Environmental Microbiology Reports (2021) 13(4), 458–463 doi:10.1111/1758-2229.12904 Brief Report Saltmarsh rhizosphere fungal communities vary by sediment type and dominant plant species cover in Nova Scotia, Canada Tyler W. d’Entremont, 1 Zoë Migicovsky,2 sediment carbon sequestration (Boesch and Juan C. López-Gutiérrez1 and Allison K. Walker 1* Turner, 1984). The term ‘blue carbon’ refers to carbon 1Department of Biology, Acadia University, Wolfville, stored in saltmarsh sinks and is greater, per unit area, Nova Scotia, Canada. than the carbon sinks of terrestrial counterparts (McLeod 2Department of Plant, Food, and Environmental et al., 2011). Despite large carbon stores, saltmarshes Sciences, Faculty of Agriculture, Dalhousie University, are declining globally, with minimal efforts targeting their Truro, Nova Scotia, Canada. conservation in Atlantic Canada (McLeod et al., 2011). Saltmarsh ecosystems in Atlantic Canada are domi- nated by two Sporobolus species in a zonal distribution. Summary Sporobolus alterniflorus (Loisel.) (Poaceae), formerly We surveyed Spartina saltmarsh sediment rhizo- Spartina alterniflora (smooth cordgrass), grows at the sphere fungal communities at three saltmarshes and tidal interface, and Sporobolus pumilus (Roth) two timepoints in coastal Nova Scotia. Based on ITS2 (Poaceae), formerly Spartina patens (saltmeadow cord- Illumina miSeq rDNA data and multivariate analysis, grass), dominates the high marsh (Bertness, 1991). neither sediment zone nor collection period corre- These grasses are adapted to growth in hypersaline sedi- lated with fungal ASV richness, but collection site ment and periodic tidal inundation. They excrete large did. However, Shannon diversity indicated that sedi- amounts of salt to regulate homeostasis, but it remains ment zone played a significant role in fungal diver- unknown how the stressors affect rhizosphere microbial sity. For unweighted and weighted UniFrac distance, communities in saltmarsh sediments. Abiotic factors such site was the major factor driving beta-diversity, with as pH, water content and nutrient loads all influence the sediment zone and collection period having smaller fungal community present at a site (Kendrick, 2017), roles. Sediment type and saltmarsh plant species which may affect coastal plant restoration success. may play important roles in structuring rhizosphere Assessing the sediment fungal diversity in different fungal assemblages, here dominated by ascomy- saltmarshes that are resistant to tidal erosion, such as cetes. To our knowledge, our study is the first to those in megatidal environments, may indicate which assess fungal sediment communities in saltmarshes species are key to a healthy ecosystem. in Atlantic Canada using metabarcoding. It provides To our knowledge, no eDNA studies of fungal assem- a biodiversity analysis of sediment fungi in a poorly blages in saltmarsh sediments have been done in Atlan- studied but highly important ecosystem and points to tic Canada. Saltmarsh sediment fungal communities their roles in nutrient cycling, blue carbon, coastal have been studied in southeastern Louisiana, USA, stability and coastal restoration. Our work will inform focusing on rhizosphere community shifts after the 2010 ongoing saltmarsh restoration in Atlantic Canada. Deepwater Horizon oil spill (Lumibao et al., 2018). Fungi associated with North American saltmarsh Sporobolus Introduction include arbuscular mycorrhizal symbiont (AMF) Funneliformis geosporum (Wilde et al., 2009; d’En- Saltmarshes are critical to sustainability of intertidal eco- tremont et al., 2018), the primary shoot decomposers systems; due to their highly productive vegetation and such as Phaeosphaeria spartinicola (Buchan et al., 2002; Walker and Campbell, 2010), and Fusarium pathogens of Sporobolus, F. palustre and F. incarnatum- Received 1 May, 2020; accepted 9 November, 2020. *For corre- spondence. E-mail [email protected]; Tel. 1-902-585-1333; equiseti (Elmer and Marra, 2011). Interestingly, a culture- Fax 1-902-585-1059 based study from New Brunswick, Canada saltmarsh © 2020 Society for Applied Microbiology and John Wiley & Sons Ltd Saltmarsh rhizosphere fungal communities 459 sediment using buried wooden baits recovered a different Our study provides the first analysis of sediment fungal fungal community, dominated by ascomycete fungi eDNA data from Atlantic Canada saltmarshes. Known (Mansfield and Bärlocher, 1993). Our study addresses a terrestrial, amphibious and marine fungi were detected in knowledge gap and will inform saltmarsh restoration sediments at all saltmarsh sites. This may result from fungi practices throughout Atlantic Canada to combat current in root tissues or spore deposition from wind, freshwater habitat degradation and loss due to