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,grow bags for gardening 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 system specific 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. Organic networks had lower normalized betweenness centrality values than conventional networks . 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. Future work could explore the genomes of these ASVs to discern why they are important in their respective agricultural systems and test the hypothesis that they serve as keystone species using synthetic communities. Concluding whether adaptive plant-microbe feed backs result in an M × R interaction leading to shifts in other rhizosphere processes is complicated by the importance of poorly understood fungal communities and methodological limitations of this study. Numerous fungal taxa respond to the M × R interaction according to our differential abundance analysis , yet knowledge of these taxa remains limited due in part to the constraints of culture-dependent methods prevalent in the past. Nonetheless, fungi influence inter-kingdom interactions and agriculturally relevant processes in the rhizosphere, and novel molecular biology tools could be used to improve our understanding of key fungal regulators identified in these analyses.

Metagenomics and -transcriptomics would facilitate a much more comprehensive analysis of potential functional shifts. A highly useful starting point would be to delve into dynamic variation in microbial genes involved in carbon metabolism and nitrogen cycling in the rhizosphere, in combination with root exudate metabolomics and measurements of root N uptake. Stable isotope labeling and in situ visualization methods could further complement our understanding of how management, plant roots, and their interactive effects shape rhizosphere processes. The scope of this study was intentionally restricted to a single genotype of one crop in two management systems to limit the main sources of variation to the management and rhizosphere effects that were of primary interest, but the limits to inference of this small-scale study must be considered. Other studies in maize have found that strong legacy effects of soil managementhistory are generally acted upon in a similar manner by two maize cultivars and that rhizosphere bacterial community composition varies only slightly among hybrids from different decades of release. Testing whether these limited effects of plant selection hold true for additional contrasting genotypes and genetic groups of maize would further complement this work. Furthermore, variation in root system architecture across crop genotypes might interact with tillage and soil properties responsive to management effects. Management practices such as the inclusion of forage or cover crops planted in stands rather than rows might affect the differentiation of bulk and rhizosphere soil uniquely from systems based on perennial crops, successive plantings of row crops in the same locations,garden grow bags and/or minimal tillage. Study designs incorporating more genotypes, management systems, and cultivation environments would therefore be highly useful to test how results of this study may be extrapolated to other settings. Future studies should also identify functional genes that are upregulated or downregulated in the rhizosphere under specific agricultural management practices. Whether such functional shifts are adaptive will provide insight into the relationship between agroecology and ecology. Positive eco-evolutionary feedbacks resulting in adaptive microbial communities have been described in unmanaged ecosystems, for example, habitat-adapted symbiosis in saline or arid environments. If similar adaptive recruitment can occur with annual crops in the context of agroecosystems, maximizing this process should be added to the list of rhizosphere engineering strategies and targets for G × E breeding screens. Finally, while our results provide evidence that management and plant influence interact to shape microbial communities at one sampling point, we highlight the need to reframe the M × R interaction as a dynamic process. Rhizosphere communities may be more different from one another than bulk soil communities because roots develop right after tillage and fertilization, when management systems are most distinct. Plants are not static entities, but active participants in the ongoing process of rhizosphere recruitment. As an alternative to the “rhizosphere snapshot,” we propose a “rhizosphere symphony” model that acknowledges the active role of root exudates in orchestrating the composition and function of microbial communities.

Altered root exudation during development and in response to water and nutrient limitation can upregulate or downregulate microbial taxa and functions, as a conductor brings together different sections of instruments in turn during a symphony. Although it is unknown whether this plasticity in exudate composition occurs in response to agricultural management, observations of changed exudate quantity and quality in response to soil type and long-term N fertilization suggest that it is possible. Differences in the timing of nutrient availability between management systems, such as delayed N release from cover crop mineralization compared to mineral fertilizer, could thus result in management-system-specific exudate dynamics and rhizosphere microbial communities, i.e., an M × R interaction. If true, this mechanism suggests that we may be able to manipulate the sound of the symphony by talking to the conductor: plant-driven strategies may be instrumental in maximizing beneficial rhizosphere interactions throughout the season.The Elkhorn Slough is located in the Central Monterey Bay area and feeds into the head of the Monterey Submarine Canyon in the newly designated Monterey Bay National Marine Sanctuary. The slough is described by the Department of Fish and Game as “one of the most ecologically important estuarine systems in California” . Elkhorn Slough was designated as an environmentally sensitive habitat in the 1976 California Coastal Plan and over 1400 acres of the slough are in the National Estuarine Research Reserve System. Water quality in the Elkhorn Slough is heavily influenced by both past and present human activities on the land surrounding the slough. This is especially true of agriculture. Non-point source pollutants from farm use of chemical fertilizers and pesticides have been identified as a primary cause of water quality degradation in the Elkhorn Slough. Agriculture is one of the main land uses in the slough watershed with about 26% of the local watershed in agricultural production. Of this land, strawberry production accounts for the greatest area under production . Field testing and monitoring of alternative farming practices that decrease dependence on synthetic chemical inputs has been extremely limited. What is needed is the development of farming systems that are economically as well as environmentally sustainable. The Azevedo Ranch site encompasses 137 acres, approximately 120 of which are currently in strawberry cultivation. The land is jointly owned by The Nature Conservancy and the Monterey County Agricultural and Historical Land Conservancy, whose stated goal is to keep this property in open space in perpetuity. The property will be divided into a wetlands buffer zone surrounding three “pocket marshes,” and an upland agricultural zone.They are connected to tidal water by culverts through the berm, making each independent. The buffer zone, which is currently in cultivation, will be restored with native vegetative cover including native bunch grasses, Coast Live Oaks, and maritime chaparral. The upper agricultural zone will encompass 83 acres and will eventually be converted to low-input sustainable agriculture.