Leaves: Approximately 5g of whole, healthy leaves were collected, placed into a clean Ziploc bag and kept in refrigeration. All subsequent processing occurred within 12 hours of collection. Edges were removed from leaves and leaves chopped into smaller chunks. Leaves were treated with bleach and ethanol to reduce the presence of microbes on the outside of the tissue. Using sterile technique in a biosafety cabinet and following the methods of Kandalepas et al. (2015), leaf pieces were submerged for 10 seconds in 95% ethanol, 2 minutes in 0.525% sodium hypochlorite (10% bleach) followed by 2 minutes in 70% ethanol and frozen in a CTAB buffer (2% CTAB, 0.02M EDTA, 0.1M Tris, 1.4M NaCl final concentrations) at -20 °C until DNA extraction. Roots: Roots were processed similarly to leaves, however the treatment to remove external microbes was slightly different. Root pieces were submerged for 10 seconds in 70% ethanol, 2 minutes in 2.625% sodium hypochlorite (50% bleach), followed by 3 rinses in sterile DI water.
Growth protocol
The plants were germinated in the spring of 2016 and the experiment begun on May 1, 2016. It was sampled every 6 months for the next two years, and completely harvested in June 2018. Of 10 replicates for each treatment, only three replicates from June 2018 were anlyzed with GeoChip. Spartina alterniflora seeds were collected in November 2015 in southern Louisiana and placed at 4 °C to stratify. In February 2016 the microbial community on the surface of the seeds was reduced by subjecting them to a 95% EtOH bath for 3 minutes, then 30%min in 0.825% bleach and rinsed in sterile DI water for 10 seconds. The seeds were then germinated in DI water and transplanted to trays in a Conviron Model GR48 Plant Grow Room (Controlled Environments Ltd., Winnipeg, Canada). The growing substrate was a 1:1 mixture of organic humus and vermiculite that was autoclaved three times at 121 °C for 60 min to reduce the microbial community in the substrate. Seedlings were grown under conditions meant to mimic conditions in southern Louisiana, after the methods of Krauss et al. (1998). Seedlings were watered with deionized water and given 1 tablespoon of Osmocote fertilizer (14:14:14 NPK). In April 2016 the plantlets were transferred to the greenhouse and allowed to acclimate to greenhouse conditions for about three weeks. On May 1st the plantlets were planted into pots filled with inoculum soil. The inoculum soil was collected from Bay Jimmy, LA at sites that had been heavily oiled (Google maps: 29.44464, -89.88959) or relatively unoiled (Google maps: 29.44006, -89.88583) in the DWH oil spill (Zengel et al., 2015). Within 60 hours of collection the soil was sieved through a 1cm screen, placed in 1-gallon trade pots and either planted or left unplanted. The pots were randomized in 5 blocks on an approximate East-West axis on 4 tables oriented approximately North-South. The plants grew in the inoculum for one month and then the entire plant-soil plug was placed in a 5-gallon mesocosm. Each mesocosm consisted of a 3-gallon trade pot nested in a water-filled 5-gallon bucket. The soil/plant plug was placed inside of the 3-gallon trade pot containing 2.5 gallons of a 2:3 organic humus to sand mixture. Half of the mesocosms also received 1.6 L of oil naturally weathered oil skimmed off the ocean during the DWH oil spill and distributed by the Gulf of Mexico Research Initiative (GoMRI). To reduce sporefall germination, 0.5 gallons of clean sand were placed in an even layer around the top of the plant. Three weeks after mesocosm construction, 8 grams of Scott’s Osmocote Plus (Marysville, Ohio) was added to the