4.3: Laboratory Studies
Karen McKee and Irv Mendelssohn, the same investigators who were among the first to establish study sites on the ground, are heading up a multi-faceted laboratory task which, by virtue of its experimental nature and the broad range of disciplines involved, has to be considered one of the cornerstones of the entire Brown Marsh Project.
The effort is split between LSU and NWRC, and the experiments draw on the expertise of plant ecologists, plant physiologists and plant pathologists in a coordinated effort to first examine variables in isolation and, subsequently, in combination. At first glance, those unfamiliar with the project would likely find it odd that, as Mendelssohn puts it, "a group of experts was assembled in order to kill plants in a variety of ways."
While the scientists, of course, are mostly interested in learning what it takes to kill smooth cordgrass, they also included blackneedlerush (Juncus roemerianus) and black mangrove (Avicennia germinans) as controls representative of other plant species found in the same natural settings as smooth cordgrass yet did not die back or even show signs of stress. In brief, the experimental design calls for ever-increasing levels of stressors (e.g., salinity, acidification, moisture reduction, aluminum, and iron) to be applied to the three species and measurements of their responses. Pathogenic fungi are being investigated by Ray Schneider (LSU) in separate experiments.
Each stressor is being escalated at 2-week intervals while all other factors are held constant (e.g., aluminum levels would be gradually increased while salinity, pH, and the other variables are held at optimal constants). In addition to an unmanipulated control group for each of these isolates, a third group has been created to mimic any chronic conditions that smooth cordgrass may have been exposed to in the wild.
As Mark Hester, a coastal plant ecologist with UNO working on the experiment, explains it, while one experimental group continued to have stress placed upon it until mortality, the other experimental group's stressor level was held at the point at which the visible onset of plant stress was observed, helping the researchers understand the function of time with respect to stress levels that are otherwise seemingly nonlethal. Moreover, this design fits neatly with the task's mission to examine both short- and long-term growth responses.
Hester's role in the McKee-Mendelssohn trials amounts to measuring photosynthetic response in the short-term, measures that will then be compared to plant biomass responses. As far as photosynthetic response is concerned, Hester uses a "photosystem," a closed-chamber instrument that, given a known leaf area, uses light, water vapor, and carbon dioxide to calculate the rate of photosynthesis occurring within a sample. The amount of photosynthetic activity, in turn, provides a good index of overall plant health.
Echoing the findings of other researchers, Hester's preliminary results suggest that salinity alone is not the culprit. "Salt is an important stressor in coastal plant communities and may have been involved," he states, "but it cannot the only factor behind the dieback." During the salinity stressor experiment blackneedle rush succumbed to the increasing salinity levels before the smooth cordgrass. Hester explains that "this is not in agreement with what was observed in the field where black needlerush was often observed to be relatively healthy among the dead stems of smooth cordgrass in the dieback areas. Therefore, some factor(s) beyond salinity stress appear(s) to be involved in the sudden marsh dieback."
 The picture above demonstrates the responses of the three species in the McKee-Mendelssohn trials to a variety of salinity regimes. The effects of increased exposure to salinity upon black needlerush (Juncus) and smooth cordgrass (Spartina) are clearly visible. On the other hand, the black mangroves (Avicennia) show no outward signs of salinity-induced stress. |
Hester's previous research-where he subjected 25 different genotypes of smooth cordgrass to increasing salinity stress--is in agreement with the current findings. Although he found significant variation between genotypes, the salinity level that resulted in 50% death of aboveground tissue (similar to an LD50) ranged from 83 ppt to 115 ppt, with a median above 100 ppt. Therefore, Hester feels confident that salinity stress was not the only factor involved in the sudden marsh dieback.
According to Mendelssohn, the role of soil acidification is different from that displayed by salinity. In the isolate pH trials, the team began with a pH level of 6.5 and lowered it (i.e., made it more acidic) by 0.5 on the scale every 2 weeks. The sublethal stress level in black needlerush, black mangrove, and smooth cordgrass became apparent at a pH of 2.5, and that became the chronic experimental level they would hold while the acute group continued to drop.
One theory from the outset had been that drought had led to soil acidification, an effect that increased the concentration and "bioavailability"of toxic metals such as aluminum and iron in the soils.
