Integrated metagenomic assessment of multiple pre-harvest control points on lettuce resistomes at field-scale

An integrated understanding of factors influencing the occurrence, distribution, and fate of antibiotic resistance genes (ARGs) in vegetable production systems is needed to inform the design and development of strategies for mitigating the potential for antibiotic resistance propagation in the food chain. The goal of the present study was to holistically track antibiotic resistance and associated microbiomes at three distinct pre-harvest control points in an agroecosystem in order to identify the potential impacts of key agricultural management strategies. Samples were collected over the course of a single growing season (67 d) from field-scale plots amended with various organic and inorganic amendments at agronomic rates. Dairy-derived manure and compost amendment samples (n= 14), soil samples (n= 27), and lettuce samples (n= 12) were analyzed via shotgun metagenomics to assess multiple pre-harvest factors as hypothetical control points that shape lettuce resistomes. Pre-harvest factors of interest included manure collection during/post antibiotic use, manure composting, and soil amendment soil with organic (stockpiled manure/compost) versus chemical fertilizer. Microbial community resistome and taxonomic compositions were unique from amendment to soil to lettuce surface according to dissimilarity analysis. The highest resistome alpha diversity (i.e., unique ARGs, n=642) was detected in amendment samples prior to soil application, while the composted manure had the lowest total ARG relative abundance (i.e., 16S rRNA gene-normalized). Regardless of amendment type, soils acted as an apparent ecological buffer, i.e., soil resistome and taxonomic profiles returned to background conditions 120 d-post amendment application. Effects of amendment conditions surprisingly re-emerged in lettuce phyllosphere resistomes, with the highest total ARG relative abundances recovered on the surface of lettuce plants grown in organically-fertilized soils (i.e. compost- and manure-amended soils). Co-occurrence analysis identified 55 unique ARGs found both in the soil amendments and on lettuce surfaces. Among these, arnA and pmrF were the most abundant ARGs co-occurring with mobile genetic elements (MGE). Other prominent ARG-MGE co-occurrences throughout this pre-harvest lettuce production chain included: TetM to transposon (Clostridiodies difficile) in the manure amendment and TriC to plasmid (Ralstonia solanacearum) on the lettuce surfaces. This suggests that, even with imposing manure management and post-amendment wait periods in agricultural systems, ARGs originating from manure can still be found on crop surfaces. This study demonstrates a comprehensive approach to identifying key control points for the propagation of ARGs in vegetable production systems, identifying potential ARG-MGE combinations that could inform future surveillance. The findings suggest that additional pre-harvest and potentially post-harvest interventions may be warranted to minimize risk of propagating antibiotic resistance in the food chain

Groundwater influence on water budget of a small constructed floodplain wetland in the Ridge and Valley of Virginia, USA

Study region: A floodplain in the headwaters of a tributary to the Chesapeake Bay, Ridge and Valley of the Eastern United States. Study focus: This study investigated the influence of groundwater exchange in the annual wetland hydrologic budget and identified spatial and temporal variability in groundwater hydraulic gradients using an array of nested piezometers. New hydrological insights for the region: Data showed that the created wetland met hydrologic success criteria, and that the wetland storage was fully connected with the groundwater table. Water-surface storage fluctuation was not fully explained by precipitation and evapotranspiration, suggesting that storage was highly influenced by groundwater inputs. The potentiometric surface showed that hillslope seep recharge was the dominant groundwater vector. However, during the summer and fall months, the adjacent stream channel was a losing system, and storm-driven rise in stream stage affected wetland storage. The complex hydrology of this relatively small wetland indicates that predicting the fluctuations of storage for design of unconfined floodplain wetlands is challenging, and that if the influence of groundwater seepage is negated, then fluctuations may be underestimated to the point of harming vegetation

Quantifying the Spatial Variability of Annual and Seasonal Changes in Riverscape Vegetation Using Drone Laser Scanning