surface and covered with an additional 0.25 gal sand layer. The fertilizer is a patterned-release complete nutrient fertilizer containing 15% N, 9% P, and 12% K (releasing at maximum 1.00 g·m-2 nitrate per month). Drip irrigation supplied water to the surface of each pot, and pots were inserted into buckets in order to maintain a high water table. Drainage tubes were installed near the top of each bucket, allowing some flow through. To prevent reactivity to oil, the 3-gallon trade pots were lined with Teflon bags (P-00113, Welch Flurocarbon, Inc, Dover, NH, USA). The bottom of each bag was punctured to permit drainage. Over the course of the experiment, plants received water from a timer-based drip irrigation, so that some portion of the water would turn over every day. If necessary, during hot summer months the watering was augmented by hand to maintain the height of the water table. Once a month, the mesocosms received 1 liter of 5 ppt solution of Instant Ocean salt (Instant Ocean Spectrum Brands, Blacksburg, VA). Every two months mesocosms were augmented with an additional 4 grams of fertilizer and 1 liter of inoculum, a soil slurry from the same marsh sites used in the original inoculation. Whenever salt built up on the plant leaves, mesocosms (planted and unplanted) were misted to wash salt off the leaves.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from root and leaf samples using liquid nitrogen grinding and QIAGEN RNEasy PowerSoil Total RNA kit (Qiagen, Germantown, MD). The extracted RNA was converted immediately into cDNA using High Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA). cDNA was then sent to Glomics Inc. (Norman, OK) for GeoChip analysis. Glomics Inc. amplified samples using whole community genomic amplification. Kandalepas, D., Blum, M.J., and Van Bael, S.A. (2015). Shifts in Symbiotic Endophyte Communities of a Foundational Salt Marsh Grass following Oil Exposure from the Deepwater Horizon Oil Spill. PLoS One 10, e0122378. Krauss, K.W., Chambers, J.L., and Allen, J.A. (1998). Salinity effects and differential germination of several half-sib families of baldcypress from different seed sources. New Forests 15, 53-68. Zengel, S., Bernik, B.M., Rutherford, N., Nixon, Z., and Michel, J. (2015). Heavily Oiled Salt Marsh following the Deepwater Horizon Oil Spill, Ecological Comparisons of Shoreline Cleanup Treatments and Recovery. PLoS ONE 10, e0132324.
Label
Cy3
Label protocol
amplified product labeled with cy3 using klenow and random primers at 37C
reference: Synthetic oligonucleotides complementing GeoChip-specific probes for signal normalization.
Treatment protocol
Leaves: Approximately 5g of whole, healthy leaves were collected, placed into a clean Ziploc bag and kept in refrigeration. All subsequent processing occurred within 12 hours of collection. Edges were removed from leaves and leaves chopped into smaller chunks. Leaves were treated with bleach and ethanol to reduce the presence of microbes on the outside of the tissue. Using sterile technique in a biosafety cabinet and following the methods of Kandalepas et al. (2015), leaf pieces were submerged for 10 seconds in 95% ethanol, 2 minutes in 0.525% sodium hypochlorite (10% bleach) followed by 2 minutes in 70% ethanol and frozen in a CTAB buffer (2% CTAB, 0.02M EDTA, 0.1M Tris, 1.4M NaCl final concentrations) at -20 °C until DNA extraction. Roots: Roots were processed similarly to leaves, however the treatment to remove external microbes was slightly different. Root pieces were submerged for 10 seconds in 70% ethanol, 2 minutes in 2.625% sodium hypochlorite (50% bleach), followed by 3 rinses in sterile DI water.
