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Phenotypically anchored transcriptomics across diverse agrichemicals reveals conserved pathways and unique gene expression signatures in zebrafish

Agrichemicals such as herbicides, fungicides, insecticides, and biocides are widely used in agriculture, yet some are associated with adverse effects in humans and the environment. While many of these chemicals have been extensively studied in vitro and are included in the EPA’s ToxCast program, comprehensive in vivo comparisons using RNA sequencing across structurally diverse agrichemicals, in a single screening platform, are lacking. In this study, we examined structurally diverse agrichemicals found in the U.S. Environmental Protection Agency’s (EPA) Toxcast Phase I and II library by statically exposing early life stage zebrafish at 6 h post fertilization (hpf) until 120 hpf at concentrations ranging from 0.25 to 100 µM. Morphological outcomes were assessed at 120 hpf across 10 endpoints, including yolk sac edema, craniofacial malformations, and axis abnormalities. Chemicals that produced robust concentration-response relationships were selected for transcriptomic profiling. For transcriptomic analysis, zebrafish were statically exposed to each chemical and sampled at 48 hpf, prior to the onset of morphological effects observed at 120 hpf. Differential expression analysis identified between 0 and 4,538 differentially expressed genes (DEGs) per chemical, with no clear correlation to morphological severity. Both DEG and co-expression network analyses revealed chemical-specific expression patterns that converged on shared biological pathways, including neurodevelopment and cytoskeletal organization. Key regulatory genes such as mylpfa and krt4 were identified within co-expression modules, suggesting their potential role in conserved toxicity mechanisms. Semantic similarity analysis of enriched gene ontology (GO) terms, when compared to existing datasets, highlighted gaps in the annotation of neurodevelopmental processes, indicating that some in vivo effects may not be fully captured by current curated resources. The results provide new insights into the modes of action of diverse agrichemicals and establish a framework for understanding how agrichemical structure relates to biological function in a vertebrate model.

agrichemical↗

Nuclear Data: From Measurement, Evaluation, Processing to Practical Applications [Slides]

Any analysis, design of a nuclear system will be as good as the knowledge of the nuclear data used whether differential and integral. ORNL is engaged in generating high level nuclear data for reactor, shielding, and safety applications. Nuclear data remains a major source of data uncertainties. Uncertainties on nuclear data rely upon measurements and evaluations. Tight connections with experimental facilities around the world have helped ORNL to address issues on ND and uncertainty. It is extremely important for ORNL to retain expertise on nuclear data in support of the research and expertise activities.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

PETSc/TAO Users Manual (Rev. 3.19)

This manual describes the use of the Portable, Extensible Toolkit for Scientific Computation (PETSc) and the Toolkit for Advanced Optimization (TAO) for the numerical solution of partial differential equations and related problems on high-performance computers. PETSc/TAO is a suite of data structures and routines that provide the building blocks for the implementation of large-scale application codes on parallel (and serial) computers. PETSc uses the MPI standard for all distributed memory communication. PETSc/TAO includes a large suite of parallel linear solvers, nonlinear solvers, time integrators, and opti mization that may be used in application codes written in Fortran, C, C++, and Python (via petsc4py; see Getting Started). PETSc provides many of the mechanisms needed within parallel application codes, such as parallel matrix and vector assembly routines. The library is organized hierarchically, enabling users to employ the level of abstraction that is most appropriate for a particular problem. By using techniques of object-oriented programming, PETSc provides enormous flexibility for users. PETSc is a sophisticated set of software tools; as such, for some users it initially has a much steeper learning curve than packages such as MATLAB or a simple subroutine library. In particular, for individuals without some computer science background, experience programming in C, C++, python, or Fortran and experience using a debugger such as gdb or lldb, it may require a significant amount of time to take full advantage of the features that enable efficient software use. However, the power of the PETSc design and the algorithms it incorporates may make the efficient implementation of many application codes simpler than “rolling them” yourself. For many tasks a package such as MATLAB is often the best tool; PETSc is not intended for the classes of problems for which effective MATLAB code can be written. There are several packages, built on PETSc, that may satisfy your needs without requiring directly using PETSc. We recommend reviewing these packages functionality before starting to code directly with PETSc. PETSc can be used to provide a “MPI parallel linear solver” in an otherwise sequential, or OpenMP parallel code. This approach cannot provide extremely large improvements in the application time by utilizing large numbers of MPI processes but can still improve the performance. Certainly all parts of a previously sequential code need not be parallelized but the matrix generation portion must be parallelized to expect true scalability to large numbers of MPI processes. See PCMPI for details on how to utilize the PETSc MPI linear solver server. Since PETSc is under continued development, small changes in usage and calling sequences of routines will occur. PETSc has been supported for twenty-five years; see mailing list information on our website for information on contacting support.

