Background. Eelgrass (Zostera marina) is a marine angiosperm and foundation species that plays an important ecological role in primary production, food web support, and elemental cycling in coastal ecosystems. As with other plants, the microbial communities living in, on, and near eelgrass are thought to be intimately connected to the ecology and biology of eelgrass. Here we characterized the microbial communities in eelgrass sediments throughout an experiment to quantify the rate of ammonification, the first step in early remineralization of organic matter, or diagenesis, from plots at a field site in Bodega Bay, CA.
Methods. Sediment was collected from 72 plots from a 15 month long field experiment in which eelgrass genotypic richness and relatedness were manipulated. In the laboratory, we placed sediment samples (n= 4 per plot) under a N2 atmosphere, incubated them at in situ temperatures (15 oC) and sampled them initially and after 4, 7, 13, and 19 days to determine the ammonification rate. Comparative microbiome analysis using high throughput sequencing of 16S rRNA genes was performed on sediment samples taken initially and at 7, 13 and 19 days to characterize the relative abundances of microbial taxa and how they changed throughout early diagenesis.
Results. Within-sample diversity of the sediment microbial communities across all plots decreased after the initial timepoint using both richness based (observed number of OTUs, Chao1) and richness and evenness based diversity metrics (Shannon, Inverse Simpson). Additionally, microbial community composition changed across the different timepoints. Many of the observed changes in relative abundance of taxonomic groups between timepoints appeared driven by sulfur cycling with observed decreases in sulfur reducers (Desulfobacterales) and corresponding increases in sulfide oxidizers (Alteromonadales and Thiotrichales). None of these changes in composition or richness were associated with ammonification rates.
Discussion. Overall, our results showed that the microbiome of sediment from different plots followed similar successional patterns, which we surmise to be due to changes related to sulfur metabolism. These large changes likely overwhelmed any potential changes in sediment microbiome related to ammonification rate. We found no relationship between eelgrass presence or genetic composition and the microbiome. This was likely due to our sampling of bulk sediments to measure ammonification rates rather than sampling microbes in sediment directly in contact with the plants and suggests that eelgrass influence on the sediment microbiome may be limited in spatial extent. More in-depth functional studies associated with eelgrass microbiome will be required in order to fully understand the implications of these microbial communities in broader host-plant and ecosystem functions (e.g. elemental cycling and eelgrass-microbe interactions).
tl;dr – The microbes (bacteria) on plant parts (root, leaf) and near-by sediment were different from each other. We did not find a difference between the microbes on eelgrass leaves or roots at the edge of a patch versus the middle of the patch. However, the microbes in sediments from different locations in the patch (middle, edge, outside of the patch) differed and these differences correlated with eelgrass density.
Zostera marina (also known as eelgrass) is a foundation species in coastal and marine ecosystems worldwide and is a model for studies of seagrasses (a paraphyletic group in the order Alismatales) that include all the known fully submerged marine angiosperms. In recent years, there has been a growing appreciation of the potential importance of the microbial communities (i.e., microbiomes) associated with various plant species. Here we report a study of variation in Z. marina microbiomes from a field site in Bodega Bay, CA.
We characterized and then compared the microbial communities of root, leaf and sediment samples (using 16S ribosomal RNA gene PCR and sequencing) and associated environmental parameters from the inside, edge and outside of a single subtidal Z. marina patch. Multiple comparative approaches were used to examine associations between microbiome features (e.g., diversity, taxonomic composition) and environmental parameters and to compare sample types and sites.
Microbial communities differed significantly between sample types (root, leaf and sediment) and in sediments from different sites (inside, edge, outside). Carbon:Nitrogen ratio and eelgrass density were both significantly correlated to sediment community composition. Enrichment of certain taxonomic groups in each sample type was detected and analyzed in regard to possible functional implications (especially regarding sulfur metabolism).
