < 0.05).Meanwhile, the TN content of CK decreased after experiencing freeze-thaw, but not significant (p > 0.05).
The freeze-thaw change rate of the qMBN in each plot was Ta (309.92%)> Ta + Ms (119.08%)> Ms (100.09%)> Ta + Vr (80.31%) > Vr (3.75%) > CK (−64.76%).The freeze-thaw change rate of the qMBN was consistent with that of the SMBN.The freeze-thaw change rate of the qMBN in Ta was significantly higher than that in other plots (p < 0.05).Other cover crops plots did not show significant differences in their qMBN before and after freeze-thaw (p > 0.05).In addition, they were all higher than the CK.

RDA between Carbon, Nitrogen, and Influence Factors
As seen in Figure 10, the RDA results of the SOC, TN, and C/N ratio to environmental factors indicated that axis 1 and 2 explained 56.64% and 9.72% of the variation seen, respectively.Alkali-hydrolysable nitrogen, the soil saturation degree, soil bulk density, volume water content, soil total porosity, mass water content, and pH explained 53.0%, 5.1%, 3.7%, 3.0%, 0.7%, 0.6%, and 0.2% of the soil organic carbon, total nitrogen, and C/N ratio, respectively.Alkali-hydrolysable nitrogen and the soil saturation degree were significant factors affecting the SOC, TN, and C/N ratio (p < 0.05).As seen in Figure 11, the RDA results of the SMBC, SMBN, and SMBC/SMBN ratio as environmental factors indicated that axis 1 and 2 explained 55.86% and 8.27% of the variation seen, respectively.The soil saturation degree, mass water content, volume water content, alkali-hydrolysable nitrogen, pH, soil total porosity, and soil bulk density explained 41.7%, 7.9%, 6.6%, 6.1%, 1.7%, 1.5%, and 0.3% of the soil microbial biomass carbon and nitrogen and SMBN/SMBC ratio, respectively.The soil saturation degree, volume water content, mass water content, and alkali-hydrolysable nitrogen were significant factors affecting the SMBC, SMBN, and SMBC/SMBN ratio (p < 0.05).

Responses of Soil Microbial Biomass C and N Content of Winter Cover Crops to Seasonal Freeze-Thaw Was Greater Than Total Soil C and N
The soil carbon and nitrogen contents were the key factors affecting the bacterial growth in soil, while soil microorganisms also affect soil carbon and nitrogen [34].A study by Fang et al. [35] found that the mineralization and decomposition of nitrifying bacteria resulted in a decrease in SOC content.Furthermore, the growth and multiplication of nitrifying bacteria affected the process of the conversion of ammonium nitrogen to nitrate nitrogen, thus altering the soil nitrogen fraction [36].In our study, the regulation of change in SOC, TN and SMBC, SMBN was not consistent.Overall, the SOC and TN content of the soil surface were slightly elevated after seasonal freeze-thaw due to winter cover crops, while their rate of change was smaller compared to the SMBC and SMBN.Novara, A. [37] et al.'s research findings indicated that the effect of cover cropping on total soil organic carbon is not pronounced, which is consistent with the conclusions drawn in our study.It is commonly believed that the SMBC and SMBN in soil only account for a minor portion of the SOC and TN, ranging from 1% to 5% and 2% to 6%, respectively [38,39].The results obtained in this study were close to this.In addition to microbial biomass carbon, soil organic carbon components also include dissolved organic carbon, easily oxidizable organic carbon, recalcitrant organic carbon, etc.A large percentage of the organic carbon fraction is recalcitrant organic carbon.The proportion of recalcitrant organic carbon in Chinese paddy soils reaches 65% to 68% [40].Xie et al. [41] demonstrated that recalcitrant organic carbon accounted for about 1/4 of the soil carbon in tropical forests.In addition to microbial nitrogen, soil nitrogen components also include dissolved organic nitrogen, nitrate nitrogen, ammonium nitrogen, etc.The total nitrogen content is a composite of the contents of each nitrogen component.Therefore, even if the rate of change of the SMBC and SMBN before and after freeze-thaw was large, they did not have a major impact on the SOC and TN due to them being a small percentage of the SOC and TN.The turnover time of soil organic carbon is 5 to 10 years [42].SOC and TN are closely related, and both are affected by soil type, erosion conditions, etc.In this study, the rate of change of SOC and TN before and after freeze-thaw were smaller than those of the SMBC and SMBN.The reason for this may be that, on the one hand, the SMBC and SMBN accounted for a small percentage of the SOC and TN; additionally, they had a weak effect on recalcitrant organic carbon.On the other hand, the turnover time of soil organic matter is generally greater than 5 years, so it did not respond substantively to the cover crops after so short a time as one winter.The turnover time of soil microbial biomass is 1 to 3 years, or even less than 1 year, which enables it to respond rapidly to environmental changes in the soil over a short time [9,10,43,44].This can be attributed to the fact that the activities of microorganisms are directly affected by environmental factors, such as temperature, humidity, and oxygen content; at the same time, microorganisms have a short life cycle and a rapid rate of reproduction, as well as a rapid response to environmental changes in short periods.
Before seasonal freeze-thaw, the SMBC and SMBN contents of each plot were all less than that in the CK.This may be due to the fact that the cover crops need to take up a lot of nutrients from the soil when growing, and as the soil's physicochemical properties will change with the planting of cover crops.The SMBC and SMBN are affected by a combination of environmental factors, and further research needs to be carried out.The SMBC and SMBN contents in the CK after freeze-thaw were smaller than those of the different cover crop planting plots.The reason for this is that the cover effect formed by the crops on the surface can effectively reduce water evaporation, and the root system of the cover crops can also improve the soil structure and enhance its water storage capacity [45,46].Root secretions and litter from cover crops provided fresh organic matter for microorganisms' growth [47].These factors provided a favourable condition for the growth and reproduction of microorganisms compared to bare plots.The rate of change of the SMBC in each cover crop plot before and after freeze-thaw was negative, while that of the SMBN was positive.The regulation of change in the SMBC and SMBN was inconsistent.The research findings of Manral, V. [48] et al. indicated that the trends in microbial biomass carbon and nitrogen are generally consistent, which is inconsistent with the results of our study.This may be due to the inconsistent carbon-to-nitrogen ratio released by different microbial communities after being killed by low temperatures.Generally, the carbon-to-nitrogen ratio of bacteria is around 5:1, that of fungi is around 10:1, and that of actinomycetes is around 6:1 [49].Further research needs to be performed to analyse the composition of soil microbial colonies.
The rate of change of the SOC and TN before and after freeze-thaw was not large, while the rate of change of the SMBC and SMBN was large, and the regulation of change in the qMBC and qMBN were basically the same as those of the SMBC and SMBN, respectively.Although the rate of change of the SMBC and qMBC before and after freezethaw was negative for each cover crops' plot, the degree of decrease was small relative to the CK, suggesting that the cover crops' planting had a protective effect on the SMBC in the soil surface layer.Covering crops can reduce the decrease in the SMBC content and qMBC.Interestingly, the change rates of the SMBN and qMBN before and after freezethaw were both positive, indicating that the planting of covering crops had a positive effect on the SMBC, SMBN, qMBC, and qMBN of the surface soil, which can help to maintain soil ecological balance, promote nutrient cycling, and organic matter decomposition.The change rate of the C/N ratio before and after freeze-thaw was small, and all cover crops' plots before and after freeze-thaw were slightly higher than the CK.This shows that winter cover crops have a certain effect that increases the soil C/N ratio.The increase in the C/N ratio was beneficial for increasing nitrogen fixation, reducing nitrogen loss, improving soil quality and fertility, and promoting plant growth [50].
Compared to the CK, cover crops reduced the SMBC/SMBN ratio of the soil surface, which may be a result of an increase in the bacterial communities in the soil microbial community [51].