anthropogenic inputs or ocean currents. Some fungi detected are unlikely destruction and natural stressors. to be metabolically active in these ecosystems, although With this ITS2 rDNA metabarcoding study we: we did not test for this. Ascomycete fungi were the most (i) compared the rhizosphere sediment fungal diversity at common fungi found, which correlates with data from two time points within three different saltmarshes border- saltmarshes of Rhode Island, USA (Mohamed and ing the megatidal Minas Basin, Nova Scotia to test the Martiny, 2011) and Louisiana, USA (Lumibao et al., 2018). fi hypothesis that fungal communities are site dependent Unclassi ed fungi were also detected, having no DNA and (ii) determined whether saltmarsh plant zonation sequence matches in the publicly available UNITE data- (sediment zone) influences total sediment fungal base (version 7.0)(Kõljalg et al., 2005); tidal wetland sedi- diversity. ment is a source of novel fungal diversity. It is worth noting that ITS metabarcoding can miss some fungal classifica- tions due to missing reference sequences in public data- Results and discussion bases (Heeger et al., 2019). Fungi identified in this study such as Lulworthia Saltmarsh rhizosphere fungal community composition sp. Phaeosphaeria halima, Phaeosphaeria spartinicola, 3Taxonomy was assigned using the UNITE version 7.0 Scheffersomyces spartinae,andFunneliformis geosporum database (Kõljalg et al., 2005) and relative abundance are known from other saltmarshes in the USA and Canada was assessed using QIIME2 (Bolyen et al., 2019) for (Filip and Alberts, 1993; Walker and Campbell, 2010; 65 saltmarsh sediment samples. Saltmarsh rhizosphere Kurtzman et al., 2011; d’Entremont et al., 2018). fungal assemblages, as revealed by ITS2 metabarcoding, Phaeosphaeria halima and P. spartinicola have both been showed differences by site and sediment zone, as well as isolated from the leaves of S. alterniflorus and play much unclassified diversity (Fig. 1, Appendix S1). Number important roles in the decomposition of Sporobolus litter of reads per sample are given in Appendix S2, and rare- and nutrient cycling in saltmarsh habitats (Filip and faction curves are provided in Appendix S3. Of the fungi Alberts, 1993; Buchan et al., 2002; Walker and able to be classified, ascomycetes dominated the sedi- Campbell, 2010). Scheffersomyces spartinae is only ment fungal communities at all sites and sediment zones, known from aquatic environments and it is unknown including obligate marine genus Lulworthia. Some fungal whether a true interaction between Sporobolus species plant pathogens, such as Gaeumannomyces graminis, and S. spartinae exists. Occurrence may be from suspen- were more abundant at some sites and sediment zones sion in the water column (Kurtzman et al., 2011). than others. One salt-tolerant arbuscular mycorrhizal fun- Funneliformis geosporum forms a mutualistic relationship gus (AMF), Funneliformis geosporum, was common to all with both Sporobolus alterniflorus and Sporobolus pumilus sites. Three other AMF species (a Claroideoglomus sp., in Nova Scotia saltmarshes (d’Entremont et al., 2018). Archaeospora trappei,andRhizophagus irregularis)were Funneliformis geosporum is an arbuscular mycorrhizal fun- also detected. It is important to note that eDNA-based gus that colonizes Sporobolus roots, although the strength assessments of sediment fungal diversity can include of the interaction is different for S. pumilus and detections of dormant or nonliving fungi. Sequences S. alterniflorus; the former is more extensively colonized obtained may be from fungal hyphae, spores, or other fun- than the latter. gal reproductive structures; future detection of fungal RNA from coastal sediments (metatranscriptomics) is needed to Species richness among Minas Basin saltmarsh sites determine which fungi are metabolically active in these and sediment zones habitats (Amend et al., 2019). Diversity of saltmarsh sediment fungal communities in We estimated alpha diversity using observed ASV rich- megatidal environments such as the Bay of Fundy, ness for samples taken from three locations, two Canada, have been poorly studied compared with terres- collection periods, and two sediment zones (Fig. 2, trial soil fungal communities. Both provide critical ecosys- Appendix S1). Considering all three variables, the total tem services such as decomposition, nutrient cycling and number of samples was insufficient to test all possible symbioses forming the basis of most food webs and are interactions and therefore we only performed pairwise essential for nutrient turnover and ecosystem sustainabil- comparisons for location, collection period, and sediment ity (Dighton, 2016). zone independently. Using a Kruskal–Wallis test, we © 2020 Society