Growth protocol
The plants were germinated in the spring of 2016 and the experiment begun on May 1, 2016. It was sampled every 6 months for the next two years, and completely harvested in June 2018. Of 10 replicates for each treatment, only three replicates from June 2018 were anlyzed with GeoChip. Spartina alterniflora seeds were collected in November 2015 in southern Louisiana and placed at 4 °C to stratify. In February 2016 the microbial community on the surface of the seeds was reduced by subjecting them to a 95% EtOH bath for 3 minutes, then 30%min in 0.825% bleach and rinsed in sterile DI water for 10 seconds. The seeds were then germinated in DI water and transplanted to trays in a Conviron Model GR48 Plant Grow Room (Controlled Environments Ltd., Winnipeg, Canada). The growing substrate was a 1:1 mixture of organic humus and vermiculite that was autoclaved three times at 121 °C for 60 min to reduce the microbial community in the substrate. Seedlings were grown under conditions meant to mimic conditions in southern Louisiana, after the methods of Krauss et al. (1998). Seedlings were watered with deionized water and given 1 tablespoon of Osmocote fertilizer (14:14:14 NPK). In April 2016 the plantlets were transferred to the greenhouse and allowed to acclimate to greenhouse conditions for about three weeks. On May 1st the plantlets were planted into pots filled with inoculum soil. The inoculum soil was collected from Bay Jimmy, LA at sites that had been heavily oiled (Google maps: 29.44464, -89.88959) or relatively unoiled (Google maps: 29.44006, -89.88583) in the DWH oil spill (Zengel et al., 2015). Within 60 hours of collection the soil was sieved through a 1cm screen, placed in 1-gallon trade pots and either planted or left unplanted. The pots were randomized in 5 blocks on an approximate East-West axis on 4 tables oriented approximately North-South. The plants grew in the inoculum for one month and then the entire plant-soil plug was placed in a 5-gallon mesocosm. Each mesocosm consisted of a 3-gallon trade pot nested in a water-filled 5-gallon bucket. The soil/plant plug was placed inside of the 3-gallon trade pot containing 2.5 gallons of a 2:3 organic humus to sand mixture. Half of the mesocosms also received 1.6 L of oil naturally weathered oil skimmed off the ocean during the DWH oil spill and distributed by the Gulf of Mexico Research Initiative (GoMRI). To reduce sporefall germination, 0.5 gallons of clean sand were placed in an even layer around the top of the plant. Three weeks after mesocosm construction, 8 grams of Scott’s Osmocote Plus (Marysville, Ohio) was added to the surface and covered with an additional 0.25 gal sand layer. The fertilizer is a patterned-release complete nutrient fertilizer containing 15% N, 9% P, and 12% K (releasing at maximum 1.00 g·m-2 nitrate per month). Drip irrigation supplied water to the surface of each pot, and pots were inserted into buckets in order to maintain a high water table. Drainage tubes were installed near the top of each bucket, allowing some flow through. To prevent reactivity to oil, the 3-gallon trade pots were lined with Teflon bags (P-00113, Welch Flurocarbon, Inc, Dover, NH, USA). The bottom of each bag was punctured to permit drainage. Over the course of the experiment, plants received water from a timer-based drip irrigation, so that some portion of the water would turn over every day. If necessary, during hot summer months the watering was augmented by hand to maintain the height of the water table. Once a month, the mesocosms received 1 liter of 5 ppt solution of Instant Ocean salt (Instant Ocean Spectrum Brands, Blacksburg, VA). Every two months mesocosms were augmented with an additional 4 grams of fertilizer and 1 liter of inoculum, a soil slurry from the same marsh sites used in the original inoculation. Whenever salt built up on the plant leaves, mesocosms (planted and unplanted) were misted to wash salt off the leaves.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from root and leaf samples using liquid nitrogen grinding and QIAGEN RNEasy PowerSoil Total RNA kit (Qiagen, Germantown, MD). The extracted RNA was converted immediately into cDNA using High Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA). cDNA was then sent to Glomics Inc. (Norman, OK) for GeoChip analysis. Glomics Inc. amplified samples using whole community genomic amplification. Kandalepas, D., Blum, M.J., and Van Bael, S.A. (2015). Shifts in Symbiotic Endophyte Communities of a Foundational Salt Marsh Grass following Oil Exposure from the Deepwater Horizon Oil Spill. PLoS One 10, e0122378. Krauss, K.W., Chambers, J.L., and Allen, J.A. (1998). Salinity effects and differential germination of several half-sib families of baldcypress from different seed sources. New Forests 15, 53-68. Zengel, S., Bernik, B.M., Rutherford, N., Nixon, Z., and Michel, J. (2015). Heavily Oiled Salt Marsh following the Deepwater Horizon Oil Spill, Ecological Comparisons of Shoreline Cleanup Treatments and Recovery. PLoS ONE 10, e0132324.
Label
Cy5
Label protocol
amplified product labeled with cy3 using klenow and random primers at 37C
Hybridization protocol
labeled DNA and universal standard mixed with hybridization buffer (Agilent) and 10% formamide; hyb 22 hr at 68C
Scan protocol
Scanned on a Nimblegen MS400, images were quantified using Agilent Feature Extraction Software (version 12.1.1.1).
