Covering Ground: Southern Native Leguminous Summer Tepary Beans to Boost Productivity of Organic Spinach

Materials and Methods

This project was conducted on a certified organic farm of the Texas A&M AgriLife Research and Extension Center, Uvalde, Texas, during the 2024-25 and 2025-26 growing seasons.

Objective 1: To evaluate the USDA tepary bean (Phaseolus acutifolius) accessions under organic field conditions and identify genomic regions associated with agronomically and nutritionally important traits, using genome-wide association studies (GWAS).

Tepary bean evaluation panel: The tepary bean evaluation panel consisted of 206 USDA germplasm accessions (Phaseolus acutifolius) obtained from the USDA-ARS Western Regional Plant Introduction Station, Pullman, Washington. In addition, four commercially available tepary bean cultivars: Sacaton Brown, Sonoran White, Blue Speckled, and Black tepary bean, were included as check varieties to provide reference standards for agronomic performance under organic production conditions.

Field Design

Research plots were established in June 2024 at the certified organic research farm of the Texas A&M AgriLife Research and Extension Center, Uvalde, Texas. The experiment was conducted using an augmented block design, with the tepary bean panel distributed across five blocks. The four commercial check varieties were replicated and randomized within each block to facilitate comparison among accessions. Standard organic production practices, including minimum tillage, drip irrigation, and OMRI-approved biopesticides for insect management, were followed throughout the growing season to ensure crop growth and reliable phenotypic evaluation under organic conditions.

Phenotyping traits

Phenotypic evaluations were conducted throughout the growing season and at physiological maturity to characterize agronomic and nutritional traits associated with tepary bean performance under certified organic production.

Days to Flowering: Flowering time was recorded as the number of days from sowing to at least 50% flowering for each accession.

Biomass Production: Above-ground biomass was measured at the flowering stage to evaluate the potential of each accession as a summer cover crop and source of organic matter. Fresh biomass was recorded immediately after harvest, and samples were oven-dried to determine dry biomass accumulation.

Nitrogen-Use Efficiency (NUE): Nitrogen-use efficiency was evaluated by measuring plant nitrogen accumulation and soil nitrogen availability. Plant tissues were analyzed for total Kjeldahl nitrogen (TKN) and nitrate concentrations, while soil nitrogen data were integrated to calculate nitrogen-use efficiency indices for each accession.

Leaf Amino Acid Analysis: Leaf samples collected at flowering were freeze-dried using a lyophilizer, finely ground, and analyzed for amino acid composition using Water’s UPLC-MS/MS and established protocols in Joshi Lab. These analyses provided insight into nitrogen metabolism and physiological responses among tepary bean accessions.

Seed Protein and Amino Acid Analysis: Mature seeds were harvested at physiological maturity and analyzed for total soluble protein content using the Bradford assay. Seed amino acid composition was quantified using UPLC-MS/MS following established laboratory protocols. These analyses were conducted to evaluate nutritional quality and identify accessions with superior seed composition for future breeding efforts.

Protein and amino acid extraction and analysis

Total soluble protein was quantified from harvested seeds. 15–18 mg of finely ground seed meal per accession was extracted in 0.1 M NaOH, subjected to ultrasonication (15 min, Model: Branson 5200), and clarified by centrifugation at ≈ 16,000 × g for 15 min at 4 °C (Eppendorf Centrifuge 5425R). Supernatants were stored at −80 °C until analysis. Extracts were diluted 10X, and 20 μL aliquots were incubated with 180 μL Bradford reagent (Thermo Scientific™ Pierce™ Bradford Plus Protein Assay Kit) for 10 min in 96‑well plates. Absorbance at 595 nm was read on a Thermo Fisher Varioskan LUX. A BSA standard curve (0.125–2 mg mL⁻¹; **R² ≥ 0.99) was used to compute protein concentration (mg mL⁻¹), which was converted to total soluble protein.

