Over the course of the past two decades, Phragmites. australis has been successful of suppressing substantial native plant species. Our goal is to eradicate P. australis without the use of harmful pesticides and to eventually re-establish the native plant species in Piermont Marsh, NY. In order to better understand the thermal tolerance of P. australis and its response to photo-deprivation, three experimental 6m quadrats were created. Quad 1 was cut to ground level and monitored for regrowth, Quad 2 was cut and covered with black visqueen to block the sun and to heat the rhizosphere, while Quad 3 served as our control. Rhizome samples were heated from 30°C to 100°C in 10°C increments, stained with Evans blue dye, and examined under a microscope to test for viability. Results from the heating experiment were inconclusive due to variability in our sampling and slide-making process. Initial observations indicated substantial regrowth in Quad1 and Quad 2, but the new P. australis growth under the black visqueen was reddish and structurally different from the new growth in Quad 1. These results suggest that the blocking of light has some impact on the regrowth of P. australis. Temperature readings regularly taken of the ambient air temperature and the temperature under the black visqueen suggest that an increase in the water table due to tidal forces may prevent the temperature under the black visqueen from reaching levels necessary to negatively impact the rhizosphere. Additional samples of the rhizosphere were collected from all three Quads, dried, and ground into a powder in order to measure the total non-structural carbohydrate (TNC), which will help us determine the impact of cutting, heating, and photo-deprivation on P. australis. Measurement of the TNC levels using a spectrophotometer will be conducted at a later date. Our research this summer is foundational and will greatly influence the research we conduct in subsequent summers, enabling us to better understand P. australis and the natural forces that may aid in its eventual eradication.

Our team found it difficult to regulate the temperature of the marsh water in our thermal-tolerance experiment. It was difficult to slice rhizome samples thin enough to visualize clearly under the microscope, and the poor quality of our slides caused them to fall apart after a few days. When examined under a standard microscope through high power, the results were inconclusive. All samples contained some cell structure, but no nuclei, stained or unstained, were visualized. When compared to the samples of A. wakegi successfully processed in our trial-run, the samples of P. australis exhibited a radically different anatomy. The cells of P. australis appeared much smaller and were tightly grouped inside larger rectangular structures that appeared to provide rigidity to the sample.

The temperature beneath the black visqueen in Quad 2, while higher than the ambient air temperature, did not approach the high levels reported in previous research (Boone, 1987). We were surprised to find new growth under the black visqueen. Small, reddish shoots of P. australis filled the area between the soil and the black visqueen, which different in appearance significantly from the small green shoots of new P. australis on Quad 1. The rhizome samples for our photo-deprivation experiment are securely stored for processing next summer.

Since this is the first year of a multi-year investigation, and our team is still in the process of building the necessary skills to carry-out our experiments, we were not able to reach any formal conclusions regarding the eradication of P. australis. Our extensive literature search at the beginning of the summer was productive and will continue to inform our research for years to come and our experiences in the marsh and the lab helped to clarify and focus our efforts for any future research. We were able to reach the following conclusions:● The protocol for the thermal tolerance portion of our research can be

fine-tuned to produce more reliable, meaningful results.● We must learn how to make more meaningful, permanent slides.● The cellular structure and layout of P. australis differs significantly from

A. wakegi.● Not all parts of the rhizome from P. australis contain meristematic cells.● We must first verify the existence of living cells in our rhizome samples

before we can test for cell viability.● LDEO has everything we need to conduct the TNC experiment next year

Abstract Results

MethodsThermal Tolerance ExperimentA 0.5% working volume of Evans blue dye was made using distilled water. Then, a trial-run of our staining protocol was performed using Allium wakegi (onion skin) to determine an appropriate staining and rinsing time. Three minutes of staining followed by 3 minutes of rinsing provided optimum results. Our team then dug a 30cm trench 2m long and 0.25m wide into an area adjacent to our established quadrats. Eight 15cm long rhizome samples with observable growth-nodes were selected from the trench. Rhizome samples were heated together in a large pot of marsh water from 30°C to 100°C. The temperature of the water was held for 15 minutes at each 10°C increment and then a random sample was taken, placed in a plastic bag, and refrigerated. Samples of rhizome were peeled from the inside of each heated sample, stained with Evans blue, and examined under a microscope to test for viability. When none of the samples exhibited the kind of nuclei staining found in A. Wakegi, additional trials were completed using boiled samples of different parts of the rhizome (tips, root hairs, and growth-nodes). None of the additional samples resulted in the kind of nuclei staining found in our trial run. Treated samples were photographed and replanted in their original trench. At a later date they will be checked for any additional growth.