97 MATHEMATICS AND COMPUTING↗

PETSc Users Manual (Rev. 3.13)

This manual describes the use of PETSc for the numerical solution of partial differential equations and related problems on high-performance computers. The Portable, Extensible Toolkit for Scientific Computation (PETSc) is a suite of data structures and routines that provide the building blocks for the implementation of large-scale application codes on parallel (and serial) computers. PETSc uses the MPI standard for all message-passing communication. PETSc includes an expanding suite of parallel linear solvers, nonlinear solvers, and time integrators that may be used in application codes written in Fortran, C, C++, and Python. PETSc provides many of the mechanisms needed within parallel application codes, such as parallel matrix and vector assembly routines. The library is organized hierarchically, enabling users to employ the level of abstraction that is most appropriate for a particular problem. By using techniques of object-oriented programming, PETSc provides enormous flexibility for users. PETSc is a sophisticated set of software tools; as such, for some users it initially has a much steeper learning curve than a simple subroutine library. In particular, for individuals without some computer science background, experience programming in C, C++, python, or Fortran and experience using a debugger such as gdb or dbx, it may require a significant amount of time to take full advantage of the features that enable efficient software use. However, the power of the PETSc design and the algorithms it incorporates may make the efficient implementation of many application codes simpler than \rolling them" yourself.

97 MATHEMATICS AND COMPUTING↗

Toward Muon Neutrino Charged-Current Neutral Pion Cross Section Measurements at ICARUS

At ICARUS and the broader Short-Baseline Neutrino Program, few neutrino interaction channels offer as much utility as those producing neutral pions, which are an important background to electron neutrino searches and provide a standard candle for calibrating the electromagnetic shower energy scale. Beyond this, measurements of neutrino-induced neutral pions offer a probe of resonant interactions that are relevant to future accelerator neutrino experiments like the Deep Underground Neutrino Experiment. In this document, a measurement of charged-current muon neutrino interactions in liquid argon with a single neutral pion in the final state is presented, making use of neutrino interaction events recorded with the ICARUS T600 detector at Fermilab while exposed to the Booster Neutrino Beam. Event selection is carried out with a fully-automated machine learning reconstruction framework, allowing for the extraction of single-differential cross section measurements as a function of muon and neutral pion kinematic observables.

Carber, Dan [Colorado State U.]↗

Chapter Six - Derivation, cross-validation, and comparison of analytic formulas for electrostatic deflector aberrations

Here, we derived first and second order analytic aberration formulas in the horizontal transverse plane for an electrostatic deflector specified by the reference orbit radius, the central angle spanning the deflector, and its inhomogeneity coefficients. The derivation was performed using an iterative order-by-order perturbation method in a Frenet–Serret beamline coordinate system. We produced a C program edabrt for calculation of the first and second order aberrations using the formulas that we derived. The electrostatic deflector aberration formulas from Wollnik (1965) disagree with COSY INFINITY and the aberration formulas that we derived here, and they also deviate from the first and second order symplecticity conditions. The reason for this discrepancy is that the aberration formulas from Wollnik (1965) consider only motion in the main field of the deflector and do not properly account for fringe field effects, in particular the necessarily occurring change of kinetic energy due to the change in potential. The code GIOS uses electrostatic aberration formulas from Wollnik (1965) and exhibits the same discrepancy. We performed a comparison of first and second order electrostatic deflector aberrations for (1) the analytic formulas derived in this work, (2) differential-algebraic (DA) numerical integration of the equations of motion using the code COSY INFINITY, (3) the aberration formulas from Wollnik (1965), (4) aberrations computed using the code GIOS, and (5) the aberration formulas from Wollnik (1965) adjusted to account for the occurring change in potential. An electrostatic spherical deflector and an electrostatic cylindrical deflector were used as test cases for this comparison. There is excellent agreement between methods (1), (2), and (5), and these three methods satisfy the first and second order symplecticity conditions.

