Denise M. Seliskar

Root-secreted Allelochemical in the Noxious Weed Phragmites Australis Deploys a Reactive Oxygen Species Response and Microtubule Assembly Disruption to Execute Rhizotoxicity

Phragmites australis is considered the most invasive plant in marsh and wetland communities in the eastern United States. Although allelopathy has been considered as a possible displacing mechanism in P. australis, there has been minimal success in characterizing the responsible allelochemical. We tested the occurrence of root-derived allelopathy in the invasiveness of P. australis. To this end, root exudates of two P. australis genotypes, BB (native) and P38 (an exotic) were tested for phytotoxicity on different plant species. The treatment of the susceptible plants with P. australis root exudates resulted in acute rhizotoxicity. It is interesting to note that the root exudates of P38 were more effective in causing root death in susceptible plants compared to the native BB exudates. The active ingredient in the P. australis exudates was identified as 3,4,5-trihydroxybenzoic acid (gallic acid). We tested the phytotoxic efficacy of gallic acid on various plant systems, including the model plant Arabidopsis thaliana. Most tested plants succumbed to the gallic acid treatment with the exception of P. australis itself. Mechanistically, gallic acid treatment generated elevated levels of reactive oxygen species (ROS) in the treated plant roots. Furthermore, the triggered ROS mediated the disruption of the root architecture of the susceptible plants by damaging the microtubule assembly. The study also highlights the persistence of the exuded gallic acid in P. australis’s rhizosphere and its inhibitory effects against A. thaliana in the soil. In addition, gallic acid demonstrated an inhibitory effect on Spartina alterniflora, one of the salt marsh species it successfully invades.

Persistent Differences in Two Forms of Spartina Alterniflora: A Common Garden Experiment

Tall- and short-form Spartina alterniflora plants were transplanted from the Delaware marsh to common garden plots during the summer of 1978 and irrigated with saltwater three times a week during that and each subsequent growing season. Nine years later plant biomass, culm height, density, and diameter and flowering frequency remained distinct. Underground regrowth reserves and root profiles likewise remained distinct for the two forms. Since the two growth forms had been living in the same environment for 9 yr and had retained many morphological and physiological differences, some genetic control of the morphology and physiology of the two growth forms is highly probable.

Rhizome growth dynamics of native and exotic haplotypes ofPhragmites australis (Common reed)

Phragmites australis (common reed), a clonal grass, has expanded from a minor component of the mid-Atlantic wetlands to a dominant species. It has been suggested that invasive populations ofPhragmites are an exotic haplotype responsible for the dramatic increase in the distribution of the species. We used field observations and measurements and a greenhouse assay to compare native (haplotype F) and exotic (haplotype M) populations, growing adjacent to one another in a brackish marsh near Odessa, Delaware. In the marsh, shoots of the exotic strain emerged from the rhizomes earlier than those of the native and by March there was an order of magnitude more new shoots of the exotic strain than the native. In August, the exotic strain was 30% taller than the native, had twice the leaf biomass, and twice the total biomass. Nine of ten morphological and biomass characteristics measured differed significantly between the native and exotic strains. A greenhouse assay was conducted by planting rhizomes collected in March in shallow trays and growing them for 70 d followed by shoot harvest (Harvest 1). Rhizomes were measured, replanted, and grown for 35 d after which time they were measured and shoots were harvested (Harvest 2). At Harvest 1, shoot height was approximately 80% greater in the exotic strain, shoot biomass was three times higher, aboveground to belowground biomass ratio was twice as high, and rhizome internode length was 50% greater in the exotic strain than the native. These traits, in addition to number of shoots, were also greater in the exotic strain at Harvest 2. The number of rhizome buds at Harvest 1 was three times greater in the native than in the exotic strain. The greater number of rhizome buds in the native would seem to be an advantage, but it did not result in more shoot production. Buds were maintained in an inactive state that does not allow this strain to compete well in a wetland environment inhabited by a more efficient spreader. The earlier emergence of new shoots from the rhizomes, the greater aboveground structure, the greater rhizome internode length, and the quick transition of rhizome buds to shoot or rhizome explain in part the exotic strains advantage over the native and the mechanisms for its invasive nature.

