Methods

Mesocosm setup and sampling

The hypothesis tested was that there is an optimal salinity for productivity that differs among the three marsh species, and productivity under saline stress is influenced by the soil microbiome.

The experimental setup involved three species of marsh plants (Spartina alterniflora, Juncus roemerianus, and Schoenoplectus americanus) maintained at freshwater (0 ppt), brackish (5-10 ppt), and marine (20-40 ppt) salinity levels (Figure 1). Plants for this study were randomly selected from the general population in the USM Gulf Coast Research Laboratory botany nursery. They were planted in 15-cm diameter pots filled with 1:1 mix of topsoil and sand and placed into tubs with water (ten replicate pots per tub). Plants were treated to the assigned salinity condition by adding artificial sea salts (Instant Ocean, Blacksburg, VA) at the beginning of the 6 month experiment. Tap water was added 2-3 times per week over the duration to maintain the water level and salinity condition. Plants were fertilized monthly by adding one tablespoon of Miracle-Gro Water Soluble Plant Food 20:20:20 (Scotts Miracle-Gro, Marysville, OH) to the water in the tubs.

At the end of the experiment, each species’ survival was assessed by counting the number of remaining pots with at least one green plant. Morphology measurements were taken (n = 3) to record the number of stems per pot, the length of the two tallest leaves/stems per pot to nearest 0.5 cm accuracy, and the width of two mature green leaves per pot to nearest 1 mm accuracy. To measure the aboveground (AGM) and belowground (BGM) biomass, plants were destructively harvested, rinsed, and processed to separate stems, leaves, and flowering structures from roots and rhizomes. The AGM material was then separated into alive and dead portions with green and brown/dark material considered, respectively, alive and dead. Following separation, all tissue samples were dried in a 70°C oven for 3 days and weighed to record the mass (g). Replicate mature green leaf samples per pot (n = 3) were excised at random from each species and salinity treatment to measure the chlorophyll content index (CCI) and fluorescence. Both measurements were taken at locations approximately 2.5 cm from the bottom, the mid-point, and about 2.5 cm from the leaf tip (or green to brown transition area if the distal end of the leaf was not green). The leaf was marked immediately after collection to ensure that all measurements were taken at the same location. Both light-adapted (F’/Fm’) and dark-adapted (Fv/Fm) leaf fluorescence measurements were performed using a miniPEA continuous excitation fluorimeter (Hansatech Instruments, Norfolk, UK), with a minimum of 30 minutes dark adaptation time. A blank fluorescence value was recorded at the beginning of every set of leaves by salinity treatment. The chlorophyll content was measured with a CCM-300 meter (Opti-Sciences, Hudson, NH) using default settings. The instrument accuracy was checked against a known solid standard at the beginning of every set of leaves by salinity treatment.

Figure 1: Spartina, Juncus, and Schoenoplectus growing in salinity treatment tubs.