43 PARTICLE ACCELERATORS↗

Challenge Solutions for a Significant Water Cut Reduction History Match on Heavy Oil Polymer EOR

Milne Point Field is conducting the first-ever active polymer flood pilot program designed to enhance oil recovery (EOR) of heavy oils on the Alaska North Slope (ANS). The experimental field program consists of two pairs of horizontal injection and production wells deployed in an isolated fault block in the Schrader Bluff reservoir. The EOR method combines polymer flooding and low salinity water flooding to improve recovery. Prior average water cut reduction in polymer EOR fields ordinarily ranges between 10% to 15% for most light oil and heavy oil reservoirs. When comparing other field applications, the long-lasting (24 months) and much lower water cut poses a challenge for numerical history matching when applying typical simulation methods. This abstract discusses two practical new approaches used for history matching of the long-lasting ultralow water cut. These new applications include 1) multiple relative permeability curves with J-function incorporation with low endpoints of relative permeability of water (Krw). This approach integrated the permeability differential adjustment in the geometries of horizontal and vertical directions based on geological review. 2) A specific permeability channel strips along with the horizontal or vertical planar assumption. The current history matching results indicate success in achieving an excellent agreement with the field observed low water-cut.

Namie, S.↗

Methodology for physics-informed generation of synthetic neutron time-of-flight measurement data

Accurate neutron cross section data are a vital input to the simulation of nuclear systems for a wide range of applications from energy production to national security. The evaluation of experimental data is a key step in producing accurate cross sections. There is a widely recognized lack of reproducibility in the evaluation process due to its artisanal nature and therefore there is a call for improvement within the nuclear data community. This can be realized by automating/standardizing viable parts of the process, namely, parameter estimation by fitting theoretical models to experimental data. This automation effort could greatly benefit from a synthetic data resource. This work leverages problem-specific physics, Monte Carlo sampling, and a general methodology for data synthesis to generate unlimited, labelled experimental cross-section data that is statistically indistinguishable to the observed data. Heuristic and, where applicable, rigorous statistical comparisons to observed data support this claim. The demonstration is based on/limited to transmission measurements at Rensselaer Polytechnic Institute (RPI) and energy-differential cross sections in the resolved resonance region (RRR). An open-source software is published alongside this article that executes the complete methodology to produce high-utility synthetic datasets. The goal of this work is to provide an approach and corresponding tool that will allow the evaluation community to begin exploring more data-driven, ML-based solutions to long-standing challenges in the field.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

A new form of the Saint-Venant equations for variable topography

The solution stability of river models using the one-dimensional (1D) Saint-Venant equations can be easily undermined when source terms in the discrete equations do not satisfy the Lipschitz smoothness condition for partial differential equations. Although instability issues have been previously noted, they are typically treated as model implementation issues rather than as underlying problems associated with the form of the governing equations. This study proposes a new reference slope form of the Saint-Venant equations to ensure smooth slope source terms and eliminate one source of potential numerical oscillations. It is shown that a simple algebraic transformation of channel geometry provides a smooth reference slope while preserving the correct cross-section flow area and the total Piezometric pressure gradient that drives the flow. The reference slope method ensures the slope source term in the governing equations is Lipschitz continuous while maintaining all the underlying complexity of the real-world geometry. The validity of the mathematical concept is demonstrated with the open-source Simulation Program for River Networks (SPRNT) model in a series of artificial test cases and a simulation of a small urban creek. Validation comparisons are made with analytical solutions and the Hydrologic Engineering Center's River Analysis System (HEC-RAS) model. The new method reduces numerical oscillations and instabilities without requiring ad hoc smoothing algorithms.

54 ENVIRONMENTAL SCIENCES↗

Bubble Mass Transport Measurement in the Large-Scale Test Loops at Oak Ridge National Laboratory