The Primary Factors Driving Soil Total and Microbial C and N Change were Inconsistent
The RDA results indicated that alkali-hydrolysable nitrogen and soil saturation degree were significant factors affecting the SOC, TN, and C/N ratio (p < 0.05).The alkalihydrolysable nitrogen had the highest degree of explanation.The soil saturation degree, although at a significant level, was relatively poorly explained compared to the alkalihydrolysable nitrogen.Alkali-hydrolysable nitrogen, also known as quick-acting nitrogen, is the nitrogen that is quickly available for plant growth.It corresponds to the plants, ability to supply soil nitrogen.On the one hand, alkali-hydrolysable nitrogen plays an instrumental role in plant growth and enhances soil fertility [52].On the other hand, the promotion of plant root growth by alkaline nitrogen decomposition affects microorganisms and the decomposition of organic matter, in which microorganisms are involved [53,54].
The soil saturation degree, volume water content, mass water content, and alkalihydrolysable nitrogen were significant factors affecting the SMBC, SMBN, and SMBC/SMBN ratio (p < 0.05).The soil saturation degree had the highest degree of explanation.The soil saturation degree is the ratio of the volume of pore water in the soil to the total volume of pores.The soil saturation degree describes the degree to which pores in the soil are filled with water.A higher saturation degree can lead to insufficient oxygen supply in the soil, slow down the metabolic activity of microorganisms, and decrease the decomposition rate of organic matter.Previous results [55] have shown that an excessive soil saturation degree can limit the life activities of soil microorganisms, thereby affecting the soil content of microbial biomass carbon and nitrogen.Meanwhile, a higher soil saturation degree can limit the nitrification and reduction reactions of nitrogen, directly affecting the ability of soil microorganisms to utilize nitrogen and influencing the amount of microbial biomass nitrogen.Although the volume water content, mass water content, and alkali-hydrolysable nitrogen had reached a significant level, their explanatory power was relatively small compared with the soil saturation degree.Winter cover crops can reduce water evaporation.The electrostatic effect of the water film increases adhesion between soil particles, effectively minimizing the negative effects of wind and water erosion, reducing the loss of organic matter and preserving nutrients for soil microorganisms [56].This has a positive impact on microbial biomass.Meanwhile, the coordinated relationship between water content and voids is relaxed, facilitating the growth of microorganisms.
The results of the RDA showed that the total organic carbon, total nitrogen, microbial biomass carbon, and microbial biomass nitrogen had common influence factors (soil saturation degree, alkali-hydrolysable nitrogen).Alkali-hydrolysable nitrogen, which had a greater impact on soil organic carbon and total nitrogen, also had an impact on microbial biomass carbon and nitrogen, but this impact was relatively smaller.The soil saturation degree, which had a greater impact on microbial biomass carbon and nitrogen, also had an impact on total carbon and nitrogen, but this impact was relatively smaller.

