Bacterial community structures significantly affected by tillage practices
In this study, irrespective of the tillage practice, soil bacterial communities were divided into two major groups according to the bulk and rhizosphere soil samples, suggesting that rhizosphere effort rather than tillage practice was the dominant factor in regulating the bacterial communities (Fig. 3a). However, the bacterial communities were also significantly affected by the tillage practices in the bulk and rhizosphere soils (Fig. 3b and c). This finding is corroborated by Tyler (2019) and Xia et al. (2019), who observed that underlying soil properties varied under different tillage practices and were the dominant factors in shifting microbial communities. Intriguingly, we observed that the variations in the bacterial community structures in the bulk soils were larger than those in the rhizosphere soils, which were likely because the stronger determinant of root exudates released from the roots onto the bacterial communities concealed the variation between tillage practices (Philippot et al., 2013).
Soil chemical properties had significant effects on bacterial community structure in the bulk and rhizosphere soils (Fig. S1). Soil NH4+-N played a dominant role in driving differentiation across all soil samples (Fig. 3d). Soil available nitrogen observably affects plant growth, and its uptake by plants is strongly dependent on rhizosphere microbial guilds (Moreau et al., 2019). In this study, the NH4+-N content ranged from an average of 1.75 mg kg− 1 in the bulk soils to 4.36 mg kg− 1 in the rhizosphere soils (Table S1); thus, it is reasonable that a large variation in soil NH4+-N contributed the most to shaping the bacterial community structures of all the samples across the bulk and rhizosphere soils. Additionally, soil pH had contributed the most to regulating the bacterial community structures in the bulk and rhizosphere soils (Fig. 3e and f), although soil pH varied by only 0.3 units among the three tillage practices. This finding suggested that even a small pH range change induced by tillage practices was still the most important factor determining soil bacterial community compositions (Degrune et al., 2015). This result occurred because of the narrow optimum soil pH range for bacteria, and when pH values exceed a certain range, they can directly cause a physiological constraint on soil bacteria that will change the competition outcomes or decrease the net growth of the individual groups that cannot survive (Lauber et al., 2009).
Variations in soil bacterial community compositions induced by different tillage practices
We discovered that the tillage practices displayed species-specific effects on the bacterial communities in both the bulk and rhizosphere soils. Although the degree of differentiation in the soil bacterial community structures among the tillage practices in the bulk soils was larger than that in the rhizosphere soils, more unique species induced by tillage practices occurred in the rhizosphere soils than in the bulk soils (Fig. 2a and b). This result is understandable because the rhizosphere has a more resource-enriched environment owing to the variety of carbon and nutrients provided by root exudates, which reduces microbial competition and thus allows more species to maintain their lives freely (Costello et al., 2012). However, fewer changes in OTU numbers were observed in the rhizosphere soils than in the bulk soils (Fig. 2c and d). The results are consistent with those of Thebault and Fontaine (2010), who showed that in comparison to bulk soils, rhizosphere soils have greater ecological stability. This may be explained by plant roots only selectively enriching microbes with specific functions (Berendsen et al., 2012; Yan et al., 2017), which leads to a strong carbon mineralization capacity and rapid nutrient cycling in rhizosphere soils (Fan et al., 2017; Turner et al., 2013).
The responses of the functional bacteria involved in carbon and nitrogen cycling to different soil disturbances were diverse (Tables S2 and S3). NT and RT in the bulk and rhizosphere soils significantly reduced the relative abundances of OTU1829 and OTU846, which were affiliated with Blastococcus that can degrade soil organic matter (Wang et al., 2020c) and Sphingomonas that can degrade polycyclic aromatic hydrocarbons (Macchi et al., 2018). OTU3343 (Streptomyces adustus) and OTU3407 (Streptomyces yanglinensis) can effectively degrade lignocellulose (Noda et al., 2012) and had lower abundances in the bulk soils with RT and in the rhizosphere soils with NT. These results indicate that intensive disturbance may accelerate the decomposition of carbon sources (Wang et al., 2020c) because the increased disturbance would increase the soil surface area by forming more microaggregates (Six et al., 2000).
In contrast, Nitrospirae, which contains many nitrifiers, was enriched in the bulk soils with NT and RT and likely enhanced the nitrogen turnover capacity of conservation tillage (Xue et al., 2020). In brief, the representative sequences of OTU3445 and OTU2114 had 97.12% and 99.53% similarity to Burkholderia singularis and Mesorhizobium gobiense, respectively. Burkholderia and Mesorhizobium have been widely reported to have nitrogen fixation capabilities (Fan et al., 2019; Soe et al., 2020), and their abundances were significantly higher in the rhizosphere soils with NT and RT than in those with MP. OTU2806 (Nitrosospira multiformis) and OTU3942 (Nitrosospira tenuis), which play a pivotal role in the nitrification process (Lin et al., 2018), were enriched in the bulk soils with RT and rhizosphere soils with NT. OTU4404 (Bradyrhizobium japonicum) and OTU2109 (Rhizobium etli) are widely documented to facilitate nodulation and biological nitrogen fixation of legumes (Kalita and Malek, 2020; Rivas et al., 2009; Roper et al., 2020), and they increased in the rhizosphere soils with NT and RT. Therefore, given the changes in the relative abundances of the bacterial taxa associated with carbon and nitrogen cycling in the soils with NT and RT compared with that with MP, we speculate that conservation tillage might enhance the soil nitrogen cycle and weaken soil carbon degradation. This concept was reconfirmed by the FAPROTAX annotation; NT and RT reduced the relative abundance of the functional groups associated with aromatic compound degradation while increasing nitrogen fixation in the rhizosphere soils (Fig. 4).