Molten salts are complex fluids that incorporate multi-phase behavior depending on the chemistry and the physical properties of the entrained components. These components include the carrier salt, the actinide fuel, and fission, activation, and corrosion products, the concentrations of which depends on the burnup history of the salt. Radionuclide transport from the salt into the cover gas / off-gas system depends on volatility as predicted by thermochemistry, but data from the Molten Salt Reactor Experiment (MSRE) conducted in the late 1960s suggest that bubble formation and transport are also important. Anomalously high fractions of noble metals were found in the off-gas system and were attributed to transportation with parent salt aerosols and their association with rising noble gas bubbles. The prediction of such phenomena requires coupled neutronic, thermal hydraulic, and chemical equilibrium calculations, the framework of which is being developed within the Nuclear Energy Advanced Modeling and Simulation (NEAMS) program supported by the US Department of Energy (DOE) Office of Nuclear Energy (NE). However, separate effects tests and models of experiments using tools such as SAM and Thermochimica can guide model developers through important processes. Conversely, model development guides the choice of experiments and systems to provide data that are relevant for validation. This report describes several experiments that tracked gas transport in molten salts, ranging from small-scale systems to large-scale loops. Gas transportation in a molten salt, LiCl-KCl, has been studied using the shadowgraph technique. Sensors such as residual gas analysis, Raman spectroscopy, and laser-induced breakdown spectroscopy have been tested for off-gas measurements. These data were used to interpret how the gases move through the upstream salt / cover gas and the interface between them. Differential pressure measurements were able to detect individual gas bubbles as they popped at the liquid–gas interface. Salt aerosols were collected on a cascade impactor. Their formation was also observed directly via the shadowgraph method, and most of these aerosols were launched ballistically into the cover gas. Fine mists could also be observed. Convection currents through the salt were visualized and can be used to calculate the thermophysical properties of the salt itself. The apparatuses described in this report and in a previous work (McFarlane et al. 2023) have been commissioned and are available for use in making further measurements of salt/surrogate fission product behavior. Novel approaches using neutron imaging are planned for the study of fluoride salts, which cannot be contained in quartz, so shadowgraph visualization is not available. Bubble transport in convective flow and in a slow-moving salt column are planned. The mobile laser-induced breakdown spectroscopy (LIBS) system is available for several applications, including iodine capture in a molten hydroxide scrubber, H2 transport though molten salts to complement Raman analysis, and online tracking of salt aerosol generation, transport, and deposition. This report summarizes the findings from FY24 and the plans for FY25. The work completes milestone M2AT-24OR0702013 of the DOE-NE Advanced Reactor Technology, Molten Salt Reactor Campaign, DOE-NE-5.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

Status of the CERBERUS Evaluation for the International Criticality Safety Benchmark Evaluation Project (ICSBEP) Handbook

Modeling & Simulation (M&S) tools are used to analyze advanced reactor designs and the safety of current nuclear operations. As computers continue to improve, we are able to enhance resolution in our calculations. Therefore, the limitations of simulation capability are in the quality of data that is being used, including our ability to quantify the uncertainty and sensitivity of that data. In order to model systems of interest with increasing accuracy, the industry must improve key nuclear data measurements. The International Criticality Safety Benchmark Evaluation Project (ICSBEP) compiles and evaluates experiment data in a handbook that can be used by criticality safety engineers and others to validate computer codes and cross section libraries at nuclear facilities. Both critical and subcritical experiments are included in the handbook. These experiments, along with differential measurements, can help improve the quality of nuclear data. Concerns regarding the accuracy of Cu nuclear data have been published. The large values and trend of C-E for the Zeus intermediate energy benchmark, being one of the primary examples. Furthermore, very few experiments have been designed to be sensitive to Cu (as shown in Figure 1), so an integral, critical experiment is needed to help resolve these differences.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

PD-L1 induces macrophage polarization toward the M2 phenotype via Erk/Akt/mTOR

Highlights: • PD-L1 treatment upregulates CD206 but not CD86 expression. • PD-L1-induced DEGs are related to the immune response, mitochondrial function, and metabolism. • Nivolumab, LY294002, U0126, and rapamycin inhibit the PD-L1-stimulated CD206 expression. • PD-L1 promotes M2 polarization via Erk/Akt/mTOR. PD-L1 (programmed death-ligand 1) is the ligand of PD-1 (programmed cell death protein 1) and regulates inhibitory immune responses. It is well known that PD-L1 suppresses T cell function via binding to PD-1. However, little is known about the role of the PD-1/PD-L1 axis in macrophage polarization. According to previous studies, the function of the PD-1/PD-L1 axis in macrophage polarization is controversial, and the underlying mechanism has not been fully elucidated. Thus, we treated THP-1-derived macrophages with human PD-L1 Fc to determine the role of the PD-1/PD-L1 axis in macrophage polarization. To further explore the mechanism, we performed RNA sequencing and used specific inhibitors to identify the implicated signalling pathways. In this study, we found that PD-L1 induces the upregulation of CD206 expression, which is inhibited by nivolumab, LY294002, U0126, and rapamycin. Evaluation of differentially expressed genes (DEGs) and bioinformatics analysis indicated that PD-L1 also induces the upregulation of the expression of genes that maintain mitochondrial function and mediate metabolic switching. In addition, we did not detect PD-L1-induced CD86 alterations, indicating that PD-L1 treatment has no significant influence on M1 polarization. Taken together, these results suggest that PD-L1 binds to PD-1 and promotes M2 polarization accompanied by mitochondrial function enhancement and metabolic reprogramming via Erk/Akt/mTOR. This study elucidates the role of PD-L1 in macrophage polarization and verifies the underlying mechanisms for the first time. Considering that aberrantly upregulated PD-L1 expression contributes to a wide variety of diseases, targeting PD-L1-mediated macrophage polarization is a prospective therapeutic strategy for both neoplastic and nonneoplastic diseases.