Riverscapes are complex ecosystems consisting of dynamic processes influenced by spatially heterogeneous physical features. A critical component of riverscapes is vegetation in the stream channel and floodplain, which influences flooding and provides habitat. Riverscape vegetation can be highly variable in size and structure, including wetland plants, grasses, shrubs, and trees. This vegetation variability is difficult to precisely measure over large extents with traditional surveying tools. Drone laser scanning (DLS), or UAV-based lidar, has shown potential for measuring topography and vegetation over large extents at a high resolution but has yet to be used to quantify both the temporal and spatial variability of riverscape vegetation. Scans were performed on a reach of Stroubles Creek in Blacksburg, VA, USA six times between 2017 and 2019. Change was calculated both annually and seasonally over the two-year period. Metrics were derived from the lidar scans to represent different aspects of riverscape vegetation: height, roughness, and density. Vegetation was classified as scrub or tree based on the height above ground and 604 trees were manually identified in the riverscape, which grew on average by 0.74 m annually. Trees had greater annual growth and scrub had greater seasonal variability. Height and roughness were better measures of annual growth and density was a better measure of seasonal variability. The results demonstrate the advantage of repeat surveys with high-resolution DLS for detecting seasonal variability in the riverscape environment, including the growth and decay of floodplain vegetation, which is critical information for various hydraulic and ecological applications.https://doi.org/10.3390/drones503009

Drone Laser Scanning for Modeling Riverscape Topography and Vegetation: Comparison with Traditional Aerial Lidar

Lidar remote sensing has been used to survey stream channel and floodplain topography for decades. However, traditional platforms, such as aerial laser scanning (ALS) from an airplane, have limitations including flight altitude and scan angle that prevent the scanner from collecting a complete survey of the riverscape. Drone laser scanning (DLS) or unmanned aerial vehicle (UAV)-based lidar offer ways to scan riverscapes with many potential advantages over ALS. We compared point clouds and lidar data products generated with both DLS and ALS for a small gravel-bed stream, Stroubles Creek, located in Blacksburg, VA. Lidar data points were classified as ground and vegetation, and then rasterized to produce digital terrain models (DTMs) representing the topography and canopy height models (CHMs) representing the vegetation. The results highlighted that the lower-altitude, higher-resolution DLS data were more capable than ALS of providing details of the channel profile as well as detecting small vegetation on the floodplain. The greater detail gained with DLS will provide fluvial researchers with better estimates of the physical properties of riverscape topography and vegetation

Seasonal Variation in Floodplain Biogeochemical Processing in a Restored Headwater Stream

Stream and river restoration activities have recently begun to emphasize the enhancement of biogeochemical processing within river networks through the restoration of river-floodplain connectivity. It is generally accepted that this practice removes pollutants such as nitrogen and phosphorus because the increased contact time of nutrient-rich floodwaters with reactive floodplain sediments. Our study examines this assumption in the floodplain of a recently restored, low-order stream through five seasonal experiments. During each experiment, a floodplain slough was artificially inundated for 3 h. Both the net flux of dissolved nutrients and nitrogen uptake rate were measured during each experiment. The slough was typically a source of dissolved phosphorus and dissolved organic matter, a sink of NO₃^–, and variable source/sink of ammonium. NO₃^– uptake rates were relatively high when compared to riverine uptake, especially during the spring and summer experiments. However, when scaled up to the entire 1 km restoration reach with a simple inundation model, less than 0.5–1.5% of the annual NO₃^– load would be removed because of the short duration of river-floodplain connectivity. These results suggest that restoring river-floodplain connectivity is not necessarily an appropriate best management practice for nutrient removal in low-order streams with legacy soil nutrients from past agricultural landuse

Effect of Manure Application on Abundance of Antibiotic Resistance Genes and Their Attenuation Rates in Soil: Field-Scale Mass Balance Approach

The development of models for understanding antibiotic resistance gene (ARG) persistence and transport is a critical next step toward informing mitigation strategies to prevent the spread of antibiotic resistance in the environment. A field study was performed that used a mass balance approach to gain insight into the transport and dissipation of ARGs following land application of manure. Soil from a small drainage plot including a manure application site, an unmanured control site, and an adjacent stream and buffer zone were sampled for ARGs and metals before and after application of dairy manure slurry and a dry stack mixture of equine, bovine, and ovine manure. Results of mass balance suggest growth of bacterial hosts containing ARGs and/or horizontal gene transfer immediately following slurry application with respect to <i>ermF</i>, <i>sul1</i>, and <i>sul2</i> and following a lag (13 days) for dry-stack-amended soils. Generally no effects on <i>tet</i>(G), <i>tet</i>(O), or <i>tet</i>(W) soil concentrations were observed despite the presence of these genes in applied manure. Dissipation rates were fastest for <i>ermF</i> in slurry-treated soils (logarithmic decay coefficient of −3.5) and for <i>sul1</i> and <i>sul2</i> in dry-stack-amended soils (logarithmic decay coefficients of −0.54 and −0.48, respectively), and evidence for surface and subsurface transport was not observed. Results provide a mass balance approach for tracking ARG fate and insights to inform modeling and limiting the transport of manure-borne ARGs to neighboring surface water