Alfalfa (Medicago sativa L.) is a leguminous, perennial plant of great importance for livestock and agriculture and is therefore widely grown in many countries and regions (Han et al., 2005; Raiesi, 2007). The arid regions of northeast China are the main areas where alfalfa is grown. Due to the climatic specificity of the long winters in northeast China, livestock in the region rely heavily on summer pasture storage for forage (Chen et al., 2013). Alfalfa has a high yield and a comprehensive range of nutrients and can therefore reduce forage shortages for herbivores in winter (Su, 2007; Chen et al., 2013). As a result, perennial alfalfa is grown year after year in the region to meet the winter demand for fodder and to increase livestock productivity (Dong et al., 2003). However, this type of agricultural intensification has led to a loss of biodiversity (Sala et al., 2000; Romdhane et al., 2022). Moreover, the number of pathogenic microorganisms increases with the continuous planting of alfalfa, eventually leading to a decrease in yield, a phenomenon that is closely linked to soil microorganisms (Yan et al., 2012; Yao et al., 2019; Liu et al., 2020).

The number of years that alfalfa is grown is related to its productivity. Generally, alfalfa yields increase with the number of years planted; however, yields begin to decline when they reach a certain critical year, usually considered to be the 9^th year (Jiang et al., 2007; Li and Huang, 2008). In addition, continuous alfalfa cultivation significantly alters the physicochemical properties of the soil, which is significantly associated with yields (Ren et al., 2011). Previous studies have shown that planting alfalfa increases the organic matter and nitrogen content of the soil compared to virgin sandy soils. In addition, soil nutrients such as organic matter, nitrogen, and phosphorus increase with the number of continuous planting years. However, previous research has shown that soil nutrients tend to decrease after 10 continuous years of alfalfa cultivation (Jiang et al., 2007; Dong et al., 2016; Luo et al., 2018).

Soil microorganisms serve a crucial function in the maintenance of plant health by interacting with plants, participating in nutrient uptake and resisting stress, and responding rapidly to changes in the physical and chemical characteristics of the soil (Song et al., 2021). Differences in tillage systems, soil types, crop species, and cropping systems greatly influence the structure of the soil's microbial community (Zhou et al., 2018; Yao et al., 2019; Yuan et al., 2021). For example, one study observed that soil microbial biomass declined in the short term, but ultimately increased over the long term, in a context of continuous alfalfa cultivation (Jiang et al., 2007). Another study reported that continuous cultivation of alfalfa changed the microbial diversity by altering the physicochemical properties of the soil (Luo et al., 2018). Some studies have shown that continuous alfalfa cultivation can increase the relative abundance of Paecilomyces phaeomycocentrospora and Fusarium sp. and decrease the relative abundance of Penicillium sp. (Xu et al., 1995; Yao et al., 2019). However, other studies have found no effect of continuous alfalfa cultivation on soil microbiota structure (Hu and Wang, 1996). These different results might be attributed to heterogeneity among the soil types, sample collection times, and tillage systems used in these studies. Therefore, there is a need for more in-depth studies to investigate the barriers to continuous alfalfa cultivation under intensive tillage patterns.

Co-occurrence network analysis is a useful tool for exploring microbial associations and obtaining key information on microbial co-abundance communities associated with soil functions (Banerjee et al., 2018; Fan et al., 2021). One recent study used symbiotic networks to demonstrate that members of network modules were significantly associated with the genes involved in nutrient cycling after long-term fertilization, and that the number of members (operational taxonomic units, OTUs) in each module, rather than overall microbial diversity, influenced soil function (Fan et al., 2021). This raises the question of whether different land use practices alter the topological structures of networks, and what potential effects an altered network structure may have on soil function.

Here, we examined the influence of continuously cultivating grass, maize, and alfalfa (for 6, 10, 14, 20, and 30 years) on soil microbes and soil characteristics. Because grass grows without human intervention, we hypothesized that alfalfa and maize would have higher microbial diversity than grass, and that continuous planting of alfalfa would increase the complexity of the co-occurrence. The aims of this study were to explore the various changes in the structures of soil fungal communities across different cropping systems, and to assess the association between the physical characteristics of soil and the characteristics of its fungal community.

In the present study, fungal diversity was higher in the AC treatment than in the Me or Ma treatments, and the fungal diversity of alfalfa soils increased with subsequent years of planting. This result is in line with our first hypothesis, which suggests that alfalfa has more microbial species than grass and maize, and that continuous alfalfa planting is more beneficial to fungal diversity maintenance and soil sustainability, at least in terms of fungal diversity. Previous studies have found that continuously planted soybean has less soil microbial diversity than corn–soybean rotation systems (Liu et al., 2020). The number of years of continuous planting is also related to the microbial diversity of the soil (Liu et al., 2020). However, other studies have found no difference in microbial diversity between soils farmed continuously with soybean and soils farmed continuously with soybean–corn rotations (Li et al., 2010). These inconsistent results might be based on the soil types and the number of years of repeated harvests. Alterations in plant genotype may also be responsible for this result, given that microbial diversity has been shown to exhibit diverging trends in the context of successive cultivation of resistant and vulnerable cultivars (Yuan et al., 2021). Organic acids, phenols, and other compounds found in plant root exudates have an influence on microbial diversity in a range of agricultural situations (Tan et al., 2017; Lian et al., 2019; Liu et al., 2020; Shi et al., 2020). Furthermore, soil pH influences other soil characteristics that can directly or indirectly affect microbial diversity (Lian et al., 2019).

