Research Project: Improving Resilience of Dryland Legume Cropping Systems through Enhancement of Beneficial Microbiomes

Location: Grain Legume Genetics Physiology Research

2024 Annual Report

Objectives
Grain legumes, including Autumn-sown winter pea (WP) and pinto bean, are important crops for the U.S. Pacific Northwest (PNW) and Northern Plains. Both crops can produce nitrogen fertilizer through the colonization of their roots by nitrogen-fixing soil bacteria (rhizobia). Recently, our research unit has released several new food-grade WP and spring-sown pinto bean cultivars. However, little is known about the ability of these new cultivars to establish symbiotic associations with rhizobia native to PNW soils or the extent to which these new cultivars contribute to nitrogen budgets. To improve understanding of biological nitrogen fixation (BNF) and increase the amount of nitrogen produced by BNF this project has the following objectives: Objective 1: Increase environmental resilience and profitability of WP production systems through employment of strategies that improve plant and soil microbiomes. Sub-objective 1.A: Define native components of WP root microbiomes and ability for native rhizobia to compete with commercial inoculants in dryland production systems. Sub-objective 1.B: Isolate and characterize bacteria from nodule microbiome. Sub-objective 1.C: Identify WP cultivar/rhizobia combinations and management practices, which have the most beneficial effect on soil microbiomes, health, and soil N availability. Objective 2: Enhance symbiosis, fertility management, and resilience to environmental stresses in new dry bean cultivars. Sub-objective 2.A: Evaluate the link between new pinto bean cultivars, symbiotic performance, and environmental stress tolerance.

Approach
Sub-objective 1A: Goal: Establish the best native or commercial rhizobia and microbial partners for symbiotic nitrogen fixation (SNF) in winter pea (WP) under dryland environments. The diversity and structure of WP microbiomes and plant symbiotic preferences will be evaluated for food-grade WP cultivars across Pacific Northwest (PNW) locations for several years. Plots treated with commercial inoculums will assess the ability for native rhizobia to compete with introduced rhizobia. Plant growth parameters and disease development will be corelated with nodule and root microbiome diversity and composition. Sub-objective 1B: Hypothesis: Native soils will provide WP endophytes to improve plant growth and production under dryland-stressed environments. Isolation protocols for bacterial groups will be customized. Isolated bacteria will be tested individually and as synthetic communities for abilities to form effective symbiosis with food-grade WP cultivars and abilities to protect against cold, drought, and root rot resistance. The isolated bacteria will be tested for plant growth promotion (PGP) functions. Sub-objective 1C: Hypothesis: Incorporation of WP with effective N-fixing symbiosis with rhizobia and partners in crop rotations will improve soil health, fertility and add soil N credits. Soils from fields used for identification components of WP root microbiomes (Sub-objective 1.A) will be collected. Soil microbiome and chemical composition will be evaluated to identify changes in soil N, carbon and biology caused by WP rotations. These data will be correlated with WP genotype, field management, plant growth parameters, and WP nodule microbiome composition to identify the most efficient WP genotype/rhizobia/management combinations. Sub-objective 2A: Goal: Develop and identify pinto bean cultivars with high SNF capacity under diverse production environments. A select pinto panel (SPP) of 16 cultivars will be used to examine SNF and fertility management in pinto beans. Low N field trials will focus on the effect of legume genotype (LG) on microbiome selection. Once LGs with differences in endophyte preferences are identified, their endophyte preferences will be tested under drought stress. Plant growth parameters and N fixation will be correlated with nodule microbiome profiles to identify potential PGP.