60 APPLIED LIFE SCIENCES↗

Characterization of carbonaceous aerosols during TRACER-CAT

Absorbing aerosols (AA) have an important impact on the global radiation budget and cloud properties. The composition and properties of AA can vary substantially throughout the atmosphere, depending on the particle source and the influence of chemical aging. Uncertainties associated with the radiative effects of AA remain substantial. A key contributor to this uncertainty is understanding the extent to which coatings in general, and water uptake especially, alters absorption by AA particles and how this depends on particle composition. We deployed new and existing experimental tools during the Tracking Aerosol Convection Interactions Experiment (TRACER) campaign in Houston, TX as part of the Carbonaceous Aerosols Thrust (CAT) to provide detailed characterization of aerosol optical, chemical, and physical properties. Our TRACER-CAT measurements complemented and expanded on the planned TRACER instrumentation, allowing for more detailed characterization of aerosol properties of relevance to cloud development (a core focus of TRACER), such as the composition of particles that can act as cloud condensation nuclei, than would otherwise be available. Our measurements have allowed for assessment of the relationship(s) between AA optical properties (with a focus on absorption) and the chemical and physical characteristics (including the mixing state of black carbon (BC) containing particles). These field observations occurred in collaboration with Los Alamos National Laboratory in summer 2022 during the TRACER intensive operating period. The instrumentation we co-deployed provided for measurement of (i) multi-wavelength dry aerosol absorption, scattering, and extinction, (ii) the size-dependent composition and abundance of sub-micron aerosol, differentiating between those particles that do and do not contain BC, (iii) BC-specific concentrations and size distributions, (iv) particle size, and (v) the first field measurements at an ARM site of the influence of RH on multi-wavelength absorption by ambient AA. We have leveraged the natural variability of the atmosphere and of aerosol sources in the Houston region to (i) specifically disentangle contributions to light absorption from BC, absorbing organic carbon (brown carbon), and coatings on BC, (ii) characterize the mixing state of BC and assess the factors that give rise to compositional differences between BC-containing and BC-free aerosol, and (iii) establish how water uptake influences absorption and how any such effect depends on particle composition and BC mixing state. Overall, our study contributed to the mission of the Atmospheric System Research program in multiple ways. Through the deployment of complementary, advanced instrumentation for characterization of a wide range of aerosol properties our work helped to maximize the scientific impact of the TRACER campaign. Our work also allowed for development of new insights into the relationship(s) between aerosol composition, hygroscopicity, and the mixing state of BC with aerosol optical properties. Through this, our work has provided knowledge that can improve understanding and model representation of aerosol processes as they affect the Earth’s radiation budget.

54 ENVIRONMENTAL SCIENCES↗

Advances in Nuclear Data Evaluation Theory for NCSP [Slides]

The generalized form of the Bayes’ Theorem improves UQ for NCSP of differential and/or integral data evaluations and of applications. It is being implemented in an API for use by SCALE, SAMMY, etc. Extended R-matrix formalism improves evaluations of RRR including direct reactions, direct capture, and the Reich-Moore approx., doorway state reactions with applications to RPI data on lead isotopes. Extended R-Matrix formalism will be implemented into SAMMY post modernization.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Nuclear data sensitivity and uncertainty study of copper-reflected integral experiments [Slides]

This presentation touches on reducing uncertainties in intermediate-energy actinide nuclear data and this continues to be a high priority for many applications. The goal of PARADIGM (PARallel Approach of Differential and InteGral Measurements): accelerate efforts to reduce biases and uncertainties in nuclear data through improvements to the nuclear data pipeline. This presentation includes integral experiments and a summarization of existing copper nuclear data.

