Publication Date

5-2025

Date of Final Oral Examination (Defense)

3-24-2025

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Biology

Department

Biological Sciences

Supervisory Committee Chair

Marcelo Serpe, Ph.D.

Supervisory Committee Member

Marie-Anne de Graaff, Ph.D.

Supervisory Committee Member

Leonora Bittleston, Ph.D.

Abstract

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that form symbiotic associations with the roots of over 70% of terrestrial plants. These symbioses are crucial in plant nutrient acquisition, stress tolerance, and soil health. AMF inoculum could be valuable for ecological restoration, but challenges in culturing and multiplying AMF limit their use in natural habitats. In this project, I investigated methods to characterize and culture AMF native to the sagebrush steppe. For comparison, I also grew a non-native strain of the AMF fungus Rhizophagus clarus. First, I used trap cultures to multiply AMF present in roots and field soil collected from a sagebrush habitat, and separately the R. clarus strain. I then analyzed the germination requirements of the spores generated in the trap cultures and used the germinated spores to initiate in vitro cultures. Based on next-generation sequencing analyses, the native AMF cultures included species within the Glomerales and Diversisporales orders, with the most common species in the Rhizophagus, Claroideoglomus, Dominikia, and Otospora genera. Both native spores and those of R. clarus were dormant and required a minimum of fifteen weeks of cold storage before any germination occurred. After this period, germination in vitro was much higher in R. clarus than in the native spores, at 29.4% and 2%, respectively. Surface sterilization of native spores, while not affecting their viability, appeared to be a factor that contributed to their low germination in vitro, suggesting that biotic components may play a role in breaking spore dormancy. Using an approximately equal number of R. clarus and native germinated spores, I attempted to establish in vitro cultures. On sixteen occasions, I established in vitro cultures of R. clarus using Ri T-DNA carrot roots as a host. Also, I cultured this fungus asymbiotically in a medium with more complex organic additives. In contrast, in vitro culturing of native AMF was more difficult. Most germinated spores ceased growing before associating with the Ri T-DNA carrot roots. Others developed hyphal networks without secondary spore production, and a few are developing hyphal networks and spores very slowly, which may limit their utility for AMF multiplication. Because the native spores included taxa from various families and two orders, the results suggest that their deeper dormancy and more restricted hyphal growth were unrelated to taxonomy. Instead, these limitations may reflect characteristics that evolved independently in response to the local environment. Overall, the methods tested proved partially successful in establishing AMF in vitro. However, for native spores, identifying factors that promote aseptic spore germination and enhance hyphal growth and secondary spore formation seems necessary to make the procedure more practical and reproducible.

Comments

https://orcid.org/0009-0002-3200-1406

DOI

10.18122/td.2382.boisestate

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