The differentially abundant fungi and bacteria were evenly distributed between the two management systems. For fungi, 11 ASVs were more abundant in the rhizosphere of conventional plants and 13 were more abundant in organic. The Mortierellales were the most-represented order with four ASVs, but these were not disproportionately found in CR or OR .We asked how agricultural management and plant roots act individually and in combination to shape microbial community composition, co-occurrence patterns, and N-cycling functions, and whether this interaction leads to system-specific adaptation. In accordance with known management and rhizosphere effects on microbial community structure and N dynamics in agroecosystems, we observed conventional/organic and bulk/rhizosphere differences in many of the parameters measured. Furthermore, many of our analyses supported the hypothesis that plant selective influence varies with management to shape plantassociated microbial community composition and structure . Management, rhizosphere, and M × R effects on microbial communities are likely mediated in large part by soil physicochemical properties, which differed between management systems and soil compartments . Strong effects of management on soil physicochemical properties were visible in the higher NO3-N, P, K, Ca, Na, and SOM levels in the organic system and higher Mg and pH in the conventional system. Rhizosphere soil was depleted in NO3-N, P, and K in both management systems. M, R, and M × R effects on soil properties such as nutrient availability, pH, and organic matter likely contribute greatly to microbial community assembly in these treatments. Significant differences in the direction or magnitude of the rhizosphere effect were observed for bacterial diversity,plastic plant pot community composition, and indicator species .
Plant roots consistently imposed a strong selective filter, and similarity between rhizosphere communities was greater than similarity between bulk soil communities . Nevertheless, rhizosphere communities still reflected the impacts of management on the contributing microbial pool, and rhizosphere communities were more similar to their corresponding bulk soil communities than to one another . The direction of the rhizosphere effect varied with management for bacterial diversity, indicator species, and community structure. This M × R interaction resulted in rhizosphere bacterial communities that were more similar in diversity, composition, and structure than bulk soil bacterial communities. Rhizosphere bacterial/archaeal diversity was lower in the organic rhizosphere but higher in the conventional rhizosphere compared to bulk soil . Although roots are often thought to impose a selective filter that decreases diversity, higher species richness in the rhizosphere as observed here in the conventional system has been reported elsewhere when plants select for enrichment of certain processes . Here, however, whether functional enrichment is related to selection for increased diversity is unclear. Environmental filtering may account for the fact that bacterial rhizosphere networks were more similar than bulk soil networks. Although it has been hypothesized that niche sharing should lead to greater co-occurrence and thus more densely connected networks in the rhizosphere, this effect was seen only in the bacterial organic networks . Viewed in combinationwith previous work showing smaller, less densely connected networks in rhizosphere soil, our results suggest that rhizosphere effects on co-occurrence networks, like other metrics of microbial community structure, may well be context- and system-dependent. The magnitude of plant effects on rhizosphere communities also differed between management systems.
We generally found greater differences between bulk and rhizosphere community composition in conventional soils compared to organic . Hartman et al. attribute a similar M × R interaction observed in their study of wheat agroecosystems to the application of management practices immediately before root establishment. This explanation may apply here as well, specifically with regard to the spatial scale of cover crop and fertilizer inputs. Inorganic fertilizer and composted poultry manure were trenched in seed beds and therefore near crop roots, likely favoring divergence of bulk soil and rhizosphere microbial communities. Since cover crops were sown throughout the organic plots, covercropping-induced changes in microbial community composition were likely similar in the bulk soil and early root zone, whereas emerging roots in the conventional plots would likely have encountered a fertilizer-enriched zone already distinct from most of the bulk soil.We further hypothesized that rhizosphere communities would be enriched in system-specific beneficial taxa and functions of importance for plant adaptation to system-specific soil conditions. Although indicator species analysis revealed system-specific taxa, we cannot definitively conclude whether these taxa are beneficial based on amplicon sequencing data. Three members of the order Myxococcales and two members of the order Burkholderiales were indicators of organic environments, in line with previous studies showing these orders to be organic-system-specific. Two strains of the Anaerolineales, an order that displaces other fermenters under high-nitrate conditions, were indicators of the conventional system. Broad ecological information about soil fungi is limited in comparison to bacteria and archaea, despite extensive specialized literature on pathogens of humans and plants or AMF and other endophytes. Many fungal indicators identified here belong to genera known to be pathogenic on other host species, and these were relatively evenly distributed among environments. The significance of pathogens as indicator species in these systems is unclear, especially for pathogens such as Boeremia exigua, which causes leaf spot on diverse host crops including tomato, the other crop in this rotation, but is not known to cause disease in maize. Fewer details of metabolism and ecology are available for non-pathogenic fungal indicators. Mortierella, the most common genus among fungal indicators in this study, are known to be a large genus of saprotrophs.