Our results are mostly consistent with prior work on seagrass associated microbiomes with a few differences and additional findings. From a functional point of view, the most significant finding is that many of the taxa that differ significantly between sample types and sites are closely related to ones commonly associated with various aspects of sulfur and nitrogen metabolism. Though not a traditional model organism, we believe that Z. marina can become a model for studies of marine plant-microbiome interactions.
Plant-associated microorganisms are essential for their hosts’ survival and performance. Yet, most plant microbiome studies to date have focused on terrestrial species sampled across relatively small spatial scales. Here we report results of a global-scale analysis of microbial communities associated with leaf and root surfaces of the marine eelgrass Zostera marina throughout its range in the Northern Hemisphere. By contrasting host microbiomes with those of surrounding seawater and sediment, we uncovered the structure, composition and variability of microbial communities associated with eelgrass. We also investigated hypotheses about the assembly of the eelgrass microbiome using a metabolic modeling approach. Our results reveal leaf communities displaying high variability and spatial turnover, that mirror their adjacent coastal seawater microbiomes. In contrast, roots showed relatively low compositional turnover and were distinct from surrounding sediment communities — a result driven by the enrichment of predicted sulfur-oxidizing bacterial taxa on root surfaces. Predictions from metabolic modeling of enriched taxa were consistent with a habitat filtering community assembly mechanism whereby similarity in resource use drives taxonomic co-occurrence patterns on belowground, but not aboveground, host tissues. Our work provides evidence for a core eelgrass root microbiome with putative functional roles and highlights potentially disparate processes influencing microbial community assembly on different plant compartments.
IMPORTANCE Plants depend critically on their associated microbiome, yet the structure of microbial communities found on marine plants remains poorly understood in comparison to terrestrial species. Seagrasses are the only flowering plants that live entirely in marine environments. The return of terrestrial seagrass ancestors to oceans is among the most extreme habitat shifts documented in plants, making them an ideal test bed for the study of microbial symbioses with plants that experience relatively harsh abiotic conditions. In this study, we report results of a global sampling effort to extensively characterize the structure of microbial communities associated with the widespread seagrass species, Zostera marina or eelgrass, across its geographic range. Our results reveal major differences in the structure and composition of above- versus belowground microbial communities on eelgrass surfaces, as well as their relationships with the environment and host.
Plant-associated microorganisms are essential for their hosts’ survival and performance. Yet, most plant microbiome studies to date have focused on terrestrial species sampled across relatively small spatial scales. Here we report results of a global-scale analysis of microbial communities associated with leaf and root surfaces of the marine eelgrass Zostera marina throughout its range in the Northern Hemisphere. By contrasting host microbiomes with those of their surrounding seawater and sediment communities, we uncovered the structure, composition and variability of microbial communities associated with Z. marina. We also investigated hypotheses about the mechanisms driving assembly of the eelgrass microbiome using a whole-genomic metabolic modeling approach. Our results reveal aboveground leaf communities displaying high variability and spatial turnover, that strongly mirror their adjacent coastal seawater microbiomes. In contrast, roots showed relatively low spatial turnover and were compositionally distinct from surrounding sediment communities – a result driven by the enrichment of predicted sulfur-oxidizing bacterial taxa on root surfaces. Metabolic modeling of enriched taxa was consistent with an assembly process whereby similarity in resource use drives taxonomic co-occurrence patterns on belowground, but not aboveground, host tissues. Our work provides evidence for a core Z. marina root microbiome with putative functional roles and highlights potentially disparate processes influencing microbiome assembly on different plant compartments.
So for the last couple of years I’ve supervised a series of undergraduates who have spent some time isolating bacteria from seagrass samples… sometimes from the plants themselves and sometimes from associated sediment. We usually used non-specific aerobic media such as Marine Broth and Seawater Nutrient Agar. The result has been a series of the usual suspects; Vibrio, Shewanella, Pseudoalteromonas, etc. We’ve sequenced a number of these genomes, examples of Genome Announcements papers like this can be found here, here, here, here, here,here and here. The goals of this culturing were three-fold; cool undergraduate projects (check), add seagrass-associated genome data to the database to aid in metagenomics and such (check), and to characterize isolates that might be important in seagrass biology (unknown).
