1 Metagenomics and metatranscriptomics of the leaf- and root-associated microbiomes of Zostera marina and Zostera japonica by John Michael Adrian Wojahn A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Microbiology and Biology (Honors BS) Presented May 10, 2016 Commencement June 2016 2 3 AN ABSTRACT OF THE THESIS OF John M. A. Wojahn for the degree of Honors Baccalaureate of Science in Microbiology and Biology presented on May 10, 2016. Title: Metagenomics and Metatranscriptomics of the leaf- and root-associated microbiomes of Zostera marina and Zostera japonica . Abstract approved: _____________________________________________ Byron C. Crump A great deal of research has been focused on the microbiomes of terrestrial angiosperms (flowering plants), but much less research has been performed on the microbiomes of aquatic angiosperms (Turner et al. 2013). Eelgrass beds are extremely productive ecosystems that provide habitat for many marine organisms, such as fish, shelfish, crabs, and algae (Smith et al. 1988). Eelgrass beds contribute to storm surge damping (Spalding et al. 2009), nutrient cycling (Smith et al. 1988), and water clarification (Orth et al. 2006). We examined the metagenomics and metatranscriptomics of the leaf- and root- associated microbiomes of Zostera marina and Zostera japonica. In our study, the phylogenetic composition of plant-associated bacterial communities was not significantly different between plant species for leaf communities (ANOSIM P<0.199) and for root communities (ANOSIM P<0.091). However, leaf-, root-, and water column associated bacterial communities were significantly different from one another (ANOSIM, P<0.001). We found taxa present on leaves that are capable of metabolizing methanol and of producing agarases that cause disease and die-offs in populations of competitive red seaweed, and of producing indoleacetate, a plant hormone. Members of genus Granulosicoccus were found to be particularly abundant in our leaf samples. We also found taxa present on the roots that are capable of metabolizing sulfur compounds, of fixing nitrogen, and of degrading methanol. Key Words: seagrass, Netarts, Oregon, estuary, ocean, microbiology, molecular biology Corresponding e-mail address: [email protected] 4 ©Copyright by John M. A. Wojahn 2016 All Rights Reserved 5 Metagenomics and metatranscriptomics of the leaf- and root-associated microbiomes of Zostera marina and Zostera japonica by John Michael Adrian Wojahn A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Microbiology and Biology (Honors BS) Presented May 10, 2016 Commencement June 2016 6 Honors Baccalaureate of Science in Microbiology and Biology project of John M. A. Wojahn presented on 10 May 2016. APPROVED: Byron C Crump, Mentor, representing the College of Earth, Ocean, and Atmospheric Science Fiona Tomas Nash, Committee Member, representing the Department of Fisheries and Wildlife Ryan Mueller, Committee Member, representing the Department of Microbiology 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. John M. A. Wojahn, Author 7 Introduction A great deal of research has been focused on the microbiomes of terrestrial angiosperms (flowering plants), but much less research has been performed on the microbiomes (organism-associated microbes) of aquatic angiosperms (Turner et al. 2013). Bacteria associated with terrestrial plant leaves (phyllosphere microbiome) and roots (rhizosphere microbiome) are known to have direct impacts on the health of plants (Bakker et al. 2012). Positive effects of these microbiomes include outcompeting pathogenic soil microbes, modulating plant immunity by priming their defenses for an imminent infection, rendering nitrogen from the air usable to plants (nitrogen fixation) (Galloway et al. 2008), and neutralizing harmful products (e.g. methanol) exuded from leaves (Turner et al. 2013; Abanda-Nkpwatt et al 2006). Rhizosphere microbiomes tend to be very diverse and mainly derived from soil microbiota (Triplett et al. 2002). In contrast, phyllosphere microbiomes are much less diverse and are very different than microbes in the air (Sainis, 2012). The structure of rhizosphere microbiomes is primarily determined by the combination of chemicals exuded by the plant roots (Haichar et al. 2008), whereas the structure of phyllosphere microbiomes is determined by abiotic environmental factors, such as precipitation and light exposure (Turner et al. 2013). We believe a similar contrast likely exists for the microbiomes of submersed aquatic plants, but the factors controlling microbiome community