Exophiala equina and Didymella sp. have been reported elsewhere to be associated with plant roots. Fungi are critical drivers of C/N cycling and carbon sequestration in agricultural systems, and linking specific taxa to roles beyond pathogenic interactions will be a valuable expansion of the existing literature. With regard to N-cycling functions, we quantified six genes involved in different steps of the nitrogen cycle, all of which were affected by plant selection and only two of which were differentially selected between systems . The relative abundance of genes relative to one another was similar across treatments, suggesting that no system-specific bottlenecks in the N cycle were observed . The abundances of the nifH, amoA , nirK, nirS, and nosZ genes were higher in the bulk soil,plastic planter pot in contrast to previous studies that found the maize rhizosphere was enriched in functional genes related to nitrogen fixation , nitrification , and denitrification.That effect was also observed with the addition of artificial maize root exudates, suggesting that exudates are the main mechanisms influencing microbial N cycling independently of other physicochemical characteristics of the rhizosphere. However, mechanisms other than exudates may be responsible for the discrepancy in the direction of the rhizosphere effect between the present study and the literature: while certain root exudates inhibit nitrification in wheat, sorghum, and rice, this effect has not been shown in maize. Sampling in the present study occurred during the silking period of maize, when crop N uptake reaches a maximum. The rhizosphere may be N-depleted in comparison to bulk soil, and microbial N limitation may account for the decreased abundance of these N-cycling genes. Differences in soil organic matter or shifts in root exudation during development leading to altered rhizosphere carbon availability may also account for the change in direction of the rhizosphere effect in the present study as compared to the literature. Increased sampling frequency over the course of the growing season paired with metabolomic analysis of root exudates would provide insight into the mechanisms linking root C release and N uptake dynamics to microbial N-cycling gene abundances. We hypothesized that differences in N-cycling gene abundance between conventional and organic systems would reflect adaptive shifts, increasing the abundance of gene pathways linking system-specific N inputs to plant-available species, but this hypothesis was not supported. Only two of six genes were affected by soil management history. The abundance of the nosZ and bacterial amoA genes, the only genes affected by the M × R interaction, was higher in the organic system . The increase in abundance of the nosZ gene could potentially indicate greater conversion of N2O to N2 and decreased greenhouse gas production, while increased abundance of the amoA gene may reflect increased conversion of ammonium to nitrite and subsequent nitrification products. Higher soil carbon as a result of long-term organic matter applications at this site may contribute to higher abundances of the nosZ gene in bulk and rhizosphere soil in this system. Putz et al. found that higher soil organic carbon under a ley rotation increased expression of the nrfA and nosZ genes relative to the nirK gene as compared to a conventional cereal rotation, favoring higher rates of dissimilatory nitrate reduction to ammonium and lower rates of denitrification. However, previous work in the treatments examined in the present study found that abundances of the amoA and nosZ genes were not correlated with gross rates of N transformation processes. Prediction of cropping system impacts on microbial N cycling therefore requires a nuanced integration of gene abundances with parameters such as carbon availability, moisture content, and temperature within soil aggregate microenvironments over time.
That few differences were observed late in the growing season between N-cycling genes in systems receiving organic or inorganic N inputs is consistent with the results of a meta-analysis by Geisseler and Scow, which found that N fertilizer impacts on microbial communities tend to fade over time. Sampling occurred at silking in the present study, long after the preplant fertilizer and compost applications that likely maximize differentiation between systems. Potential N limitation in the rhizosphere in both systems may also have outweighed management effects. Co-occurrence networks, which provide insight into ecological interactions among microbial taxa, were influenced by M, R, and M × R effects. Bulk and rhizosphere bacterial networks from the conventional system had the same number of nodes but were more densely connected than networks from the corresponding soil compartment in the organic system . Other bulk soil comparisons of organic and conventional agroecosystems using networks constructed from OTU-level data have found conventional networks to have more nodes or, alternatively, fewer nodes and edges than organic networks. Clearly, predicting cooccurrence patterns of incredibly diverse microbial communities based on a conventional-versus-organic classification is too simplistic. Agricultural management is likely better represented as a continuum than discrete categories, and causal relationships between specific practices and network topological properties have yet to be determined. An M × R interaction was also observed for network properties in which size, density, and centralization were lower in the rhizosphere network from the conventional system than from the organic system . These network properties follow the same pattern as alpha diversity of bacterial communities, suggesting a shared yet perplexing cause: while the mechanism remains unclear, rhizosphere communities appear to be converging from very distinct bulk soils towards similar diversity and structural metrics. Conventional agriculture is hypothesized to disrupt the connections between bulk soil and rhizosphere networks, as tillage and mineral fertilization are proposed to disturb fungi and soil fauna that serve as a bridge between bulk soil and rhizosphere environments. While tillage does not differ between the systems we measured, fertilization effects are likely partly responsible for the observed interaction. Regardless of the mechanisms involved, the systemspecific direction of the rhizosphere effect on cooccurrence network properties suggests that management and plant influence interactively determine not only which taxa are present, but how they interact, with potential implications for agriculturally relevant functions and ecological resilience. Hub ASVs were identified in each network based on high values for normalized betweenness centrality, a metric often used to describe keystone taxa. Lower betweenness centrality values for hub taxa may indicate that network structure depends less on individual species, potentially increasing resilience to environmental stresses that could destabilize networks overly dependent on hub taxa sensitive to those specific stresses. Different hub ASVs were identified in each rhizosphere environment, but information on the ecology of these taxa is generally absent from the literature. Although it would be misleading to state that these taxa are keystone species in their respective habitats without experimental validation, the fact that many of these taxa were also identified through indicator species analysis suggests that they play important ecological roles.