In regards to the last goal, we’ve attempted to use abundance of taxa as one rough proxy of “importance” and significant changes within an experiment as another. We have several large 16S projects to work with, but in most cases the isolates that we have generated are not found at particular abundance or significance in these datasets. And perhaps that’s not surprising, non-specific media is good at isolating widely distributed generalists.
So now we plan to approach the problem from the opposite direction, we’re picking the OTUs from our 16S data that are the most interesting and attempting to selectively culture them. #1 on our hit list is Sulfurimonas which crops up over and over again.
Recently the Seagrass Microbiome group has been wrapped up in sending (and receiving!) microbiome sampling kits. These kits are part of a larger collaborative project focused on re-sequencing of Zostera marina samples in conjunction with sequencing of additional marine and freshwater Alismatid species and their microbiomes. JGI recently sequenced and released the Zostera marina genome, and we are hoping to build on their efforts and explore population level variation within Zostera marina, as well as differences in genome content and structure between Zostera and other Alismatids, in conjunction with microbiome sequencing.
The sampling kits sent by the seagrass microbiome group have focused on the microbial aspect of this project. We have asked members of the Zostera Experimental Network (ZEN) as well as additional collaborators to sample both plant tissue for sequencing (coordinated through Jay Stachowicz and Jeanine Olsen) and microbiome samples. We are extremely excited about this sample set, as it covers populations of Zostera marina across many different environments, for which we already have extensive metadata through the ZEN group! We are requesting root, sediment (within the rhizosphere), and leaf tissue, as detailed in the diagram below (courtesy of Jeanine Olsen).
Collaborators are also sampling at two depths per site (deep and shallow), so that we can examine microbiome differences that may correlate with population depth. We are sampling 24 individuals per site, 12 per depth.
So – am participating in a workshop, supported by CIFAR and the Gordon and Betty Moore Foundation over the next few days on “Marine Algal and Plant Microbiomes”. The workshop is basically trying to come up with a white paper / position paper on the future of such studies and to continue the conversation about this topic afterwards. We are asking questions like
What are the challenges and opportunities in this area?
What are the major scientific questions?
How are such systems different from fresh water or terrestrial systems?
How are they different?
How are marine systems involving other hosts (e.g., coral, sponges, dolphins) comparable (i.e., is there something about marine systems that links them together in any way).
What tools and resources could help advance work in this area?
So I am posting here asking for a few bits of information from any readers.
Are you interested in participating in follow up discussions on this topic?
Do you know of any people we should try to bring into the conversation even if they are not, well, you?
Are there any major projects in this area that would be worth engaging?
Any thoughts (on the topic that is) would be welcome.
My name is Karley Lujan and I am an undergraduate working on culturing bacterial isolates from the Seagrass microbiome. I joined this project because I am interested in learning about what information we can obtain from studying microbiomes. I think it is fascinating that although we can’t see microorganisms they are extremely prevalent and can have crucial roles in biological systems. The focus at the beginning of this project was to take Seagrass samples from Bodega Bay, create culture samples, and use Sanger sequencing of the 16S rRNA to identify what we grew. Seagrass and sediment samples were taken from Bodega Bay, CA. Then, in order to obtain isolates from the seagrass, we focused on the leaves, roots, and sediment. What we were able to successfully extract DNA from were identified as Shewanella, Pseudoalteromonas, Colwellia, Tenacibaculum, Vibrio and Alteromonas.
