Progress 08/01/23 to 07/31/24

Outputs
Target Audience: Nothing Reported Changes/Problems:Although the project officially started on08/01/2023, there has been miscommunication between the USM Office of Research Administration andthe USDA-ARS Wheat Health, Genetics, and Quality Research Unit (co-PI Paulitz) and the USDA-ARS Plant Science Research Unit (co-PI Schlatter). This resulted in a significant delay inthe processing of project subawards and the hiring of a technician and graduate students. However, the issue was ultimately resolved, and the delay did not affect our ability to commence field sampling planned for year 1. We are confident that these problems will not affect our ability to complete the entire scope of work planned under this project. What opportunities for training and professional development has the project provided?The grant supports one USM graduate student, Oleksandra Shabliy, by covering the cost of her research assistantship and providing supplies and reagents for her thesis research project.A second graduate student is being admitted through the?University of Minnesotato conduct microbiome and metagenome analysis experiments under objectives 1 and 3.The project also supports two USM undergraduate students, who will participate in the ongoing experiments and work as part-time lab helpers. Mentoring and supervision skills are essential professional development skills to attain during graduate school. The 'train-to-train' approach is applied in the personnel structure within the PI and co-PI's labs. This approach is evident in the mentoring and outreach efforts associated with this USDA-AFRI-funded project. Graduate students involved in this project are assigned to mentor and train undergraduates. Such experience is vital for the development of undergraduate and graduate students and plays a key role in the development of the next generation of scientists. With Mavrodi's guidance, Oleksandra Shabliy mentors Garrett Mans, a USM biology major who was involved in developing plant drought stress assays. Garrett Mans, is also a recipient of a grant from the Eagle Scholars Program for Undergraduate Research (Eagle SPUR), a competitive program that supports undergraduate research and creative activity at the University of Southern Mississippi. Oleksandra Shabliy also helps to supervise Elizabeth Lawrence, a biology freshman who works in the Mavrodi lab on exploring the role of plant-derived osmoprotectants in the adaptation of rhizobacteria to water stress. How have the results been disseminated to communities of interest?We are currently collecting and processing samples and perfecting plant assays and do not have the results to disseminate to communities of interest. What do you plan to do during the next reporting period to accomplish the goals?Under Objective 1, we plan to complete the field sampling and extraction of bulk soil and rhizosphere DNA of wheat and alfalfa.All DNA samples will be shipped to the University of Minnesota Genomics Center for library prep, Illumina sequencing, and data processing. We estimate that sufficient sequencing depth for profiling the irrigated and dryland metagenomes can be achieved with four lanes of NovaSeq S4 (9,000M 2x150 bp paired-end reads, or 750M reads per crop/condition/time point). To supplement short Illumina reads, high-molecular-weight DNA will be processed with a Rapid Barcoding Sequencing Kit (Nanopore) and sequenced in the Schlatter lab on an Oxford Nanopore MinION instrument. We will also complement the metagenome analysis with 16S- and ITS-based profiling of bacterial and fungal communities in the bulk soil, rhizosphere, and endosphere of dryland and irrigated alfalfa. The community analysis will involve amplification of the V3-V4 region of the 16S rRNA gene and fungal ITS1 region and subsequent Illumina sequencing (MiSeq, 2x300 bp paired-end). We will then proceed with sequence data analysis. Under Objective 2, we will establish optimal and reproducible conditions for the drought stress mesocosm assays involving wheat grown in soil from Lind, WA. We will then collect exudates of water-replete and water-stressed plants and ship them for metabolome profiling to the Analytical Resources Core - Bioanalysis and Omics (ARC-BIO) at Colorado State University.We will also collect samples of rhizosphere DNA and RNA from the mesocosms to correlate drought-induced shifts in the root exudate metabolome with changes in the metagenome content and microbial gene expression in the rhizosphere communities of wheat and alfalfa. The RNA and DNA samples will be shipped to the University of Minnesota Genomics Center for library prep, sequencing on an Illumina NovaSeq S4, and data processing. Under Objective 3, we will start greenhouse disease assays to evaluate how drought and disease interact, and how this interaction is influenced by the microbiomes that the plant selects under these stresses. We will use soil collected from a dryland field adjacent to the Lind experiment site. The non-drought treatment will be watered to keep the soil at or near field capacity. In the drought stress treatments, wheat will be grown to tillering (Zadok's growth stage 26) and alfalfa (Verticillium wilt-susceptible cv 'Saranac') will be grown to the three-leaf stage, then plants will be subjected to drought stress. For the inoculated and non-inoculated treatments, wheat will be inoculated with Rhizoctonia solani AG-8, and alfalfa will be inoculated with Verticillium alfalfae.

Impacts
What was accomplished under these goals? 1) In agreement with the proposed research timeline, in objective 1, theUSDA-ARS Wheat Health, Genetics, and Quality Research Unit (co-PI Paulitz) iscurrently collecting field samples tocharacterize metagenomes of dryland/irrigated wheat and alfalfa. The first sampled site is located at the WSU Dryland Research Station at Lind, WA (47.001°N, 118.563°W) and consists of eight adjacent plots continuously cropped to soft white spring wheat (cv. Louise). Half of these plots are irrigated from an installed sprinkler system. In May 2024, we also used the USDA Crop Layer data to survey and locate adjacent dryland and irrigated alfalfa plots for future experimental work.There are numerous irrigated crop circles of alfalfa in the Columbia Basin, but it is too dry for dryland alfalfa. On the other hand, there are extensive plantings in the higher rainfall areas close to the Idaho border that receive 20+ inches of precipitation per year. We started by identifying 7 potential locations in the intermediate precipitation area of eastern Washington and ground-truthed the potential sites identified from satellite data. Ultimately, we identified 3 paired irrigated/dryland locations, obtained permission to sample from the landowners, and completed 2 samplings this season. In the scabland area between the two precipitation extremes, growers can irrigate from groundwater and get 1-2 cuttings from dryland. The second set of samples was collected on June 28 after the first cutting, so we will also determine how this management practice affects the alfalfa rhizosphere microbiome. This second sampling was done with the co-PI Schlatter visiting the locations. All sampled sites were geo-referenced with a Global Positioning System (GPS) unit. At each location, wheat and alfalfa were sampled by selecting replicate plants at random along each of four perpendicular transects through each plot and digging with a shovel to a depth of about 25 cm. The samples of plant and bulk soil were immediately bagged, placed in a cooler, and transported to a laboratory where they were processed and frozen for future DNA extractions. The Paulitz team also continues to build a culture collection of Streptomyces spp. from the Lind site, which will be used in synthetic communities to assess the capacity of the dryland-adapted rhizobiome to reduce or delay symptoms of drought stress and/or disease. Cultures are being cataloged, stored, and sequenced for identification. 2) PI Mavrodi at The University of Southern Mississippi admitted a new graduate student to work on objective 2 of the project. They procured necessary small equipment, supplies, and soil moisture sensors and started preliminary drought stress mesocosm experiments using soil from the Lind location and soft white spring wheat cv. Louise. These mesocosm assays will be used to generate and compare root exudates of well-watered and water-stressed plants and correlate changes in the exudation patterns with shifts in the rhizosphere metagenome and metatranscriptome. 3)The USDA-ARS Wheat Health, Genetics, and Quality Research Unit (co-PI Paulitz) is currently hiring a technician for the project. They have received 9 applications and are in the process of interviewing the candidates. The USDA-ARS Plant Science Research Unit (co-PI Schlatter) is admitting a?University of Minnesota PhD student to run alfalfa experiments proposed under objectives 1 and 3 and collaborate with the broader team.

Publications