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

This project will be performed on a certified organic farm at the Texas A&M AgriLife Research and Extension Center, Uvalde, during 2024-25 and 2025-26 cropping season rotations.

Objective 1: Identify tepary bean accessions best suited for organic summer production based on (a) adaptability to organic production, (b) nitrogen-fixing potential,

Tepary Bean evaluation panel: The Tepary bean evaluation panel includes 207 Tepary bean (Phaseolus acutifolius) accessions obtained from the USDA-ARS, Western Regional Plant Introduction Station at Washington State University, Pullman, Washington. Three commercially available Tepary bean varieties were used as checks: Sacaton brown, Sonoran white, and Blue speckled, Black.

Field design:

Research plots were prepared in June 2024 at a certified organic research farm, Texas A&M AgriLife Research Station in Uvalde, Texas. The field was divided into five blocks, following an augmented block design. Four commercial varieties were used as checks, replicated in each block and randomized within the blocks. Standard organic practices were adopted, with minimal tillage to promote normal crop growth and ensure proper expression of genotypes throughout the crop season. OMRI-Certified biopesticides were used regularly to manage insects such as whiteflies.

 Phenotyping traits: Before and at physiological maturity, the following traits will be evaluated.

• Biomass Production: Measure the above-ground biomass of each accession at different growth stages (e.g., vegetative, flowering) to assess its potential as a soil cover and source of organic matter.
• Nitrogen Fixation Efficiency: Evaluate nodulation and nitrogen fixation capacity by quantifying nodule number, nodule size, and nitrogen content in plant tissues to assess the crop's contribution to soil fertility.
• Nitrogen Content: Analyze nitrogen concentration in leaves, stems, and roots to assess the legume's ability to accumulate nitrogen and redistribute it within the plant. The tissue will be oven-dried and analyzed for total TKN and free nitrates using established protocols at the Center Physiology laboratory. Soil nitrogen will be used to estimate % NUE.
• Estimate of N-15 natural abundance: Leaves from five randomly selected plants will be harvested, dried, and ground to pass a 1-mm sieve. Ground samples will be analyzed for total N and δ15N isotope contents at the Texas A&M ¹⁴Stable Isotopes for Biosphere Science Laboratory.
• Protein and non-protein amino acids: Nodulated plants accumulate ureide compounds, allantoin, and allantoic acids in the shoots. All the amino acids will be measured using Water's UPLC-MS MS and established protocols in Joshi Lab.

Sample preparation for protein and amino acid analysis: Tepary beans were collected from 143 accessions and four replicated checks at their physiological maturity. Beans were pooled from 3 to 4 plants within the accessions, and a subset of the beans was ground into a fine powder using a mortar and pestle. Two sets of 15 to 20 mg of ground powder were taken in three replicates in a 2 mL Eppendorf tube for protein and amino acid extraction. Total proteins were extracted using XX buffer, and the proteins were measured using the Bradford reagent (Deans et al., 2018). Quantification was performed by normalizing against standard curves with 0.5 mg/mL BSA as a standard. Absorbance assay is ongoing using the Varikosan LUX microplate reader, resulting in the total soluble protein concentration in the tepary bean accessions. All the amino acids will be measured using Water's UPLC-MS MS and established protocols in the Joshi Lab.

Sample preparation for leaf DNA extraction

Emerging leaf samples were collected from 189 accessions in a 2mL Eppendorf tube and stored at -80°C until further processing. Eighty to one hundred milligrams of frozen leaf tissue were homogenized into a fine powder in a Tissue Lyser using 3mm Demag stainless steel balls. Total DNA was extracted using the Qiagen DNeasy Plant Mini Kit, following the manufacturer’s protocol. The purity of the DNA was assessed using a NanoPhotometer Spectrophotometer. The University of Minnesota Genomics Center performed the construction of the DNA libraries and Illumina sequencing. For sequence analysis, low-quality reads and adapter sequences were removed from the raw fastq files using computational pipelines at the University of Minnesota Genomics Center. After trimming and removing the sequencing barcodes, only high-quality reads (score >2) with a length of 180bp will be used to align the data against the tepary reference genome, Phaseolus acutifolius v1.0. This resulted in a total of 188 samples and 132887 markers on 41228 loci.

