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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Sustainable Agricultural Systems Laboratory » Research » Research Project #440891

Research Project: Soil, Crop, and Manure Biochemistry and Molecular Ecology: Bridging Knowledge Gaps in Microbiome Response to Management and Climate Change

Location: Sustainable Agricultural Systems Laboratory

2022 Annual Report

Objective 1: Determine the dispersal and activity patterns of fungi, bacteria and archaea with depth and across environmental gradients in agricultural systems and determine their impacts and influence on soil organic matter sequestration to inform better soil health management decisions. Objective 2: Develop a quantitative understanding of the impact of crop Genetics x Environmental context x Management strategies (G x E x M) on crop productivity as influenced by enhanced biological nitrogen fixation (BNF) and a fuller understanding of the soil and plant - microbiome symbiosis in leguminous cash and cover crop systems at local, long-term study sites and through LTAR collaborations. Sub-objective 2A: Study LTAR sites where legumes are grown in rotation with commodity crops to determine the factors that control regulation and efficiency of BNF and the net contribution of BNF nitrogen (N) to agroecosystems. Evaluations of the BAU and ASP cropping systems will be conducted. Sub-objective 2B: Establish fundamental understanding of BNF in the context of plant genotype by environment interactions in the commodity crop cowpea, Vigna unguiculata, and Soybean, Glycine max. Sub-objective 2C: Develop a standardized protocol for portable and low entry cost DNA sequencing platforms to evaluate critical sources of variability and error in analyses of biological transformations of soil carbon(C) and N. Objective 3: Assess thermal and anaerobic treatment processes of manure and in water resource recovery and treatment to reduce antibiotics in wastewater streams and develop effective approaches for treatment and monitoring materials of concern. Sub-objective 3A: Measure antibiotic removal during anaerobic processing of dairy manure and biosolids with small and large-scale processing methods. Sub-objective 3B: Develop protocols for anti-microbial gene detection in agricultural systems consistent with current recommendations from the EPA and One Health Initiative. Objective 4: Improve the ability to track the loading of nitrate from agricultural sources by using time dated metabolites of metolachlor to address N management strategies and to improve environmental and water quality. Sub-objective 4A: Redesign sampling and analysis protocols for metolachlor ethane sulfonic acid (MESA) to include metolachlor oxanilic acid (MOXA) for collection and analysis of stream water as a tool to track nitrate sources from groundwater. Sub-objective 4B: Determine isomer composition of both MESA and MOXA in watershed networks in order to describe groundwater nitrate loading from agriculture sources.

A molecular ecological approach will be taken to bridge gaps in understanding of biogeochemical stocks and flows in agroecosystems. Using classic chemistry, metabolomics, molecular biology, and plant physiology for analysis of samples from different cropping systems at the Farming Systems Project site critical issues in soil carbon sequestration and soil enzyme activity, and plant-microbiome interactions in relation to nitrogen fixation in legumes will be addressed. The Farming System Project in Beltsville, MD is part of the LTAR network and is a platform for comparison of long-term impacts of five cropping systems (conventional chisel till, conventional no-till, and three organic crop production rotations) commonly used in the Mid-Atlantic region of the US and elsewhere. New techniques will be developed to investigate how to improve manure anaerobic digestion systems for increased degradation of antibiotics and other compounds of concern in animal production waste streams to minimize the effect of their release into the environment. This research will also leverage the development of novel, passive sampling devices that detect breakdown products of the pesticide metolachlor as a surrogate for nitrate release from crop production fields. This improved technique will allow quantification of conservation practices directed towards reduction of agricultural waste in the nation’s water resources. In considering the connectivity and entirety and outcomes of the efforts of this project, this project will develop best management practices that improve water resources and soil quality in the Mid-Atlantic region helping to improve the sustainability of small, mid-sized, and large farms.

