Geographic variation in the seed mycobiome of Coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) By Gillian E. Bergmann A THESIS Submitted to Oregon State University Honors College In partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in BioResource Research, Sustainable Ecosystems (Honors Scholar) Presented May 24, 2019 Commencement June 2019 AN ABSTRACT OF THE THESIS OF Gillian E. Bergmann for the degree of Honors Baccalaureate of Science in BioResource Research presented on May 24, 2019. Title: Geographic variation in the seed mycobiome of Coastal Douglas-fir (Pseudotsuga menziesii var. menziesii). Abstract Approval: ______________________________________________________________ Posy E. Busby Seeds are an essential component of plant life histories, and seed endophytes have the potential to influence germination, seedling establishment and development. That said, seed endophytes are a relatively new area of study, both in the factors that influence which taxa are present and how these microbes alter plant function. The objectives of my thesis were to characterize the fungal endophytes present in native and introduced populations of Coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) seeds, and to test whether some of these endophytes affect seedling survival and growth in response to drought. Using culture-based techniques, endophytes were isolated from eight native populations of Douglas-fir seeds in the United States and from three introduced populations in New Zealand. All seeds had zero or one fungal endophyte; total endophyte isolation frequency was 5.3% in the United States populations and 9.2% in the New Zealand populations. These results are consistent with previous work documenting a bottleneck in the plant microbiome at the seed stage. Based on DNA sequence data (ITS), the dominant taxa present in the sampled seed populations were Trichoderma spp. and Sydowia polyspora. Endophyte community composition differed among US seed provenances; future work could further investigate how these communities vary along environmental and plant genetic gradients. To test the hypothesis that endophytes confer drought tolerance, Douglas fir seedlings were inoculated with one of two isolates of seed-borne Trichoderma spp., or sterile water for controls, and grown under drought conditions. Drought reduced the survival of seedlings (i.e., increasing the time to death compared to un-inoculated controls); however, inoculation with endophytes did not significantly alter this result. Further study is needed to more fully characterize the functional roles of seed endophytes in Douglas-fir response to abiotic stresses, with applications in mitigating plant stress due to climate change. KEY WORDS: Ascomycota, endophytes, Primary Symbiont Hypothesis, provenance, Pseudotsuga menziesii, Sydowia polyspora, Trichoderma, maternal transmission Corresponding email address: [email protected] ©Copyright by Gillian E. Bergmann May 24, 2019 All Rights Reserved Geographic variation in the seed mycobiome of Coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) By Gillian E. Bergmann A THESIS Submitted to Oregon State University Honors College In partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in BioResource Research, Sustainable Ecosystems (Honors Scholar) Presented May 24, 2019 Commencement June 2019 Honors Baccalaureate of Science in BioResource Research project of Gillian E. Bergmann presented on May 24, 2019 APPROVED: Posy E. Busby, Mentor, representing Department of Botany and Plant Pathology Joseph W. Spatafora, Secondary Mentor, representing Department of Botany and Plant Pathology Katharine Field, Committee Member, representing BioResource Research Interdisciplinary Program Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. Gillian E. Bergmann, Author INTRODUCTION Plant-associated microbes are pivotal for their effects on plant survival and function, and one life stage where they could be especially important is in seeds. Of course, the primary purpose of seeds is the reproduction of plant species. Seeds provide population stability through their high genetic variability, and their ability to survive harsh environmental conditions through dormancy (Fenner and Thompson 2005). Seeds are also major agents of dispersal, which can benefit plants through reducing competition between parents and offspring, reducing density-dependent risks of pathogens and predators, and allowing species to colonize new areas (Willson and Traveset 2000). Given their importance, it is critical to note that seed germination and early seedling establishment represent a significant bottleneck