Population structure and Genetic diversity analysis:

Population structure analysis will assess population stratification and help reduce false positive discoveries in GWAS. STRUCTURE 2.3.4 will infer population structure using SNPs associated with traits of interest identified through GWAS. GWAS will be conducted using filtered SNPs with a Bayesian Information and Linkage Disequilibrium Iteratively Nested Keyway (BLINK) model to identify candidate gene(s) based on the computed LD decay. Based on augmented design statistical analysis (R script) of the organic tepary beans, SNP markers linked to higher nutritional traits and superior accessions will be identified as deliverables. This analysis is ongoing, and the final project report will provide more detailed results.

Objective 2: Identify the best combinations of leguminous cover crops (tepary beans, Sun hemp, cowpeas) and fall cash crop spinach based on spinach productivity, nitrogen use efficiency (NUE), and soil microbiome diversity.

• Experimental design: A split-plot design is selected to accommodate six tepary bean varieties, and two varieties of regionally available cowpea and Sun hemp varieties will be grown in 4 replications. For normalization, fallow plots between the primary and control plots in each replication will be given.
• Terminating cover crops: After flowering, cover crops will be terminated by crimping down to the soil, which ceases further growth of cover crops without being completely cut down.
• Cultivating Spinach crop: After crimping/incorporating the cover crops, a window period of at least 4-8 weeks would be given for the cover crop decay process. Organic spinach seeds of 4 commercial varieties would be sourced from approved agencies. Spinach seeds will be sown in all the plots, leaving fallow plots unseeded. Regular agronomic practices will be carried out by following organic farming protocols and guidelines.
• Plant and Soil Analysis: The Solvita soil respiration system will measure soil respiration. An overall soil health assessment would consider soil pH, organic matter content, soil texture, wet aggregate stability, total carbon and nitrogen, and electrical conductivity. For plant analysis, fresh weight will be measured following oven-drying and freeze-drying for other sets of samples to determine total nutrient composition and total nitrogen.
• Bulk soil DNA extraction and Sequencing: Soils collected before cover plants sowing, after terminating cover crops, and at the end of spinach harvest will be used to extract the DNA using Qiagen Power Soil kit and will be outsourced for amplicon sequencing to identify the structure and diversity of microbial populations.                                                                                                                           

As a deliverable, this objective would assess the potential of tepary beans as a summer crop to impact soil health and organic spinach productivity.

Field design

Research plots were prepared in June 2024 at a certified organic research farm, the Texas A&M AgriLife Research station in Uvalde, Texas. The field was divided into three rows, representing three replications. Each replication plot was further divided into smaller split plots to accommodate four commercially available Tepary bean cultivars (TV1: Sacaton brown, TV2: Sonoran white, TV3: Blue speckled, TV4: Vermont Black), three Cowpea cultivars (CV1: TX001, CV2: TX002, CV3: CB46), and one Sunn hemp cultivar (SH: Crescent sunn). Control plots were randomly assigned in each replication where no cover crop was grown. Cover crop seeds were broadcast onto the designated plots and covered with a layer of soil. Cover crops were grown on the research plots until the flowering stage, approximately 40-45 days after sowing, and then incorporated into the soil using a mechanical tiller. They were allowed to decompose for 30-50 days prior to sowing the fall cash crop.

Soil chemical analysis

Soil samples were collected from each replication to analyze properties such as pH (7.7-7.9), electrical conductivity (345 µmho/cm), TKN (0.0899%), NO3 (0.0013%), and NH4 (0.0005%). A follow-up analysis was performed after the cover crop decomposition, revealing a significant increase in NH4, now at 0.335%.

Fall vegetable crops

For the following cash crop, commercially available organic seeds of spinach (Spinacia oleracea; Variety: Space) and kale were obtained from Johnny Seeds and sown with a seed-to-seed spacing of 6 to 8 inches in both the cover crop incorporated plots and the control plots. Organic agronomic practices were implemented throughout to keep the plots free from weeds and pests. Spinach was harvested at its vegetative developmental stage, approximately 65-75 days, to analyze biomass accumulation. Root and bulk soil samples were collected to examine variations in microbial diversity across three niches: the root endosphere, rhizosphere, and bulk soil. Roots from three randomly selected plants were cut and stored in a Ziplock bag at –80°C until further processing. Similarly, bulk soil samples were collected from three soil cores, and a pooled representative sample was stored in 50mL Eppendorf tubes at –80°C. This project is ongoing, and a brief report on microbial analysis will be submitted in the final report.