Sample preparation: Dilutions of sediment with PBS, PBS rinse of roots and leaves, ground and crushed leaves with PBS
Culturing: Plated the PBS sample solutions onto two of each of the following plate types; one plate for 25℃ and the other at 4℃
Agar Plates/Liquid Media
Difco Marine Broth
Selected Colonies: After there was significant growth on the plates we selected various interesting colonies and isolated them by dilution streaking. Single colonies were then grown overnight in the appropriate liquid media and at the appropriate temperature
DNA Extraction: Genomic DNA extractions were performed and glycerol stocks were made using the successful liquid cultures. Extracted DNA then went through 16S rRNA gene PCR and gel electrophoresis in order to confirm that enough DNA was present for Sanger Sequencing
Sanger Sequencing: 16S rRNA sequences for each isolate were ran through BLAST and phylogenetic trees were built in order to obtain tentative identifications for the isolates
Results: After Sanger sequencing, the data was ran through BLAST to obtain a tentative identification and determine whether or not the microbe was a good candidate for sequencing.
Shewanella:Electrogenic- An electron generator that can be used in microbial fuel cells.
Vibrio:Some species of Vibrio can go through morphogenetic changes after going from a liquid to a solid surface. This leads them to change from swimmer cells to swarmer cells.
Pseudomonas:Two bacterial isolates were cable of growing on Nitrogen-Free agar plates at 25⁰C. Identified as part of the genera Pseudomonas, there are some species of this genera capable of aerobically fixing nitrogen. These are of particular interest as we will be further investigating which nitrogen-fixing bacteria are essential for seagrass health.
Currently I am beginning to look at the genomes of the bacteria we decided to sequence and I am also working with bacteria that are capable of growing on the nitrogen-free agar plates. At first it was difficult to extract the DNA from these bacteria but now both have been tentatively identified as Pseudomonas through sanger sequencing of the 16S gene. This is interesting because there aren’t many Pseudomonas that can fix nitrogen which is what these two must be doing in order to survive on the nitrogen-free plates. These two bacteria also have different morphologies which means they could be different species in the genus Pseudomonas. Due to their morphological similarities yet ability to grow on nitrogen-free agar, I think these two bacteria are very interesting and we will be finding out more about them by sequencing and analyzing their genomes.
A paper on the genome of Z. marina was released early this year. This is the first marine angiosperm genome to be sequenced, and since it’s our main host organism, we are fairly excited. This opens up a couple research possibilities, like studying host-microbe coevolution between Z. marina and its microbiome. We can also use the plant reference genome with RNA-seq data to filter out reads from the microbes, which will make it easier to look at the gene functions represented in the seagrass microbiome. Having a reference genome for our host will definitely come in handy, and has opened some exciting new doors for us.
Some of us at team Seagrass participated in a workshop on SEM (scanning electron microscopy) sponsored by the Electron Microscopy Lab at UC Davis. For this week only Hitachi Tabletop SEM was made available to researchers to test out. SEM is a great tool to produce high resolution images, especially for samples for which preparation techniques would otherwise alter the sample. We decided this would be a great opportunity to visually explore microbial diversity on seagrass roots/leaves/rhizomes. The amount of sample and preparation is minimal relative to other techniques. We’ve been particularly concerned with our previous FISH images, as FISH requires many washes, and may be removing microbes from the surface of our samples.
We put a minimal amount of sample of root (top), fresh leaf (right), decaying leaf (bottom), and rhizome cross section (left) on a piece of carbon conductive paper (sticky on both sides), which was stuck on top of the SEM sample mount. We then inserted the mount into the SEM and turned on the vacuum. The vacuum for this particular SEM is actually a partial vacuum, which maintains a small amount of air molecules within the chamber, which produces a better image in the absence of coating your sample with conductors.
The results were great! We were able to see a lot of diatoms spread out across the leaf surface, as well as some interesting plaque (?) formations on the root tips. We also noticed differences in diatom abundance between the live and decaying leaves, with the live leaf being completely covered with diatoms. Diatoms are marine microbial eukaryotes (Heterokonts) that form silica based outer layers, often resembling complex geometric patterns. Diatom assemblages have previously been characterized in Thalassia testudinum and in Zostera marina. We’re excited to use the SEM to explore microbial diversity on additional seagrass species and freshwater relatives within the Alismatales. Hopefully these results can help inform our future culturing experiments.