Triticum aestivum L. Showed the Best Performance
In this study, the positive effect of the Triticum aestivum L. monoculture on the SOC, TN, and SMBC and SMBN content was the best, possibly because of its long growing period, which allowed its root system to remain in the soil for a longer period of time during winter freezing and thawing compared to other cover crops.This persistent root system helped to protect the soil structure, promoted the activity of soil microorganisms, and it was also conducive to the input of organic matter, providing carbon sources and nutrients for microorganisms.Studies by Abdalla, M et al. [57] and Meisinger JJ et al. [58] have concluded that, whether a monoculture of leguminous plants, non-leguminous plants, or both, cover crops can effectively inhibit the loss of soil nitrogen, which was consistent with the conclusion of our study.

Conclusions
In the black soil area of Northeastern China, winter cover crops are conducive to mitigating the adverse effects of drought and low temperatures.They also provide nutrients for soil microorganisms during seasonal freeze-thaw cycles.Winter cover crops can mitigate the reduction of soil microbial biomass carbon caused by freeze-thaw in winter and also increase the soil microbial nitrogen content.The freeze-thaw change rate of total soil organic carbon was smaller compared to that of microbial biomass carbon, as well as the freeze-thaw change rate of total nitrogen compared to that of microbial biomass nitrogen.Alkali-hydrolysable nitrogen had the greatest impact on the SOC, TN, and C/N ratio.The soil saturation degree had the greatest impact on soil microbial biomass carbon and nitrogen, as well as on the SMBC/SMBN ratio.The monoculture of Triticum aestivum L. showed the best improvement effect on the soil total organic carbon, total nitrogen, microbial biomass carbon, and nitrogen.This can serve as a reference when choosing winter cover crops for the black soil of Northeast China.

Figure 1 .
Figure 1.SOC content before and after seasonal freeze-thaw.Different capital letters indicate significant differences of SOC content between before and after seasonal freeze-thaw in the same plots (p < 0.05).Different lowercase letters indicate significant differences of SOC content before and after seasonal freeze-thaw in different plots (p < 0.05) and indicate significant differences of SOC content's

Figure 2 .
Figure 2. SMBC content before and after seasonal freeze-thaw.Different capital letters indicate significant differences of SMBC content between before and after seasonal freeze-thaw in the same plots (p < 0.05).Different lowercase letters indicate significant differences of SMBC content before and after seasonal freeze-thaw in different plots (p < 0.05) and indicate significant differences of SMBC content's freeze-thaw change rate in different plots (p < 0.05).Abbreviations: Triticum aestivum L. (Ta), Medicago sativa L. (Ms), Vicia villosa Roth.(Vr), Triticum aestivum L. and Medicago sativa L. mixed planting (Ta + Ms), Triticum aestivum L. and Vicia villosa Roth.mixed planting (Ta + Vr), winter fallow fields (CK).

Figure 3 .
Figure 3. TN content before and after seasonal freeze-thaw.Different capital letters indicate significant differences in TN content between before and after seasonal freeze-thaw in the same plots (p < 0.05).Different lowercase letters indicate significant differences in TN content before and after seasonal freeze-thaw in different plots (p < 0.05) and indicate significant differences in TN content's freeze-thaw change rate in different plots (p < 0.05).Abbreviations: Triticum aestivum L. (Ta), Medicago sativa L. (Ms), Vicia villosa Roth.(Vr), Triticum aestivum L. and Medicago sativa L. mixed planting (Ta + Ms), Triticum aestivum L. and Vicia villosa Roth.mixed planting (Ta + Vr), winter fallow fields (CK).

Figure 7 .
Figure 7. SOC/TN before and after seasonal Freeze-Thawfreeze-thaw.Different capital letters indicate significant differences in SOC/TN between before and after seasonal freeze-thaw in the same plots (p < 0.05).Different lowercase letters indicate significant differences in SOC/TN before and after seasonal freeze-thaw in different plots (p < 0.05) and indicate significant differences in SOC/TN's Freeze-Thawfreeze-thaw change rate in different plots (p < 0.05).Abbreviations: Triticum

Figure 10 .
Figure 10.RDA of SOC, TN, and C/N ratio as influence factors.

Table 1 .
Basic physicochemical properties of soil (average value of before and after freeze-thaw cycles).