Progress Report
This report documents FY 2024 progress for project 2090-21600-040-000D, “Improving Resilience of Dryland Legume Cropping Systems through Enhancement of Beneficial Microbiomes”, which began in October 2023. In support of Sub-objectives 1A and 1C, winter pea (WP) soil, root, and nodule samples from nine locations (21 sites) across Idaho, Washington, and Oregon were collected and processed. Chemical analyses were conducted on the soils collected from five locations. DNA from 1,236 samples (2022 collection) and 848 samples (2023 collection) was isolated. The early root nodulation rate was assessed at most locations. Less than 50% of sites exhibited nodule formation in the Fall, indicating that WP development stage might be a major factor affecting early nodulation. A trial to evaluate the effect of soil pH on WP microbiome and nodulation was conducted. The soil at the trial had a pH between 5 and 6 and an aluminum concentration between 1.3 ppm and 21ppm. Soil and plant samples were processed for further analysis, and the isolation of soil microbes is in progress. Under Sub-objective 1A, 16S rRNA and inter-transcribed spacer (ITS2) sequencing of 136 root, nodule and soil DNA samples from the 2022 collection (three locations) was completed and are in the process of analysis. Our data indicate that a diverse population of native rhizobia can colonize WP roots cultivated in Washington soils. However, a substantially smaller subset of these bacteria can colonize winter pea nodules, which are where biological nitrogen fixation by rhizobia occurs in nodules. Interestingly, three rhizobial Amplicon Sequence Variants (ASVs) were dominant nodule-associated bacteria in all tested locations regardless of the variation in soil microbiome diversity and structure between locations. These ASVs had relatively low abundance in soils, indicating their strong attraction to host-plant roots and high competitiveness for nodulation. ARS researchers in Pullman, Washington, also tested custom primers for nodD and nodC genes for rhizobial identification based on amplicon sequencing. Both set of primers were proven to be robust in the analysis and will used in future research. For Sub-objective 1B, the efficiency of nodulation across all locations and sites was evaluated. ARS researchers in Pullman, Washington, identified two sites, one in Moscow, Idaho, and one in Pendleton, Oregon, with very efficient nodulation, resulting in formation of extra-large (mega) nodules. The plants at the Moscow location exhibited improved vigor and less root rot severity than plants at Pendelton. The mega-nodules from the Moscow, Idaho, site were used to isolate nodule-forming rhizobia and other plant growth promoting bacteria. In total, 74 rhizobia isolates were obtained and stored for future analysis. DNA from these bacteria was isolated and sequenced for strain identification. All strains were confirmed to be Rhizobium leguminosarum. The ability of three random rhizobial isolates to form nodules on WP was tested and confirmed. Additionally, 20 rhizobia from normal size nodules from the same site were isolated and stored for analysis. Sixty-five Pseudomonas strains were isolated from mega-nodules and stored for analysis. Twenty random bacteria from WP roots were isolated and stored for assessment of plant growth promoting and antifungal properties. DNA from all isolated bacteria was isolated for future genetic identification. In support of Sub-objective 1B, two isolates of Fusarium acuminatum and five isolates of Fusarium redolens were isolated from the roots of WP grown in Washington soils. They were genetically identified and tested for pathogenicity and aggressiveness. These isolates will be used to evaluate interactions between rhizobia and different Fusarium species to determine the ability of rhizobia to help control Fusarium root rots. They will also be used to evaluate the antibiotic control of different species of bacteria found in nodules that may help to manage Fusarium spp. Under Sub-objective 1C, in collaboration with the ARS scientists in Adams, Oregon, a trial was established comparing three WP cultivars rotated with winter wheat (WW) to plots with continuous WW. Soil and plant samples were collected from the trail in the spring of 2024. Samples were processed, soil chemical analysis was performed, and DNA from soil samples was isolated and sequenced using 16S rRNA and ITS2 amplicon sequencing. The trial has entered the winter wheat rotation stage and next Fall the soil and WW plant parameters, such as soil chemical composition, plant and soil microbiome, plant disease development, and WW yield will be evaluated. For Objective 2, a panel of 16 pinto bean cultivars were grown in a replicated field trial with moderate intermittent drought stress conditions. Agronomic traits including seed yield, seed size, harvest maturity, plant height, and lodging score were recorded for 64 plots. Twenty roots from each cultivar were collected across the four replications prior to flowering and rated for size and number of nodules. Thereafter, root and nodule tissue from the 16 pinto beans cultivars were processed for DNA extraction. DNA from the samples was then isolated and sent for 16S rRNA and ITS2 amplicon sequencing. Harvested seed samples from each plot were sent for determining seed protein content based on percent nitrogen and for amount of nitrogen fixed from the atmosphere based on percent nitrogen-15 isotope compared to the non-nodulation dry bean control. The results indicate that higher nodule scores are correlated with higher amounts of fixed nitrogen and both traits contributed to increased seed yield. These preliminary findings indicate that breeding for increased nitrogen fixation should improve yield potential in pinto bean.

