Dataset: Seagrass Microbiome Data

We collected Z. marina tissue from the 3rd-youngest leaf of individual shoots exhibiting wasting disease lesions and, from nearby (within 1 m), a 3rd-youngest leaf free of lesions from a different shoot at haphazard locations along each transect.  We selected tissue samples from Z. marina as described below. L. zosterae is an opportunistic endophyte that forms an ectoplasmic net, moving through host tissue by degrading cell walls, with the greatest pathogen density at the leading edge of the infection before tissue browning (17, 61). Thus, we sampled lesion tissue as well as green tissue at the leading edge of lesions to determine how microbiomes may change with lesion development (i.e., effects of “tissue type” on the same leaf). Sampling the leading edge of an infection may allow us to determine early microbial interactors in eelgrass wasting disease versus opportunistic microbes that colonize or increase in abundance following pathogen degradation of host tissue. We also sampled green tissue from a different shoot nearby whose third youngest leaf did not exhibit lesions (lesion-free leaves) to compare these to green tissue at the leading edge of infection from lesioned leaves (effects of “lesion status”). Lesion status allows us to test how wasting disease, identified by the presence of characteristic lesions on young (nonsenescing) leaves, affects the green phyllosphere microbiome adjacent to lesions. To minimize cross-contamination, we wore nitrile or similar gloves and cleaned metal tweezers and scissors with 70% ethanol wipes between each tissue sample. We immediately placed samples into 1.5 mL microcentrifuge tubes containing DNA/RNA shield (Zymo Research Cat. R1100) upon collection.

We also sampled seawater microbial communities to assess whether drivers of eelgrass microbiome structure differ from those of free-living microbial communities in the water column surrounding

eelgrass beds. We collected three bottles of water, using 500 mL sterile bottles (VWR Cat. 76299-562) from 3 haphazardly selected locations within the meadow that were approximately 20

m apart. We kept water samples on ice until filtration within 4–6 h of collection with Nalgene analytical filtration units (Cat. 130-4020), which contained 0.22 mm pore size, 47 mm cellulose nitrate filters. Final volumes of water filtered varied (200–500 mL) due to high turbidity at some sites that reduced the rate of filtration. Upon completion of water filtration, the filter was preserved in 2 mL of DNA/RNA shield (Zymo Research Cat. R1100).

Samples were shipped to the University of California, Davis within 3 weeks of sampling and stored at 280°C until processing. We extracted DNA from our samples with ZymoBIOMICS DNA Microprep Kit (Cat. D4301). Upon thawing, we vortexed the 1.5 mL tubes containing leaf tissue in DNA/RNA shield for 60 s and transferred 500 mL of supernatant to a ZymoBIOMICS bead beating tube. We added 500 mL of ZymoBIOMICS lysis solution to the bead beating tube, vortexed tubes for 20 min on a Vortex-Genie 2 with horizontal microtube holder, and performed DNA extractions for phyllosphere samples according to the manufacturer’s instructions following the bead beating step. To extract DNA from filtered seawater samples, we aseptically cut cellulose nitrate filters into 1–2 mm wide sections, transferred slices plus the 2 mL of DNA/RNA shield used to preserve the filter into two bead beating tubes, and vortexed these on a Vortex-Genie for 20 min. All other DNA extraction steps followed manufacturer instructions, except for final elution of DNA in 40 mL rather than 20 mL of DNase/RNase-free water and further dilution (1 part DNA to 9 parts DNase/RNase-free water) so that DNA concentrations would be similar for both eelgrass tissue and seawater samples, commonly falling between 1 and 10 ng/mL. We processed six negative controls similarly. Four negative controls originated from ZymoBIOMICS DNA/RNA shield and two from sterile cellulose nitrate filters following 100 mL filtration with molecular grade water (Sigma-Aldrich Cat. W4502) preserved in ZymoBIOMICS DNA/RNA shield. Following extraction, negative control samples underwent library prep with all biological samples.

We shipped DNA samples to Dalhousie University's IMR facility for library prep and Illumina MiSeq sequencing according to (92). Briefly, Nextera fusion primers F515 and R926 (93) amplified the V4–V5 region of the 16S rRNA gene with high-fidelity polymerase and 2 mL of template DNA in 25 mL PCR volumes. PCR products from two technical reactions per biological sample were verified with Invitrogen 96-well E-gels. Pooled technical replicates were cleaned and normalized with Invitrogen Sequal-Prep plates. Cleaned and normalized PCR amplicons underwent paired-end

300 bp sequencing on an Illumina MiSeq.