Tillage practices affect the stability of the soil bacterial network and the ecological function of keystone taxa and candidate biomarkers
Network analysis offers deep insight into the complex interactions of bacterial communities under soil perturbations (Barberan et al., 2012). Our study observed that tillage practices had impacts on bacterial network structures (Fig. 6, Table S6). The network of NT with the largest edge displayed more complex interactions among the OTUs in the bulk and rhizosphere soils, even though the NT network possessed relatively fewer nodes than the MP and RT network in the rhizosphere soils. This finding was in line with the results of Banerjee et al. (2019), who observed that the complexity of microbial networks is determined by the number of associations rather than the number of taxa. Additionally, the highest avgK of the NT network in the bulk and rhizosphere soils also demonstrated that in comparison to MP, NT created a higher density of interactions among OTUs (Ma et al., 2020). Bouizgarne et al. (2014) showed that closely coupled OTUs share the same habitat preferences, and their simultaneous occurrence markedly alleviates environmental stresses and stimulates crop growth. Additionally, a network with a greater average path distance (GD) can enable environmental fluctuations to propagate more slowly to the whole network, thus playing a better role in buffering environmental perturbations (Wang et al., 2014). Simultaneously, higher modularity (M) was conducive to enhancing the stability of network structures (Kitano, 2004; Krause et al., 2003). Therefore, the higher values of GD and M in the bulk and rhizosphere soils of the RT network indicated that in comparison to MP, RT was more robust to environmental disturbances.
As gatekeepers of ecosystem function, keystone species provide important contributions to biogeochemical cycling (Banerjee et al., 2018; Jiang et al., 2017). It has been widely reported that generalists with important topological roles and hub nodes with the highest degree exert a considerable influence on microbial network structure and function (Banerjee et al., 2019; Herren and McMahon, 2018; Shi et al., 2020). In this study, it was common for the Acidobacteria OTUs to be identified as keystone taxa because Acidobacteria can occupy a broad niche and have traits related to degrading soil organic carbon (Jiang et al., 2017; Mannisto et al., 2013). Additionally, half of the biomarkers were also tied to the soil carbon cycle, including Methylotenera, Gemmatimonas, Methylobacterium, Blastococcus and Mycobacterium (Guo et al., 2016; Kalyuzhnaya et al., 2012; Ren et al., 2017; Sanjenbam et al., 2020; Wang et al., 2020c). More notably, some keystone taxa were simultaneously identified by generalists and hub nodes, which might be explained by their particularly important ecological functions. For instance, the keystone taxa OTU2160 (Brevundimonas) and OTU3445 (Burkholderia) in rhizosphere soils with NT have been reported to be diazotrophic and can enhance crop growth and root colonization (Fan et al., 2019; Naqqash et al., 2020). The OTU2500 and OTU2736 in the rhizosphere soils with RT were classified as Nitrosospira and Rhizobacter, respectively, and these bacteria are well known to play a crucial role in nitrification and nitrogen fixation (Lin et al., 2018; Tong et al., 2020). We also found that the nitrogen-fixing bacterium Burkholderia was identified as a biomarker based on random forest analysis (Fig. 5). Overall, the soil bacteria closely involved in soil carbon and nitrogen cycling were integrated as keystone taxa and biomarkers, indicating that tillage practices changed microbial-mediated carbon and nitrogen cycling, thereby affecting ecological functions.
Assembly of soil bacterial communities influenced by tillage practices
In this study, we revealed that stochastic and deterministic processes played a role in the assembly of bacterial communities by null models (Ofiteru et al., 2010). Soil bacterial communities were more closely phylogenetically clustered in the bulk soils than in the rhizosphere soils (Fig. 7a). The weak phylogenetic clustering in the rhizosphere soils might be tied to the reduced relative contribution of deterministic processes, owing to less variable environmental filtering in the rhizosphere soils, which can cause phylogenetic overdispersion of bacterial communities (Fan et al., 2017; Goberna et al., 2014). Additionally, the increasing relative importance of stochastic processes in rhizosphere soils was well associated with higher soil fertility, which can weaken niche selection by promoting the growth of soil microorganisms and reducing competition for resources (Chen et al., 2017; Zhou et al., 2014). We found that stochastic processes, especially homogenizing dispersal, had a higher relative contribution to bacterial community assembly processes in the bulk and rhizosphere soils with MP than in those with NT and RT (Fig. 7c). This could be explained by the strong soil disturbance in the soils with MP, which could have led to high dispersal rates and thus tended to be more similar to the soil bacterial communities (Stegen et al., 2015; Zaneveld et al., 2017).
The results of the Mantel test also showed that soil pH was markedly correlated with bacterial community assembly processes (Fig. S1), which was consistent with previous reports (Tripathi et al., 2018). The closer to neutral pH of both the bulk and rhizosphere soils with MP may have reduced the selection pressure from environmental filters (Tripathi et al., 2018) since a neutral pH environment is more suitable for the growth of most bacteria (Madigan, 2012). Moreover, niche-based processes are influenced by not only abiotic factors (environmental filtering) but also biotic interactions (e.g., competition, predation and mutualism) (Zhou and Ning, 2017). Therefore, the soil nutrients and soil pH in NT were not significantly higher than those in the soils with RT, but NT increased the relative importance of stochastic processes because more microbial interactions in soils with NT could lead to alleviation of environmental selection pressure (Bouizgarne et al., 2014; Jiao et al., 2020).