composition and structure may be very different than those of terrestrial plants. Aquatic plant leaves are often submerged in water, and their roots are anchored in saturated sediments that are often anoxic at rhizosphere depths. Seagrasses, marine flowering plants, form the base of extense and productive ecosystems that provide habitat for many marine organisms, such as fish, shelfish, crabs, and algae (Smith et al. 1988). Eelgrass beds contribute to storm surge damping (Spalding et al. 2009), nutrient cycling (Smith et al. 1988), and water clarification (Orth et al. 2006). Also, decomposition of eelgrass detritus fuels a variety of food webs, both local and distal to the beds (Ralph et al. 2002). During the 1920s and 1930s, a large die-off of eelgrass caused by the fungus Labyrinthula macrocystis occurred across much of North America and Europe, leading to the destruction of many eelgrass beds (Ralph et al. 2002). Many estuaries never fully recovered from this die-off. Eelgrass beds have also been lost to human activity, such as chemical contamination, eutrophication, and physical disturbance (Ralph et al. 2002), and are now considered 'canaries in the coal mine' for climate change and associated global and regional changes, such as ocean acidification, warming, or sea-level rise (Boudouresque et al. 2009, Carr et al. 2012). Seagrass beds on the pacific coast of the US were once dominated by Zostera marina, which is a species of eelgrass native to Northern Eurasia and North America. However, since 1957, an invasive species of eelgrass from Japan, Zostera japonica, established itself along the West coast of North America (Lovvorn et al. 1994) (See Figure 1 for a photo of a typical specimens of Z. marina and Z. japonica). Z. japonica has smaller, thinner leaves than Z. marina (<20 cm long and 2 mm wide vs. 50-120 cm long and 12 mm wide (Lovvorn et al. 1994)). Although both species can co-occur, they typically exhibit different reproductive strategies and habitat use. Z. japonica usually 8 colonizes mid to low intertidal mudflats that have been stripped of all vegetation by winter storms, and in autumn, before the mother plants are killed by winter storms, they release large quantities of seeds, some of which will survive to colonize the newly denuded mud the following year (Harrison, 1982). In contrast, Z. marina generally colonizes low to subtidal areas of the mudflats and very rarely flowers, exhibiting both sexual and asexual (vegetative growth of shoots) reproduction, usually maintaining perennial populations (Davidson et al. 1998). Eelgrass-associated microbiomes may serve important roles in the ecosystem their hosts inhabit, including facilitating the growth and survival of their hosts through many of the same mechanisms as microbiomes of land plants (Crump et al. 2008). They may protect the plants from methanol, metabolize sulfides, fix nitrogen, and produce plant hormones. However, they may sometimes also act as phytopathogens (Short et al. 1987). These eelgrass-associated microbes could also benefit from their relationship with their hosts. For example, eelgrass, being angiosperms, may exude methanol and ethanol, which the microbes around them could use as food. The plants also provide a substrate for microbial attachment and stabilize sediments, preventing mixing and loss of sediment habitat. Eelgrass also Figure 1. Z. marina is an aquatic angiosperm that presents with 30 cm enrich the water mature blades, 5 cm hair-like roots, and a segmented rhizome. Z. japonica is smaller than Z. marina and is native to the islands of Japan. and sediment around it with dissolved oxygen. Lastly, the microbes associated with the eelgrass may provide energy and substrate for higher trophic levels, since 99.8% of their mass is lost each growing season (Tornblom 1999). Koch et al. (2001) determined that sulfide is toxic to the eelgrass Thalassia testidium under high salinity and high temperature, and Goodman et al. (1995) found that the phytotoxicity of sulfide was highest under low light conditions. This agrees with Baden et al. (2003), who found that seagrass loss was highest in turbid waters that carry higher concentrations of sulfide. Jensen et al. (2007) found that microbial communities associated with roots of a tropical seagrass were dominated by the classes Epsilonproteobacteria and Gammaproteobacteria, both of which include many organisms capable of oxidizing sulfide. The dominance of these classes suggests rapid metabolism of sulfide, which would benefit plants by removing this toxic molecule. 9 Jensen et