Regarding the beta diversity, the results from the principal coordinate analysis showed that crop types and continuous tillage time were the two most important factors affecting the structure of the soil fungal community (p < 0.05). Every species of plant releases a specific set of metabolites during growth, and this in turn allows its root system to provide a unique habitat for, and host different types of, soil fungal microorganisms. These microbes may also help plants absorb nutrients and resist stresses (Lian et al., 2019).

Moreover, our results were also consistent with previous studies that found that continuous crop planting also affects soil fungal community structure (Zhu et al., 2017; Yao et al., 2019; Yuan et al., 2021). This is mainly due to the effect of root exudates on soil microorganisms. For example, long-term continuous cultivation of soybean can lead to the accumulation of organic acids in the soil and ultimately cause soil acidification. This provides an ideal environment for pathogenic fungi to survive and alters the community structure of soil fungi, ultimately leading to reduced soil quality and crop yields (Zhu et al., 2017; Lian et al., 2019; Yuan et al., 2021).

Furthermore, according to the CCA results, the physicochemical characteristics of our soil samples, such as NO3-N, available phosphorus, and soil pH, were the main factors that altered the structure of the soil fungal community in the different treatments (Figure 6). This is similar to the findings of many previous studies, in which changes in tillage practices were shown to affect the microbial environment by altering the soil characteristics (Lian et al., 2019; Yao et al., 2019). Over all, our results also suggest that some important soil parameters changed significantly through continuous cropping over time, leading to changes in the fungal community. However, this change was not unidirectional, and longer periods of continuous cropping will perhaps lead to more positive developments for the soil microorganisms, such as increased diversity and significant enrichment of the beneficial fungi.

In AC soils, the relative abundance of Ascomycota was substantially higher compared to that of soils from the Ma and Mc systems (Figure 4). Many ascomycetes are plant-pathogenic, such as rice blast, black knot, the ergot fungi, and the powdery mildews, which suggests that continuous planting of alfalfa may have increased the abundance of potential pathogens, and influence the growth of alfalfa (Yuan et al., 2021). The relative abundance of Mortierella, Gibberella, Solicoccozyma, Metarhizium, and Phaeomycocentrospora was increased in AC fields compared to Me and Ma fields. It has been reported that Mortierella can survive under very unfavorable environmental conditions and make efficient use of carbon sources contained in polymers such as cellulose, hemicellulose, and chitin, and that it can synthesize phytohormones and 1-aminocyclopropane-1-carboxylic acid deaminase through improved access to bioavailable forms of phosphorus and iron in the soil, thereby protecting agricultural plants from pathogens (Ozimek and Hanaka, 2020). However, some pathogenic microbial species, such as Gaiellales, with high relative abundance in the AC treatment, can cause sear rot, suggesting that these fungi may suppress soil diseases, suggesting that these fungi may suppress soil diseases (Gómez Expósito et al., 2015). Therefore, it is likely that the changes in these fungi caused by the different treatments are related to the soil nutrient structure and the antagonistic activity of the plant pathogens.

In the present study, co-occurrence networks have helped us explore the complex relationships between fungi in different treatments in greater depth (Xue et al., 2018; Xiong et al., 2021). Our results show that both negative network correlations and modularity were significantly higher for AC than for Ma and Mc, which is consistent with our second hypothesis. This suggests that successive plantings of alfalfa promoted cooperation between fungi, which may be beneficial for alfalfa survival (Yao et al., 2019; Liu et al., 2020). Moreover, the difference in network topology of these treatmentsmay be due to the fact that certain microbial species are enriched to help the host increase nutrient uptake or resist stress, causing the structure of the microbial community to deviate from its original equilibrium (Lian et al., 2019). However, the results for the fungal networks alone are one-sided, as functional bacteria are also present in the soil. Therefore, in future studies, combined bacterial and fungal network analysis may yield more comprehensive results and a fuller assessment of the effects of different tillage practices on soil microbes.

In conclusion, alfalfa crop cultivation increased the alpha-diversity of soil fungi compared to grass and maize cultivation, and alpha-diversity increased further in continuous cropping systems, which is of great interest to maintain soil microbial diversity. The physicochemical properties of the soil, such as NO3--N, soil Ph, and available phosphorus, were the most important driving factors affecting the soil fungal community structure across the different cropping systems. Compared to the cultivation of grass and maize, the continuous cropping of alfalfa increased the abundance of several beneficial as well as pathogenic fungal species, such as Mortierella and Gaiellales. In addition, the networks differed among plant species and also among different lengths of time of continuous alfalfa cultivation. This suggests that the continuous cropping of alfalfa results in greater cooperation among fungi, which may be beneficial to the soil as well as to the development of the alfalfa.

The data presented in the study are deposited in the NCBI repository, accession number PRJNA890435.

YX and ZY conceived of the presented idea. YX wrote the manuscript. YX, XW, HC, YW, RW, and SL verified the analytical methods. ZY supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.

This work was supported by the Heilongjiang Provincial Scientific Research Institute Scientific Research Operating Expenses Project (CZKYF2021-2-B025), Outstanding Youth Fund of Heilongjiang Academy of Agricultural Sciences (2020JCQN003), Grass-field Rotation Scientist Studio of Heilongjiang Province (202004), Heilongjiang Province Modern Agricultural Industry Technology Collaborative Innovation Promotion System Construction Project (Heilongjiang Agricultural Department Letter (2021) No. 1492), and Natural Science Foundation of Heilongjiang (YQ2022C033).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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