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. 2020 Jul 22;15(7):e0236135.
doi: 10.1371/journal.pone.0236135. eCollection 2020.

Fungi, bacteria and oomycota opportunistically isolated from the seagrass, Zostera marina

Cassandra L Ettinger  1   2 , Jonathan A Eisen  1   2   3
Affiliations

Fungi, bacteria and oomycota opportunistically isolated from the seagrass, Zostera marina

Cassandra L Ettinger et al. PLoS One. .

Erratum in

Abstract

Fungi in the marine environment are often neglected as a research topic, despite that fungi having critical roles on land as decomposers, pathogens or endophytes. Here we used culture-dependent methods to survey the fungi associated with the seagrass, Zostera marina, also obtaining bacteria and oomycete isolates in the process. A total of 108 fungi, 40 bacteria and 2 oomycetes were isolated. These isolates were then taxonomically identified using a combination of molecular and phylogenetic methods. The majority of the fungal isolates were classified as belonging to the classes Eurotiomycetes, Dothideomycetes, and Sordariomycetes. Most fungal isolates were habitat generalists like Penicillium sp. and Cladosporium sp., but we also cultured a diverse set of rare taxa including possible habitat specialists like Colletotrichum sp. which may preferentially associate with Z. marina leaf tissue. Although the bulk of bacterial isolates were identified as being from known ubiquitous marine lineages, we also obtained several Actinomycetes isolates and a Phyllobacterium sp. We identified two oomycetes, another understudied group of marine microbial eukaryotes, as Halophytophthora sp. which may be opportunistic pathogens or saprophytes of Z. marina. Overall, this study generates a culture collection of fungi which adds to knowledge of Z. marina associated fungi and highlights a need for more investigation into the functional and evolutionary roles of microbial eukaryotes associated with seagrasses.

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Conflict of interest statement