Progress Report
This Annual Report covers the first year of this project. A new Sustainable Agricultural Systems Laboratory (SASL) scientist hired to work on this NP212 project completed onboarding, new scientist orientation, and required training. The soil chemistry skillset of the new scientist will add significant breadth to the research capability of the SASL NP212 Team. The addition of soil chemistry expertise and synchrotron X-ray techniques enables analysis of the atomic composition of soils from a novel perspective. Integrating this skillset will increase the impact and utility of future research. Collaboration has been established among ARS laboratories in Ft. Collins, Colorado; Pendleton, Oregon; and Lubbock, Texas, that has resulted in multiple, multi-area projects established in support of research initiated under Objectives 1 and 2. Supporting cross-National Program initiatives, SASL scientists have served on the SciNet Scientific Advisory Committee and as liaison to a new NPS ARS Microbiome working group. Additionally, scientists on this project continue to lead the Long-Term Agroecosystem Research (LTAR) Soils Working Group which has created a common language data inventory of soils data from all LTAR sites and developed a web-based tool that enables users to harmonize datasets among sites. This web-based tool is paired with a protocols and attributes tool that collects meta-data on protocols and procedures. Both tools are currently available on-line to the LTAR community and will be utilized in the LTAR cropping systems common experiment being implemented this fall. Two SASL NP212 scientists provided leadership for the nationwide Soil Biology Network (SBGx). SBGx works with the LTAR Network to facilitate implementation of a unified set of standardized techniques and protocols across all LTAR Network sites. For Objective 1, SASL scientists began analysis of soil microbial and biochemical indicators of nitrogen fixation. Specifically, primers were designed to target genes contained within the nifH operon (gene cluster) which is responsible for microbial biological nitrogen fixation. In collaboration with the University of Maryland, SASL scientists conducted analyses of relationships among common phylogenetic marker genes such as 16S and ITS rRNA as well as more specific functional genes involved in nitrogen and carbon cycling (nifH, nosZ, nirK, lacZ, lcc1). Predictable relationships among these marker genes were shown that differed with cropping system and the presence of the herbicide glyphosate. These relationships, now validated, will be used to more accurately predict the impact of cropping systems on functional microbial activities in soil, such as soil nitrogen and carbon cycling. For Objective 2, improvement of protocols for cross-site comparison of soil biological attributes continued. DNA and RNA were extracted from contrasting soil types representing the regional diversity of the agroecosystem in the LTAR Network. Soils collected from Jornada, New Mexico; Pendleton, Oregon; Ft. Collins, Colorado; Columbus, Ohio; Ithaca, New York; and Beltsville, Maryland, were extracted and assessed for DNA quality and quantity using common protocols. For Sub-objective 3A, a new method to extract and quantitate antibiotics from various stages of processed and unprocessed manure was finalized. The method consists of two-steps involving ultrasonic and mechanical mixing with two separate solvent extractions, an aqueous EDTA-McIlvaine buffer solution followed by methanol. The resulting extract is subsequently cleaned using solid phase extraction. The final concentrate is then subjected to LC-MS/MS analysis. To date, this method has been validated for analysis of 10 antibiotics covering 4-antibiotic classes. Performance of the method achieved beta-lactam recoveries between 5% and 74%, tetracycline recoveries between 54% and 108%, sulfadimethoxine recovery of 49%, and macrolide recoveries between 50% and 97%. A manuscript detailing this method has been submitted for publication. This extraction method was applied to samples collected from a Bedding Recovery Unit (BRU) operating on a farm in upstate New York to measure antibiotic destruction after thermal treatment to recover clean bedding from cow manure. Antibiotics were measured as the manure moved through a rotary drum BRU system. Four manure types were sampled and extracted at sites along the processing line: unprocessed source material, a liquid fraction isolated by screw press separation, and the remaining solids from the screw press both before and after rotary drum heat treating to isolate clean bedding material. Three antibiotics were detected in the manure samples: tetracycline, tulathromycin, and penicillin-G. Antibiotic concentrations ranged from 0.436 – 4.10 µg/kg. With hospital farm manure containing incurred antibiotics, mass flow analysis of the sequential processing was determined by including corn kernels that followed the manure as it moved through this BRU device. Calculated mass flow rates indicated that 95% of the manure mass was fractioned with the separated liquid fraction. The remaining 5% of the manure mass was in the separated solid faction which contained 11% to 20% of tetracycline and tulathromycin antibiotics. No significant reduction of either antibiotic was found following BRU processing of the separated solids. Biochemical Methane Potential (BMP) testing was carried out at each BRU process sampling site to see if subsequent anerobic methane recovery caused destruction of antibiotics. Such methane recovery practices are popular on many dairy farms. These tests were carried out using replicated lab-scale reaction flasks containing measured samples of the manure. Samples were spiked with oxytetracycline, ampicillin, and erythromycin and sampled and analyzed at five timed intervals up to 43 days. There was a significantly higher decrease in oxytetracycline with the heated versus the room-temperature incubations. Complete destruction of erythromycin and ampicillin occurred at room temperature and not under heated incubations. Anaerobic processing assists in removal of antibiotic from contaminated manure. For Sub-objective 3B, there was significant reduction in viable antibiotic-resistant bacteria in solids recovered from bedding material processed with the BRU. There was also complete elimination of pathogens from this material. Therefore, the BRU heating process successfully eliminated pathogenic and antibiotic-resistant bacteria in bedding material. Solid, liquid separation prior to heat processing, however, resulted in most of the mass staying with the separated liquid which accumulated in a storage lagoon. Therefore, this material continues to be at risk for release to the environment. Both mesophilic and thermophilic anaerobic treatment (BMP) of the final BRU solids were effective at eliminating spiked fecal indicator species (Enterococci and E. coli) by Day 9. Gene sequencing of the 16S ribosomal RNA gene is under way for comparative analysis of bacterial community diversity following the 43-day digestion of the final bedding material. For Sub-objective 4A, trials comparing approved MESA (metolachlor ethane sulfonic acid) extraction methods for the extraction of MOXA (metolachlor oxanilic acid) were completed and a methods paper is in preparation for submission this year. MESA and MOXA are being investigated as markers for monitoring nitrogen loss from agricultural fields. These two compounds are dominant soil degradation products of the chiral herbicide metolachlor. For Sub-objective 4B, chemical separation of MESA and MOXA was improved. All 4 significant trans-isomers of both MOXA and MESA were separated. Unfortunately, attempts to reliably measure the 4 isomers in a single injection has not yet been achieved. Lacking one injection for both MESA and MOXA, two injections (~822 sample runs) were performed on 15 sub-watershed samples from the Choptank River Watershed. The goal was to quantitate and compare all 4 isomers of MESA and MOXA for three years of samples (2007 to 2009). Lag-time calculations showed that MOXA is less persistent than MESA as a dating marker for groundwater transport of nitrogen. At the isomer specific level, it was found that variations of the rotamer pairs for each of the S and R enantiomers were closely linked to hydrologic variations in the different sub-watersheds. With this knowledge, predictive models can be developed to assist in describing flow paths for nitrogen based on soil properties. Another important result from this last year is the significant reduction in grab sample collection sizes required for MESA and MOXA. The new method requires only 50 mL of sample compared to 1-L required for the prior method. The new method makes use of new technology where larger volume injections can be introduced onto LC columns. We have validated the method for 4-peak separation of the trans isomers of both MESA and MOXA. There are several advantages to using smaller sample collection sizes; ease of collections, shipping, and storage (freezing is possible). Plans are underway to collect increased quantities of these small volume samples over flood events to look for correlations of the MESA and MOXA isomers as a function of flow.