Accomplishments
1. Identification of genes associated with Fusarium root rot resistance in lentil. Fusarium avenaceum is a common Fusarium root rot root pathogen impacting lentil production in Canada and Montana. Lentil lines (238) were screened for resistance to F. avenaceum under greenhouse conditions that are well correlated with field resistance. ARS researchers in Prosser and Pullman, Washington, completed a genome mapping study and identified two genetic markers that were significantly associated with disease resistance. One gene is similar to other well characterized defense genes, and the other gene codes for a plant hormone-binding protein that is associated with reduced root rot severity and improved shoot length under disease conditions. These genes will be used by breeders in marker-assisted selection to develop new lentil cultivars with resistance to Fusarium root rots.

Review Publications
McLaughlin, M., Yurgel, S., Abbasi, P.A., Ali, S. 2024. The effects of chemical fungicides and salicylic acid on the apple microbiome and fungal disease incidence under changing environmental conditions. Frontiers in Microbiology. 15. Article 1342407. https://doi.org/10.3389/fmicb.2024.1342407.
Yurgel, S., Sallato C., B., Cheeke, T. 2023. Exploring microbial dysbiosis in orchards affected by little cherry disease. Phytobiomes Journal. 7(3):375-384. https://doi.org/10.1094/PBIOMES-10-22-0072-R.
Ajeethan, N., Yurgel, S., Abbey, L. 2023. Role of bacteria-derived flavins in plant growth promotion and phytochemical accumulation in leafy vegetables. International Journal of Molecular Sciences. 24(17). Article 13311. https://doi.org/10.3390/ijms241713311.
Lloyd, A.W., Percival, D., Langille, M.G., Yurgel, S. 2023. Changes to soil microbiome resulting from synergetic effects of fungistatic compounds pyrimethanil and fluopyram in lowbush blueberry agriculture, with nine fungicide products tested. Microorganisms. 11(2). Article 410. https://doi.org/10.3390/microorganisms11020410.
Ajeethan, N., Ali, S., Fuller, K., Abbey, L., Yurgel, S. 2023. Apple root microbiome as indicator of plant adaptation to apple replant diseased soils. Microorganisms. 11(6). Article 1372. https://doi.org/10.3390/microorganisms11061372.
Rathor, P., Borza, T., Bahmani, R., Stone, S., Tonon, T., Yurgel, S., Potin, P., Prithiviraj, B. 2023. Expression of a heat shock protein 70 from the brown alga Ectocarpus sp. imparts salinity stress tolerance in Arabidopsis thaliana. Journal of Applied Phycology. 35:803-819. https://doi.org/10.1007/s10811-022-02897-7.
Roy, J., Soler-Garzon, A., Miklas, P.N., Lee, R., Clevenger, J., Myers, Z., Korani, W., McClean, P. 2023. Integrating de novo QTL-seq and linkage mapping to identify quantitative trait loci conditioning physiological resistance and avoidance to white mold disease in dry bean. The Plant Genome. 16(4). Article e20380. https://doi.org/10.1002/tpg2.20380.
McLaughlin, M.S., Yurgel, S., Abbasi, P.A., Prithiviraj, B., Ali, S. 2022. Impacts of abiotic factors on the fungal communities of ‘Honeycrisp’ apples in Canada. Microbial Biotechnology. 16(8):1639–1656. https://doi.org/10.1111/1751-7915.14207.
Rahman, M.M., Porter, L.D., Ma, Y., Coyne, C.J., Zheng, P., Chaves-Cordoba, B., Naidu, R.A. 2023. Resistance in pea (Pisum sativum) genetic resources to the pea aphid, Acyrthosiphon pisum. Entomologia Experimentalis et Applicata. 171(6):435-448. https://doi.org/10.1111/eea.13296.