After several months of this sustained level of acidification, smooth cordgrass in the "hold" group showed a severe decline from the point at which the pH level was fixed at 2.5. Across this same period, however, black needlerush and black mangrove showed no appreciable difference in their growth response to this sustained, chronic exposure. Hence, the preliminary results show that pH stress response across the three species matches the dieback pattern observed in the field, suggesting that acidic soil may be a possible cause behind the dieback. Field surveys did indicate much lower than normal pH values in some dieback areas, although not as low a pH as that reached in the laboratory trials.
Thus, all of the single factor trials demonstrate that the three species tested in laboratory trials show different tolerances of the stresses thought to be involved in the dieback. Moreover, species response patterns support pH, but not salinity, as a major factor potentially causing smooth cordgrass dieback. However, salinity may have played a secondary role, and this interaction will be tested in upcoming experiment to be conducted at NWRC by Karen McKee (see McKee video). One theory from the outset had been that drought had led to soil acidification, an effect that increased the concentration and "bioavailability"of toxic metals such as aluminum and iron in the soils.
Soils that develop within estuarine environments may contain pyrite and other metal-sulfide compounds that can be readily oxidized, and when water levels are drawn down as severely as they were during the 2000-01 drought, the heightened aeration (i.e., exposure to air) of the soils increases opportunities for oxidation. This oxidation process, in turn, increases the acid levels in the soils.
But as pH decreases linearly, the solubility of aluminum and iron, or the rate at which these metals can dissolve into the immediate environment, is increased several fold. Hence, McKee is looking at these metals in separate isolate trials being conducted at the National Wetlands Research Center, increasing their concentrations at 2-week intervals until mortality is reached in the acute groups while holding at a stress-inducing level in the chronic group.
The role pathogens may have played in the brown marsh dieback is perhaps the most troublesome causal isolate the team is pursuing. Heading up this facet of the research trials is Ray Schneider, a plant pathologist with the LSU Agricultural Center. Because of the specific cluster of symptoms--browning, leaf death, and root rot--Schneider's experience has led him to suspect pathogenic fungi.
Part of what makes Schneider's job difficult is that even though he examined the very first samples that the McKee-Mendelssohn team brought back from the field, he was, in essence, starting too late because a whole host of secondary invaders had arrived, opportunistic invaders that can only flourish on already diseased or dying plants. Moreover, healthy smooth cordgrass, as is the case with most plant species, has a broad range of what Schneider calls "fungal microflora" active within it.
In much the same way many humans carry pneumonia pathogens within them and only become symptomatic after their immune systems become lowered while fighting off other disease agents, many of the 100-plus fungi Schneider found in diseased smooth cordgrass are present in healthy specimens as well but can explode to pathogenic levels when a plant is stressed. And to complicate the picture even further, the literature is full of fungi that appear together only in "complexes." For example, while one fungus may cause stunting, one yellowing, and one seedling disease, rarely do any of the three appear in a plant without the other two appearing as well.
Despite these complexities, Schneider is forging ahead with his work, separating the secondary invaders from those typically found in smooth cordgrass microflora and identifying those which usually appear only as part of fungal complexes. But even as he goes about the business of inoculating healthy plants with these various fungi and observing their progress, the McKee-Mendelssohn team is planning its next phase of study: combination trials.
Because more than one stress factor may have been involved in the brown marsh phenomenon, the plan is to experimentally test three factors simultaneously to see if the dieback can be produced in the lab in that fashion. Karen McKee, the team member who will manage this aspect of the task, has already decided that salinity and pH will be two factors she will include, but her final decision will be based on the outcome of the single-factor trials.
One needs only to think of the numerous combinations possible to realize the complexity of McKee's undertaking. For instance, she could place salinity at 30 ppt, pH at 4.0, and introduce pathogen x, or she could place salinity at 20 ppt, pH at 3.0, and introduce metals at a level of y. In other words, the range of possible causative combinations, while not endless because they will be restricted by the range of field measures, is certainly daunting. These short-term stress experiments will be complemented by a long-running research task in which sections of marsh are subjected to combinations of factors. The two approaches will together have a better chance of identifying the causal agents in the dieback than either one alone.