The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress

Callus cultures of the salt marsh grass Spartina patens were examined to determine changes and consistencies in membrane lipid composition in response to salt. Major membrane lipid classes remained stable at all salinity levels (0, 170, 340 mmol/L). However, the membrane protein to lipid ratio decreased significantly in response to elevated NaCl. Callus plasma membrane (PM) consisted predominantly of sterols, about 60% (mol%) of the total lipids. Glycolipid was the second largest lipid class, making up about 20% (mol%) of the total. With increasing salinity, the relative percentage of sitosterol decreased, while that of campesterol increased. The phospholipid species detected were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). When callus was grown at 340 mmol/L NaCl, PC increased significantly. PI and PS were also significantly elevated in salinity treatments. Only 24-32% of the PM fatty acids were common plant membrane fatty acids, C16, C18, C20, and C22, while over 60% were the less common fatty acids, C11 and C14. Membrane fluidity remained stable in response to growth medium salinity. The findings on membrane responses to salinity will facilitate a better understanding of this halophytes tactics for salt tolerance.

Exploiting wild population diversity and somaclonal variation in the salt marsh grass Distichlis spicata (Poaceae) for marsh creation and restoration

The salt marsh grass Distichlis spicata was regenerated from tissue culture and propagated in a greenhouse. Selected regenerants, along with selections from six wild populations, were grown for two years in a common garden flood-irrigated thrice weekly with tidal creek water. Selected wild and regenerated plants were also planted in a created salt marsh. Significant differences among regenerant and wild population selections were found in several functionally important salt marsh plant characteristics, including potential detritus production, belowground organic matter production, canopy structure, and decomposition rate. A combination of characteristics not found in the wild populations was evident in a regenerated line that exhibited both a high detritus production potential and a high decomposition rate. The amount of variation that occurred among regenerants from one parental line via somaclonal variation was similar to that which occurred among the wild population selections. Results of this study suggest that tissue culture may provide a means of producing marsh grasses with specific characteristics for directing the functional development of newly created salt marshes.

Tissue culture and plant regeneration of spartina alterniflora: Implications for wetland restoration

A tissue culture and plant regeneration protocol for the salt marsh grass, Spartina alterniflora, has been developed. Callus was efficiently induced on Murashige and Skoog (MS) medium supplemented with 1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L−1 indole-3-acetic acid (IAA). Callus initiation from 6-day-old seedlings was faster and occurred with a greater frequency than callus initiation from coleoptile-covered segments from the same age seedlings. However, only the coleoptile-covered segments produced regenerable callus, which was maintained on MS medium supplemented with 1 mg L−1 2,4-D and 1 mg L−1 naphthaleneacetic acid (NAA). The regenerable callus differentiated into shoots upon transfer to shoot regeneration medium. A high frequency of shoot regeneration was obtained when the medium contained 3 mg L−1 6-benzylaminopurine (BA) or Thidiazuron (TDZ), with or without the addition of 0.2 mg L−1 IAA. Regenerated shoots were transferred to root regeneration medium, the optimal of which was determined to be half-strength MS medium supplemented with 1 mg L−1 indole-3-butyric acid (IBA). TDZ in the shoot regeneration medium inhibited root formation in the root regeneration medium, making BA rather than TDZ the optimal hormone for the shoot regeneration medium. The mode of plant regeneration was organogenesis. Upon transfer to soil, the most successful growth of plants occurred in a mixture of commercially available potting soil and natural marsh mud. The development of a tissue culture and regeneration protocol for S. alterniflora provides the possibility of selecting lines of this species, via somaclonal variation, with characteristics desirable for wetland creation and restoration.

Plant regeneration from callus cultures of salt marsh hay, Spartina patens, and its cellular-based salt tolerance

