Document Type
Thesis - Open Access
Award Date
2025
Degree Name
Master of Science (MS)
Department / School
Biology and Microbiology
First Advisor
Senthil Subramanian
Abstract
Pea (Pisum sativum L.) is a legume, cultivated on over 25 million acres, particularly in Canada, France, China, Russia, and the United States. Its seeds harbor microbiota including bacteria and fungi, either on the surface (epiphytes) or within seed tissues (endophytes). These microbes can be pathogenic or beneficial, enhancing seedling metabolism and protection from environmental stresses. Characterizing beneficial seed microbiota enables development of microbial inoculants for seed treatment. Seeds from 12 varieties (green or yellow seed coats) were harvested from Sturgis and Dakota Lakes in 2021. Epiphytes were extracted using sonication followed by grinding the seeds to obtain endophytes. Isolates were grown on TSA and R2A media, yielding 221 epiphytes and 129 endophytes, distinguished by colony characteristics. Epiphytes were typically slimy, endophytes, sticky or brittle. Student t-test indicated that field pea variety significantly influenced (p < 0.05) the diversity of both epiphytes and endophytes across the two sites. Factorial ANOVA revealed green-seeded varieties had more endophytes, while yellow-seeded ones had more epiphytes. R2A yielded significantly more isolates than TSA (p < 0.05), emphasizing the influence of plant variety, site, and media on microbiota structure. Isolates were evaluated for IAA production; Of those, 43 isolates (31 epiphytes and 12 endophytes) exhibiting high IAA production (≥ 10 µg/mL/hr) were screened for plant growth-promoting traits. These included 22 Gram-positive and 21 Gram-negative bacteria. DNA extraction and 16S rRNA sequencing identified the 43 isolates with Bacillus (42%), Pantoea (14%), and Acinetobacter (9%) as predominant genera. Key findings included high auxin production by isolates Ep 218 (Stenotrophomonas, ~24 µg/mL/hr) and En 1067 (Bacillus, ~26.09 µg/mL/hr); phosphate solubilization indices of 4.2 (Ep 120, Pseudomonas) and 4.3 (En 1063, Acinetobacter); siderophore units of 71% (En 1066, Acinetobacter) and 69% (Ep 130, Bacillus); and notable colony size growth in nitrogen-free media by Ep 173 (Pantoea, 1.66 mm diameter) and En 1025 (Acinetobacter, 1.5 mm). Out of the 43, fifteen isolates (11 epiphytes and 4 endophytes) identified in this study, representatives from Acinetobacter, Bacillus, Paenibacillus, Stenotrophomonas, and Pantoea demonstrated positive results across all four assays, highlighting their potential as bio-stimulants and biofertilizers to enhance seed germination and seedling health.
Publisher
South Dakota State University
Recommended Citation
Njuguna, Diana Wairimu, "Isolation, Characterization & Identification of Cultivable Field Pea Seed Bacteria in South Dakota" (2025). Electronic Theses and Dissertations. 1499.
https://openprairie.sdstate.edu/etd2/1499
Rights Statement
