Labyrinthula zosterae isolation, culturing, and identification: In May 2016, we isolated L. zosterae, the causative agent of Zostera wasting disease, from the diseased leaf tissue of live Zostera from CB. Briefly, we cut a 2 centimeter section of Zostera leaf tissue at the edge of a black or brown lesion, surface sterilized and rinsed the leaf tissue section, and plated the tissue onto a 10 centimeter diameter seawater agar plate (Mckone and Tanner, 2009; Muehlstein, 1988). We confirmed the identity of the culture to be L. zosterae by microscopic examination (Mckone and Tanner, 2009). Specifically, we observed culture and cell morphology under 200x and 400x magnification on a Nikon eclipse 50i microscope. We used previously published examples of L. zosterae culture and cell morphology as a guide (i.e. branching ‘slime’ tunnels connecting cells and fusiform cell shape; Muehlstein et al., 1991). We maintained the L. zosterae culture by transferring colonized agar plugs to fresh agar plates every two weeks.

Zostera collection and preparation: In June 2016, we collected 30 Zostera plants (connected rhizome, stem, and leaves) from the same four subpopulations we surveyed wasting disease: NB (Niles Beach, Gloucester, 42° 35.8268’ N, 70° 39.3553’ W), WB (West Beach, Beverly, 42° 33.9155’ N, 70° 47.1102), LP (Lynch Park, Beverly, 42° 32.6925’ N, 70° 51.5057’ W), and CB (Curlew Beach, Nahant, 42° 25.2378’ N, 70° 54.9474’ W).

At each subpopulation location, we collected Zostera plants at 1 m intervals along a 30 m transect running parallel to shore 2-5 m from the shoreward extent of the Zostera bed at a depth of 1-2 meters MLLW. Plants from each subpopulation were kept in separate flow-through 54 liter seawater holding tanks in a glasshouse at the Northeastern University Marine Science Center (MSC), Nahant, MA. We attached the rhizome of each plant to a weight with a zip-tie so that the plants were oriented naturally in the water column and did not float on the top of the mesocosms (Mckone and Tanner, 2009). We allowed the plants to acclimate to greenhouse conditions for one month before the start of the L. zosterae inoculation experiment.

Ten days prior to inoculating Zostera with L. zosterae, we haphazardly selected 22 plants from each subpopulation and moved them from the flow-through holding tanks into 88 45 centimeter tall and 15 centimeter diameter transparent cylindrical acrylic mesocosms filled with seawater. Immediately prior to isolating each plant in a mesocosm, we used a razor blade to remove all leaf tissue within 3 centimeters of any black and brown lesions characteristic of wasting disease infection. In addition, we clipped the rhizomes of each plant to a length of 5 centimeters from the first node and removed all secondary shoots. We sterilized our workspace and tools with 10% bleach and then rinsed with deionized (DI) water in between handling each plant in order to minimize the risk of cross-contamination.

We assigned the 88 mesocosms to eleven 54 liter tanks in the MSC glasshouse such that each tank held two Zostera from each subpopulation. We randomized the location of the mesocosms within each tank. We then supplied each tank with flow-through seawater to a depth of 3-5 centimeters below the top of the mesocosms. The flow-through seawater acted as a water bath, keeping the temperature of the water in the mesocosms equivalent to ambient conditions. Finally, we added air-stones to each mesocosm to circulate the water and prevent temperature gradients from forming.

Vector preparation: One week prior to inoculating Zostera with L. zosterae, we collected lesion-free Zostera leaves from the CB subpopulation. We gently cleaned epiphytes off of the leaf tissue, rinsed the leaves in DI water for five minutes, and cut the leaves into 144 2-centimeter sections. We then haphazardly distributed the leaf sections into twenty-four 1.85 milliliter (mL) glass drams filled with 1.5 milliliter (mL) seawater (six leaf sections per dram) and autoclaved them at 121°C for 20 minutes to eliminate the possible presence of pathogens and to prevent contamination of inoculation cultures.

Next, we transferred the autoclaved leaf tissue sections onto 24 10-centimeter diameter seawater agar plates (six leaf sections per plate) (Mckone and Tanner, 2009). We arranged the leaf sections in a circle with a radius of 3 centimeters around the center of each agar plate and 1 centimeter distance maintained between each leaf section. We sealed the first twelve plates with parafilm immediately after plating on leaf sections and used these plates as control vectors. After plating leaf sections on the remaining twelve agar plates, we inoculated each plate by placing a 1 x 1 cm agar plug cut from the growing edge of the axenic L. zosterea culture isolated in May 2016 and maintained in the lab as described above. We then sealed these plates with parafilm to use as disease vectors. We stored all vectors in ambient light at 22°C in a fume hood.

We visually inspected the control and disease vectors daily for signs of contamination. For the L. zosterae-inoculated plates, we also monitored the expansion of L. zosterae across the surface of the agar. Three days following plating, we observed L. zosterae cells beginning to spread over the vectors and by the seventh day, L. zosterae cells completely covered the vectors in all twelve of the inoculated plates.

Zostera inoculation: We inoculated all 88 plants (N=22 per subpopulation) on August 2, 2016 (experiment day 0). We haphazardly selected eleven plants from each subpopulation to inoculate with a control vector and we inoculated the other eleven plants from each subpopulation with a disease vector. We first inoculated all the plants receiving the control treatment and then inoculated all the plants receiving the disease treatment to minimize the risk of contaminating the control plants with L. zosterae.

To inoculate each plant, we first gently removed the plant from its mesocosm and placed it on a 2 x 0.25 meter laminated paper surface. We used forceps to remove a single vector from an agar plate and placed it on the surface of the 2nd youngest leaf 5 centimeters above the top of the sheath. We clamped the vector to the leaf with a sterilized, rigid split Tygon tube (1 x 0.5 centimeters in diameter) (Muehlstein, 1988) and returned the inoculated plant back into its respective container. To prevent cross-contamination, we rinsed the working surface and forceps with 10% bleach and then DI water between each plant.

Monitoring and breakdown: We monitored the progression of wasting disease lesions as well as Zostera survival, growth, and morphology eight times over the course of the one-month experiment (days 0, 1, 3, 5, 7, 14, 21, and 28). We visually assessed each plant for the production of secondary shoots (stems and leaves) and noted plant mortality. We then photographed each plant using the camera application on an iPhone 5s. To prevent cross-contamination, we photographed all control plants before plants receiving the disease treatment and disinfected the working surface and all tools as described above between each plant. On day 28, we concluded the experiment by removing all plants from their mesocosms, taking photographs, measuring rhizome length, and recording biomass of above- and below-ground tissues after 48 hours drying at 70 ºC.

We quantified Zostera growth, morphology, and wasting disease infection intensity by scoring each photograph using the free ImageJ software for Mac OS X developed by the National Institutes of Health (Schneider et al., 2012). Specifically, we quantified leaf loss and leaf production for each plant over the course of the experiment by counting the number of leaves present relative to the position of the inoculated leaf (i.e., decrease in number of older leaves and increase in number of younger leaves). We used the segmented line function to measure the length of each leaf from the top of the sheath to the leaf tip. We calculated leaf growth as the change in length of the youngest leaf present at the time of inoculation, accounting for changes in sheath length. We used the polygon function to quantify leaf area and lesion area for each leaf. We used published descriptions and photographs of lesions associated with wasting disease to identify lesion area (Burdick et al., 1993; Groner et al., 2014; Groner, Burge, et al., 2016). To quantify the severity of wasting disease infection, we divided the sum of lesioned tissue area by the sum of total leaf tissue area for each plant (Burdick et al., 1993).