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####**Aim 3: Establish culture collection of microbial eukaryotes associated with _Zostera marina_ in Bodega Bay, California.**  Microeuks diverse, multiple groups, marine in every lineage. We will focus on x, y, z because XXX. However, will adjust depending on sequence data. Detailed below is description on how we will culture a subset of these based on previous experience and what we have already had success culturing (Amoeba, fungi, ciliates). We plan to expand this initial culturing effort to include representatives of all groups (list groups) as dictated by sequence data.   This summer successfully cultured amoeba from rhizosphere sediment and leaves, using X protocol.   _Culturing/Isolation_: Rhizospheric sediment as well as _Z. marina_ roots and leaves will be collected to use as isolation innoculum. A variety of culturing conditions will be used to target fungi, amoeba ...  A variety of media recipes found previously successful at isolating seagrass-associated fungi will be used here. These include potato dextrose agar, malt-dextrose agar, glucose peptone yeast agar and cornmeal agar, with all media being supplemented with salt and antibiotics \cite{Torta_2014}\cite{Vohn_k_2015}\cite{Panno_2013}. Media will also be made using Z. marina as the carbon source as was done by Torta el al \cite{Torta_2014} using the seagrass, Posidonia oceanica.  
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-modified hay infusions with seagrass for HS outreach  _Isolation/Culturing Rationale_: Microbial eukaryotes are extremely diverse, made up of multiple lineages each containing numerous marine taxa (CITE). Because of this diversity, we will initially focus isolation and culturing methods on targeted **taxa(??)** that we know exist on seagrass leaves or within the rhizosphere sediment based on microscopy. These will include diatoms, algae, amoeba, and fungi. We have previously found representatives of each of these in seagrass samples using a modified Hay Infusion, and specifically have had success isolating and culturing amoeba from leaf and sediment. Additionally, our initial sequencing results using ITS suggest high levels of fungal diversity in rhizosphere samples, so we have chosen to include them in our initial isolation and culturing efforts. Say something about diatoms and algae?   Following the acquisition of initial sequence data and microscopy we will adjust our isolation and culturing efforts to include representatives from all of the microbial eukaryotic lineages.   Detailed below is description on how we will culture a subset of these based on previous experience and what we have already had success culturing (Amoeba, fungi, ciliates).   This summer successfully cultured amoeba from rhizosphere sediment and leaves, using X protocol.  _Phenotypic and Molecular Characterization of Isolates_:  Microscopy of X-day old cultures will be used to record external morphology, and growth rate will be characterized at 10, 20, 30 deg C. Isolates will be fixed and imaged under natural light. Fungal isolates will be stained for chitin as in http://www.mycologia.org/content/104/6/1267.full.pdf+html (?) and with fungal specific rRNA probes using flourescence in situ hybridization (FISH). All non-fungal isolates will be stained for alpha tubulin in combination with FISH staining using a general eukaryotic rRNA probe as in \cite{Hirst_2011}. If necessary, a more specific rRNA probe may also be used based on the group-specific sequence obtained.