Salt marsh hay, Spartina patens (Ait.) Muhl. (Poaceae), is a perennial salt-tolerant grass common in salt marshes and sand dunes of the Atlantic and Gulf coasts of the USA, and grows vigorously at coastal seawater salinity. To study the salt tolerance mechanisms that operate in S. patens at the cellular level, a tissue culture and regeneration protocol for this species was developed. Callus was initiated from seedling mesocotyl on ADM medium (Murashige and Skoog (MS) salts + 3% sucrose + 1 mg l−1 indoleacetic acid (IAA) and 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D)). Regenerable callus was selected from the several morphotypes that developed and was maintained on BND medium (MS salts + 3% sucrose + 0.5 mg l−1 6-benzylaminopurine (BAP), 1 mg l−1 1-naphthaleneacetic acid (NAA), 0.5 mg l−1 2,4-D, and 50 ml l−1 coconut water (CW)). Shoots formed from 90% of the cultures grown on shoot regeneration medium containing BAP and IAA. Roots formed from shoots when they were transferred to root regeneration medium containing indole-3-butyric acid (IBA) and activated charcoal or reduced strength MS medium. Plants regenerated via organogenesis have flowered and set viable seeds in a saltwater-irrigated field plot. Dry weight accumulation of unadapted callus at 510 mM NaCl is similar to that at 0 mM NaCl (control), indicating that S. patens has strong salt tolerance at the cellular level.

Root and rhizome distribution as an indicator of upper salt marsh wetland limits

Four wetland plant species, Deschampsia cespitosa, Distichlis spicata, Grindelia integrifolia, and Salicornia virginica, were tested to determine the effect of soil moisture on the vertical distribution of roots and rhizomes. In an 8-month greenhouse experiment involving plants grown from seeds and rhizomes the occurrence of more than 65% of the root and rhizome mass in the upper 10 cm of soil was indicative of saturated conditions. Roots and rhizomes were more evenly distributed under field capacity and low moisture conditions. The percent of total root and rhizome biomass in the upper 10 cm of a 35 cm core may be useful in determining whether or not a plant is experiencing saturated soil conditions, a criterion often used for defining wetlands. Limited field studies support the conclusion of the greenhouse study.

THE RESPONSE OF AMMOPHILA BREVILIGULATA AND SPARTINA PATENS (POACEAE) TO GRAZING BY FERAL HORSES ON A DYNAMIC MID-ATLANTIC BARRIER ISLAND'

Ammophila breviligulata, American beachgrass, and Spartina patens, salt meadow hay, have been grazed by feral horses on the dunes of Assateague Island for hundreds of years; however, because of a significant increase in the horse population since the 1960s, overgrazing and dune erosion have become problems. Grazing was assessed on foredunes of four different morphologies along a 21-km stretch of the Maryland portion of the island using 17 exclosure plot pairs. In addition to decreased cover and biomass of the two species, plant structure was significantly affected by grazing. Leaf length and width, stem diameter, and stem density of A. breviligulata and stem diameter of S. patens were reduced in the grazed plots. Especially sensitive to grazing were reproductive characteristics. Percentage of plants in flower, height of flowering stems, and inflorescence length were all significantly reduced by grazing (nongrazed individuals measured). Species composition was not affected by horse accessibility.

Root system architecture of Kosteletzkya pentacarpos (Malvaceae) and belowground environmental influences on root and aerial growth dynamics

PREMISE OF THE STUDY Root growth and architecture of Kosteletzkya pentacarpos and its response to nutrients and soil texture were studied to facilitate domestication of this plant as a biofuel crop (seeds for oil, stems for ethanol). Because it is salt tolerant, it does not compete with food crops for land. METHODS Four soil treatments in the bottom chambers of two-chambered growing units (three nutrient levels in sandy loam and saline, dredged material) were used to test substrate influences on root structure and aerial growth in a greenhouse study. Simultaneously, underground reserves were determined by collecting aerial biomass of dark-grown plants. Root cores (15 × 50 cm) were taken to determine root architecture of 2-year-old field-grown plants. KEY RESULTS High nutrient availability increased the coarse root biomass (≥2 mm diameter) and increased capsule production. Plants grown in dredged sediment had no substantial growth differences from the controls. Underground reserves were significant. Field cores demonstrated that roots extended more than 50 cm with most of the coarse roots in the upper 20 cm and the coarse to fine root ratio decreasing with depth. Fine root surface area, measured using image-analysis software, was substantially greater than many other crops, as was root mass density. CONCLUSIONS Root architecture of K. pentacarpos and its responses to various nutrient levels and soil types demonstrate that it is a promising species for further development as a crop on coastal saline land including dredged material sites. Its perennial taproot system stores carbon and will perform buffer functions between upland and wetlands as sea level rises.