Leaf amino acids were analyzed using established protocols (Joshi et al, 2019). At flowering, fully expanded leaves were sampled on dry ice, freeze-dried using a lyophilizer, and ground with stainless steel balls (Abbott Ball Co., West Hartford, CT, USA) in a Harbil 5G-HD shaker. Approximately 16–18 mg of powder was extracted and derivatized using AccQ.Tag™ 3X Ultra-Fluor kit (Walters Corp., Milford, MA, USA), then quantified using a Waters Acquity H-class UPLC system equipped with a Waters Xevo TQ mass spectrometer with an electrospray ionization (ESI) probe, which was used to detect individual amino acids. IntelliStart software (Waters Corp., Milford, MA, USA) was used to optimize each amino acid multiple reaction monitoring (MRM) transition, collision energy values, and cone voltage. Instrument monitoring and data acquisition were performed using Waters’ MassLynx software. Data integration, calibration curves, and amino acid quantification were performed using the TargetLynx application manager.

DNA Extraction, Genotyping, and Population Structure Analysis

Young leaf samples were collected from tepary bean accessions and stored at −80°C until further processing. Approximately 80-100 mg of frozen leaf tissue was homogenized using a TissueLyser with 3mm stainless steel beads, and genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit following the manufacturer's protocol. DNA quality and concentration were assessed using a DeNovix DS-11 Series Spectrophotometer, and high-quality DNA samples were submitted to the University of Minnesota Genomics Center for Genotyping-by-Sequencing (GBS) library preparation and sequencing. The ApeKI restriction enzyme was used for library construction, and high-quality sequence reads were aligned to the Phaseolus acutifolius v1.0 reference genome for SNP discovery. After quality filtering and genotype imputation, a total of 140,936 SNP markers representing 49,384 loci were retained for downstream analyses. Population structure was evaluated using STRUCTURE v2.3.4 following linkage disequilibrium pruning with the PLINK pipeline, resulting in 2,869 high-quality, non-redundant SNPs. The optimal number of genetic subpopulations was determined using the ΔK method implemented in Structure Harvester. Principal component analysis (PCA) was also performed to evaluate genetic relationships among accessions, and the first five principal components were included as covariates in subsequent genome-wide association analyses to account for population structure. GWAS was conducted using the BLINK (Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway) model to identify significant marker-trait associations and candidate genes associated with biomass production, flowering time, nitrogen-use efficiency, protein content, amino acid composition, and seed traits.

Objective 2: To compare summer legume cover crops (tepary bean, cowpea, and sunn hemp) for their effects on soil microbial communities, soil health, and the productivity of subsequent organic spinach production.

Experimental Design

A field experiment was conducted at the certified organic farm at Texas A&M AgriLife Research and Extension Center, Uvalde, Texas, using a randomized complete block design with three replications. The study included four commercially available tepary bean cultivars (Sacaton Brown, Sonoran White, Blue Speckled, and Black tepary), three cowpea cultivars (TX001, TX002, and CB46), one sunn hemp cultivar (Crescent Sunn), and a fallow treatment without cover crops that served as the control. Standard certified organic production practices, including drip irrigation and OMRI-approved pest management products, were followed throughout the experiment.

Cover Crop Termination and Spinach Cultivation

Cover crops were grown until the flowering stage (approximately 40–45 days after sowing) and were mechanically incorporated into the soil. The residues were allowed to decompose for approximately 30–50 days before planting the fall cash crop. Organic spinach (Spinacia oleracea L., cultivar: Space) was then sown into all plots following certified organic production practices. Spinach was harvested at the vegetative stage (65–75 days after planting) to evaluate the effects of preceding cover crops on crop establishment and biomass production.

Plant and Soil Analysis

Cover crop performance was evaluated by measuring above-ground biomass, both fresh and dry, after flowering. Soil samples were collected before cover crop establishment, at cover crop harvest, and following cover crop decomposition to determine changes in soil nitrate (NO₃⁻). Spinach biomass was recorded at harvest to evaluate crop productivity following different cover crop treatments.

Rhizosphere Soil DNA Extraction and Microbiome Analysis

Rhizosphere and bulk soil samples were collected from each treatment before cover crop termination to evaluate soil microbial communities. Soil DNA was extracted using the Qiagen DNeasy PowerSoil Pro Kit, and bacterial community composition was characterized through 16S rRNA amplicon sequencing using the Illumina platform. Sequencing data were processed using bioinformatics pipelines to evaluate microbial composition and predicted functional pathways. Microbial richness and evenness, community composition, principal coordinate analysis (PCoA), differential abundance analysis (LEfSe), and PICRUSt2 functional predictions were used to determine the effects of different summer legume cover crops on soil microbial composition under organic production systems.