JAE is on the Scientific Advisory Board of Zymo Research, Inc. CLE declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Microbes isolated from the seagrass, Zostera marina.
An example of the morphological diversity of microbial isolates (bacteria, fungi and oomycota) associated with the seagrass, Z. marina. Depicted plates were arbitrarily chosen to depict the morphological diversity of the isolates cultured in this study. Putative taxonomy of isolates shown: (a) Penicillium sp. CLE73, (b) Cladosporium sp. CLE116, (c) Colletotrichum sp. CLE5, (d) Hypocreales sp. CLE105, (e) unidentified microorganism, (f) Penicillium sp. CLE130, (g) Penicillium sp. CLE68, (h) Halophytophthora sp. CLE94, (i) Pleosporales sp CLE57, (j) unidentified microorganism, (k) Pleosporales sp. CLE102, (l) Penicillium sp. CLE26, (m) Cladosporium sp. CLE118, (n) Ramularia sp. CLE122, (o) Pseudoalteromonas sp. CLE126, (p) Talaromyces sp. CLE92, (q) Colletotrichum sp. CLE4, (r) Talaromyces sp. CLE82, (s) unidentified microorganism, (t) Acrostalagmus sp. CLE7, (u) Ramularia sp. CLE1, (v) Pleosporales sp. CLE56, (w) Penicillium sp. CLE77, (x) Ramularia sp. CLE112, (y) Penicillium sp. CLE106, (z) unidentified microorganism, (aa) Streptomyces sp. CLE117, (ab) Penicillium sp. CLE114, (ac) Cladosporium sp. CLE127, and (ad) Penicillium sp. CLE110. Unidentified microorganisms were unable to be identified using molecular methods (i.e. a PCR product was not successfully generated).
Fig 2
Fig 2. Distribution of counts of fungal isolates across isolation sources.
A histogram representing the number of fungal isolates grouped by order and colored by isolation source (leaf, rhizome, root, seawater or sediment). The numbers included on each bar represent the count of isolates obtained from that particular isolation source.
Fig 3
Fig 3. Phylogenetic placement of seagrass fungal isolates in the Basidiomycota and Mucoromycota.
A molecular phylogeny of 28S rRNA genes for isolates in the Basidiomycota and Mucoromycota was constructed using Bayesian inference. This alignment was generated using MAFFT (v. 7.402) on the CIPRES Science Gateway web server, trimmed using trimAl (v.1.2) and a phylogenetic tree was inferred on the trimmed alignment with a GTR + I + G model using MrBayes (v. 3.2.2) [–77, 81]. Displayed at each node as a circle in the tree are the Bayesian posterior probabilities (e.g. a black circle represents probabilities greater or equal to 90%, a grey circle represents probabilities greater or equal to 70%, a white circle represents probabilities less than 70%). The names of fungi isolated from Z. marina are shown in green, fungi isolated from other seagrass species are shown in black, and all other fungi are shown in grey. For visualization purposes, selected clades have been collapsed and the number of sequences within that clade is indicated. Collapsed clades are shown in green if the majority of sequences in the clade are from isolates associated with Z. marina, black if the majority of isolates are from other seagrass species, and grey otherwise. Clade names that are followed by an asterisk contain sequences from multiple sources. An expanded version of this phylogeny can be found in S7 Fig. The GenBank accession numbers of the sequences used to build this phylogeny can be found in Tables 1 and S2–S4.
Fig 4
Fig 4. Phylogenetic placement of seagrass fungal isolates in the Eurotiomycetes.
A molecular phylogeny of 28S rRNA genes for isolates in the Eurotiomycetes was constructed using Bayesian inference. This alignment was generated using MAFFT (v. 7.402) on the CIPRES Science Gateway web server, trimmed using trimAl (v.1.2) and a phylogenetic tree was inferred on the trimmed alignment with a GTR + I + G model using MrBayes (v. 3.2.2) [–77, 81]. Displayed at each node as a circle in the tree are the Bayesian posterior probabilities (e.g. a black circle represents probabilities greater or equal to 90%, a grey circle represents probabilities greater or equal to 70%, a white circle represents probabilities less than 70%). The names of fungi isolated from Z. marina are shown in green, fungi isolated from other seagrass species are shown in black, and all other fungi are shown in grey. For visualization purposes, selected clades have been collapsed and the number of sequences within that clade is indicated. Collapsed clades are shown in green if the majority of sequences in the clade are from isolates associated with Z. marina, black if the majority of isolates are from other seagrass species, and grey otherwise. Clade names that are followed by an asterisk contain sequences from multiple sources. An expanded version of this phylogeny can be found in S8 Fig. The GenBank accession numbers of the sequences used to build this phylogeny can be found in Tables 1 and S2–S4.
Fig 5
Fig 5. Phylogenetic placement of seagrass fungal isolates in the Sordariomycetes.
A molecular phylogeny of 28S rRNA genes for isolates in the Sordariomycetes was constructed using Bayesian inference. This alignment was generated using MAFFT (v. 7.402) on the CIPRES Science Gateway web server, trimmed using trimAl (v.1.2) and a phylogenetic tree was inferred on the trimmed alignment with a GTR + I + G model using MrBayes (v. 3.2.2) [–77, 81]. Displayed at each node as a circle in the tree are the Bayesian posterior probabilities (e.g. a black circle represents probabilities greater or equal to 90%, a grey circle represents probabilities greater or equal to 70%, a white circle represents probabilities less than 70%). The names of fungi isolated from Z. marina are shown in green, fungi isolated from other seagrass species are shown in black, and all other fungi are shown in grey. For visualization purposes, selected clades have been collapsed and the number of sequences within that clade is indicated. Collapsed clades are shown in green if the majority of sequences in the clade are from isolates associated with Z. marina, black if the majority of isolates are from other seagrass species, and grey otherwise. Clade names that are followed by an asterisk contain sequences from multiple sources. An expanded version of this phylogeny can be found in S9 Fig. The GenBank accession numbers of the sequences used to build this phylogeny can be found in Tables 1 and S2–S4.
Fig 6
Fig 6. Phylogenetic placement of seagrass fungal isolates in the Dothideomycetes.
A molecular phylogeny of 28S rRNA genes for isolates in the Dothideomycetes was constructed using Bayesian inference. This alignment was generated using MAFFT (v. 7.402) on the CIPRES Science Gateway web server, trimmed using trimAl (v.1.2) and a phylogenetic tree was inferred on the trimmed alignment with a GTR + I + G model using MrBayes (v. 3.2.2) [–77, 81]. Displayed at each node as a circle in the tree are the Bayesian posterior probabilities (e.g. a black circle represents probabilities greater or equal to 90%, a grey circle represents probabilities greater or equal to 70%, a white circle represents probabilities less than 70%). The names of fungi isolated from Z. marina are shown in green, fungi isolated from other seagrass species are shown in black, and all other fungi are shown in grey. For visualization purposes, selected clades have been collapsed and the number of sequences within that clade is indicated. Collapsed clades are shown in green if the majority of sequences in the clade are from isolates associated with Z. marina, black if the majority of isolates are from other seagrass species, and grey otherwise. Clade names that are followed by an asterisk contain sequences from multiple sources. An expanded version of this phylogeny can be found in S10 Fig. The GenBank accession numbers of the sequences used to build this phylogeny can be found in Tables 1 and S2–S4.
Fig 7
Fig 7. Comparison of the detection of a fungal genus across methods.
A heatmap representing a comparison of the detection of the presence / absence of fungal genera isolated in this study (using a culture-dependent method) and fungal genera identified in high throughput sequencing data from Ettinger & Eisen [43] (using a culture-independent method). For each fungal genera, we visualize if it was not detected (light grey), detected using only the culture-dependent method (medium grey), detected using only the culture-independent method (dark grey) or detected by both methods (black) for each sample type / isolation source (leaf, root, rhizome, sediment).
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This work was supported by grants from the UC Davis H. A. Lewin Family Fellowship and the UC Davis Center for Population Biology to CLE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.