Cu63↗

Bi-metallic Nanoparticle Synthesis for Advanced Manufactured Melt Wires

Science Undergraduate Laboratory Internship (SULI) Program Report: Additive manufacturing (AM) based on direct-write technologies has emerged as the predominant method for the fabrication of passive sensors for the harsh operating environments seen in a nuclear reactor. Through the modification of previous methods, Idaho National Laboratory and Villanova University have improved the synthesis process for AM feedstock, which will allow for the improvement of advanced nuclear sensors and instrumentation. A major part of this work includes the synthesis process of relevant AM compatible feedstock to support the development, fabrication, and testing of AM sensors for peak temperature detection. For this report, bismuth, bismuth/platinum, tin, tin/silver, tin/zinc, indium, and indium/silver bi-metallic nanoparticles were synthesized using the polyol method, which will enhance temperature sensitivity and allow for miniaturization. To characterize the synthesized nanoparticles, we used x-ray fluorescence to evaluate the elemental composition of the nanoparticles and differential scanning calorimetry and thermogravimetric analysis to determine the melting point and mass loss of the samples. Results show that bi-metallic nanoparticles are a viable option for the fabrication of high-resolution AM melt wires. The temperature sensitivity can be brought to within 5°C and melt wires can be fabricated that are in the micrometer scale. This will expand the range of irradiation experiments melt wires can be used for and the measured temperature will be significantly more accurate.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Comparison between PARFUME and Bison Using the AGR-2 Irradiation Experiment

This report documents comparisons between Fuel Model (PARFUME) model predictions versus Bison for selected compacts from the second irradiation test of the Advanced Gas Reactor (AGR) program that occurred from June 2010 to October 2013 in the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL). PARFUME is a fuel performance analysis and modeling code, used for evaluating gas-reactor tristructural isotropic (TRISO) coated particle fuel for prismatic, pebble bed, plate, and cylindrical type fuel geometries. PARFUME is an integrated mechanistic computer code that evaluates the thermal, mechanical, and physico-chemical behavior of TRISO coated-fuel particles and the probability for fuel failure given the particle-to-particle statistical variations in physical dimensions and material properties that arise during the fuel fabrication process. Bison is a nuclear fuel performance application built using the Multiphysics Object-Oriented Simulation Environment (MOOSE) finite element library (Permann 2020). Bison is capable of modeling multiple fuel forms in a wide variety of dimensions and geometries. It solves coupled nonlinear partial differential equations, including heat conduction, mechanics, fission product species transport etc., in a fully implicit manner. Comparisons between PARFUME and Bison were performed using four compacts from the AGR-2 experiment. Selected outputs were chosen based on their impact on the probability of the SiC layer failing. In general there was good agreement between PARFUME and Bison with the exception of predicting the gap formed between the buffer and IPyC. Further comparisons between PARFUME and Bison are planned to further develop Bison’s capabilities.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

16s Amplicon Analysis of Soil Data for Interactive effects of depth and differential irrigation on soil microbiome composition and functioning

Genomic DNA was isolated from soil and rhizosphere samples using the Zymo Quick-DNA fecal/soil microbe miniprep kit (catalog no. D6010) according to the manufacturer’s instructions (Zymo Research; Irvine, CA) with the modification of eluting in 100 uL elution buffer. Sample concentrations were quantified using the Qubit dsDNA HS assay kit (Thermo Fisher). For rhizosphere samples only, DNA was subsequently purified using Zymo’s ZR-96 DNA Clean & Concentrator kit (catalog no. D4024) to account for low DNA concentrations of these samples. . In each replicate block, there were five drip irrigation treatments (T1 = 100% normal irrigation, T2 = 56.25%, T3 = 37.5%, T4 = 18.75% and T5=no irrigation. On July 20, the strength of the drought treatments was increased: T1 remained at 100%, whereas T2 changed from 75% to 56.25%, T2 changed from 50% to 37.5%, and T4 changed from 25% to 18.75%. Sequencing was performed as described previously (Naylor, Fansler, et al. 2020). Sequences were amplified on the MiSeq platform (Illumina, San Diego, CA) using 16S primers (515F and 806R) specific to the V4 region. Raw sequence data was processed with the pipeline Hundo for amplicon quality control and annotation. Downstream statistical analyses on 16S datasets were performed using the program R and the packages ‘phyloseq’ and ‘vegan’.

Soil microbiome, metatranscriptomics↗