Photo-Deprivation TNC ExperimentRhizome samples were obtained from a depth of 10cms and 30cms from each corner of each plot and the center of each plot for a total of 10 samples from each plot. Samples were dried in a 70°C oven for 12 hours to remove the moisture content. The wet and dry mass of each sample were recorded. Dried rhizomes from each plot were combined, crushed with a mortar and pestle, and then processed together through a coffee grinder to obtain a representative sample. Combined samples from each plot were then stored in a plastic container and properly labeled. After 2 weeks of natural exposure, additional rhizome samples from each quadrat were collected, processed, and stored using the same procedure previously used. Working with Dr. Andy Juhl, a suitable protocol for measuring the Total Non-structural Carbohydrate (TNC) of each sample using a spectrophotometer was chosen. At a later date, the TNC content of the before and after samples from each quadrat will be compared to measure how cutting and photo-deprivation impacts the rhizosphere of P. australis. Optimally, monthly rhizome samples will be taken and processed to verify the TNC cycle documented in previous research (Wilcox, 2013).

The Root of the Problem: Testing the Thermal Tolerance and Effects of Photo-Deprivation on the Rhizosphere of Phragmites australis in Piermont Marsh, New York

By Alondra Cruz, Liz Pratusevich, Melody Henry, Anjelle Martinez, Bing Liang, and Julissa Nuñez

Conclusions

Boone, J., E. Furbish, and K. Turner. 1987. Control of Phragmites communis: Results of Burning, Cutting, and Covering with Plastic in a North Carolina Salt Marsh. Institute of Ecology, University of Georgia, CPSU Technical Report No. 41, 15 pp.

Science & Plants for Schools, Can You Distinguish Between the Living and the Dead?, p.2

Wilcox, J. D. 2013. Response of Phragmites australis to Black Plastic Treatment. Master’s Thesis, University of Connecticut.

References

Although the research our team conducted this summer did not result in any conclusive data regarding the possible eradication of P. australis in Piermont Marsh, it did produce several clearly defined next steps for future investigation. We need to develop better slide preparation protocols that will result in more usable, consistent samples. By obtaining a hand-held microtome and learning how to construct permanent, well-crafted slides, our team will be able to draw more reliable conclusions about our experiments. Our team will redesign the rhizome heating experiment so the water temperature is more constant, perhaps using a sou vide oven, and we will heat each rhizome sample individually. We will also use more samples and verify cell viability before heating. By conducting the heating experiment earlier in the program, results from re-planting the rhizomes will have greater meaning and reliability. Using tetrazolium chloride (TTC), or some other staining agent, our team will map which parts of the rhizome containing meristematic cells and then verify cell viability before conducting our next round of hating tests (Science & Plants for Schools, 2010). A better understanding of the anatomy of P. australis will help our team make better choices about how we can eradicate it from Piermont Marsh. Once our team pinpoints which parts of the rhizome contains meristematic cells, we can better determine cell death using the Evans blue. Our team will conduct the TNC portion of our research at the beginning of next summer’s program. We will also explore the feasibility of collecting and drying rhizome samples on a monthly basis during the fall and spring to determine if the TNC concentrations in Piermont Marsh match those in previous studies. Preliminary temperature readings taken throughout the summer suggest that tidal influence on surface and sub-surface temperatures in the rhizosphere: negatively impact any heating caused by the plastic covering. By recording more frequent or continuous temperature readings of the soil under the plastic and the ambient air, our team might better understand the relationship between these events and better determine a course of action for harnessing radiant heat to negatively impact the rhizosphere. Finally, we will attempt to contact Dr. Jeremy Wilcox at the University of Connecticut for more information on how he was able to use Evans blue to identify dead cells in the rhizomes of P. australis and for advice on what part of the rhizome to use for our samples during next year’s research.

Next Steps

Phragmites australisFresh P. australis unstained (above) and boiled stained (below). Note the differences in the size of the cells and the additional structural elements in the P. australis tissue when compared to the onion samples.

Allium wakegiFresh A. wakegi unstained (above) and boiled stained (below). Note how the Evans blue was able to stain the nuclei of cells with damaged cell walls and membranes.

Re-Planting TrenchHeat-treated rhizome samples were replanted in a trench lined with weed-proof fabric to prevent incursion from native P. australis. The samples will be checked at a later date for any new growth.

Vampire Phragmites australisPhoto-deprived P. australis appeared markedly different from the fresh, green shoots growing in the uncovered quadrat.

Thermal Tolerance SamplingIt was difficult to accurately maintain the temperature of the marsh water while heating samples using a simple hot-plate. A sou vide oven would allow for more precise temperature control next year.