in the plant life cycle (Leck et al. 2008), when young plants are vulnerable to biotic and abiotic stresses, such as pathogens, predators, and drought (Bever et al. 2015). Seed-associated microbes have been known to improve plant development at this stage (e.g. Puente et al. 2009), so understanding how seed-associated microbes contribute to seed survival and fitness is of great interest. One microbial community of particular importance is the fungal endophyte community. Endophytes, defined as microbes (fungi, bacteria, archaea, protists) that live inside plant tissues without causing disease (Rodriguez et al. 2009, Hardoim et al. 2015), are of increasing interest given their effects on plant survival and fitness. While many fungal endophytes are thought to be saprotrophs or latent pathogens, some taxa have been found to modify plant resistance to disease (Busby et al. 2016a, 2016b), herbivory (Saikkonen et al. 1998), drought (Hamilton and Bauerle 2012), and soil pollution (Regvar et al. 2010, Naik 2017). Additionally, some studies have shown that endophytes can alter nutrient acquisition (Christian et al. 2019) and fecundity (Wagner et al. 2014). Given the benefits that endophytes confer to their host plants, a major research goal in basic and applied plant sciences is to evaluate the potential to integrate endophytes into sustainable agriculture (Busby et al. 2017). Seed endophytes could be particularly vital in this area because some of them are vertically transmitted from parent to offspring (Nelson 2018), transferring endophyte effects between generations (Rosenberg et al. 2009) with potential legacy effects later in life (Aleklett and Hart 2013). However, for the vast majority of plants, we know close to nothing about the abundance and distribution of fungi in seeds, and the function of those fungi (Nelson 2018). Coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) is a common tree species in the Pacific Northwest United States, and is a major timber and nursery plant domestically (Watts et al. 2015) and internationally (ie Europe and New Zealand; Maclaren 2009, Eilmann et al. 2013). This species is ecologically critical as an early seral species in forest succession (Uchytil 1991), and is one of the first to regenerate after wildfire events (Agee 1991). P. menziesii var. menziesii also effectively sequesters carbon (Watts et al. 2015), and provides habitat for a variety of plants and wildlife over its long lifespan (Uchytil 1991). In industry, P. menziesii var. menziesii is a highly productive timber crop for its quick growth and strong wood (Uchytil 1991, Watts et al. 2015), and it is one of the most commonly used species in Christmas tree production (Kruckenbuerg and Chalker-Scott 1982). Because of its importance in forest ecosystems and plantations, there is growing concern about the impacts of drought on the species due to the climate crisis (Stewart 2015, Watts et al. 2015). Under drought conditions, Pseudotsuga menziesii seedlings experience declines in photosynthesis rates (Zavitkowski and Ferrell 1968), reduced growth (Khan et al. 1996) and xylem cavitation (Kavanagh et al. 1999). Moreover, drought events are projected to become more frequent, prolonged and severe in the future (Weiskittel et al. 2012, Watts et al. 2015), posing a major threat to the survival and productivity of this species. While it is known that certain provenances of P. menziesii var. menziesii are more drought tolerant than others (St Clair and Howe 2007, Eilmann et al. 2013), there is a need to find new tools and seed sources to help land users manage and protect this species against drought (Watts et al. 2015). While there has been some study of the foliar (e.g. Daniels 2017) and root (e.g. Hoff et al. 2004) endophyte communities of Pseudotsuga menziesii, their seed endophytes are still largely unknown. So far, only one study, published over 50 years ago, has done a cursory examination of the fungal endophytes of Pseudotsuga menziesii (Bloomberg 1966). This study reported that endophytes were present in Pseudotsuga menziesii seeds, and that there were some differences in the taxa between seed lots sampled (Bloomberg 1966). However, this study only qualitatively examined two seed lots from British Columbia, and endophyte identifications were made based on morphology. As such, a large-scale characterization effort of Pseudotsuga menziesii seed endophytes and their potential effects with molecular methods has yet to be done. Given this lack of knowledge, this exploratory study aimed to characterize the fungal endophytes of P. menziesii var. menziesii seeds using culture-based methods