1. Soil depth can be a more significant determinate of soil microbial community than farming system. The soil microbial community is important in agriculture as it is responsible for transformation and storage of a large proportion of soil carbon and nutrients. Models of stocks and flows of carbon and availability of nutrients to plants currently use outdated estimates of soil metabolic kinetics that do not take into consideration how land use decisions and farming systems regulate and impact transformation of carbon and nutrients. USDA-ARS scientists in Beltsville, Maryland, extracted and analyzed DNA and RNA from fungal, bacterial, and archaeal communities in 1-meter-deep soil cores from three different farming systems at the 26-year-old Farming Systems Project in Beltsville, Maryland. Results showed differences in soil microbial community at 0 to 15 cm among farming systems, for example Organic systems contained a higher density of arbuscular mycorrhizal fungi than conventional tilled and no-till farming systems. Below 15 cm depth soil type and physiochemical attributes were more predictable determinants of soil microbial community than farming system. Results from this study are important to scientists and will be used to improve models of soil carbon dynamics which are needed to improve soil carbon sequestration and plant nutrient availability assessments.

Review Publications
Schmidt, D., Dlott, G., Cavigelli, M.A., Yarwood, S., Maul, J.E. 2022. Soil microbiomes in three farming systems more affected by depth than farming system. Applied Soil Ecology. 173:104396.
Yan, J., Tang, Z., Fischel, M.H., Wang, P., Siebecker, M.G., Aarts, M.G., Sparks, D.L., Zhao, F. 2022. Variation in cadmium accumulation and speciation within the same population of the hyperaccumulator Noccaea caerulescens grown in a moderately contaminated soil. Plant and Soil.
Kaushik, A., Roberts, D.P., Ramaprasad, A., Mfarrej, S., Nair, M., Lakshman, D.K., Pain, A. 2022. The pangenome analysis of the soil-borne fungal phytopathogen Rhizoctonia solani and development of a comprehensive web resource: RsolaniDB. Frontiers in Microbiology.
Nimis, V., Rattner, B., Lockhart, M.J., Hulse, C.S., Rice, C., Kuncir, F., Kritz, K. 2022. Toxicological responses to sublethal anticoagulant rodenticide exposure in free-flying hawks. Environmental Science and Pollution Research.