Innovative Phragmites Control Strategies
This project has provided an exceptional start on a two-pronged approach to find more sustainable invasive species control strategies for landowners and resource managers. Scientists have identified and isolated endophytic fungi in Phragmites, something not previously documented, and produced preliminary results suggesting that eliminating endophytes in Phragmites reduces the appearance of adult plants and reduces the seed bank.
The endophyte data from 2011 are very encouraging and suggest that in some invasive plants, managing fungal endophytes inside the plants may be an effective management strategy. In 2012, we will expand these efforts for Phragmites in the Great Lakes and, using complementary funds, Halogeten glomeratus in the Great Basin. In addition, we will begin testing a new class of chemicals that are nontoxic to animals but very toxic to microorganisms to characterize the involvement of soil microorganisms in the invasion process.
We continued the development of RNAi as a species-specific management tool to control invasive Phragmites and to restore native populations. Our goal is to utilize this short nucleic acid sequence specificity to silence specific genes that contribute to the competitive vigor and fitness of invasive pest species. This specificity will allow us not only to target specific genes in species of interest, but will also prevent negative effects on non-targeted native species. Recent accomplishments include:
- Obtained MSV virus and isolated and cloned genes required for viral genomic replication.
- Constructed two new potential RNAi vectors, one using a hybrid of MSV and BCTV genes and one based entirely on MSV genome.
- Initiated tests for vector replication and gene silencing in <i>Phragmites</i> and maize.
- Obtained GATEWAY based RNAi vectors. Designed and cloned GATEWAY compatible sequences from the Phragmites rbcS gene.
- Identified, isolated, and cloned Phragmites homologue of AGAMOUS, a required gene for pollen and seed production, and PISTILLATA, a gene required for pollen and lemma development. Additionally, isolated and cloned PDS from maize, a gene required for normal chlorophyll production.
These results and the overall direction of this project directly support the broader Integrated Pest Management (IPM) approach being developed for Phragmites and many other invasive plants. There are many opportunities to work with the existing partners (e.g., SEMCOG, USFWS, TNC, GLC, DU) to build on this work, integrate with IPM efforts, and target our field trials in AOCs or other high priority sites.
Invasive species cost the United States billions of dollars annually, disrupt natural ecosystems, and consequently threaten not only native species but also the ecosystem service humans obtain from these ecosystems. Phragmites, a tall wetland grass, has been a component of wetlands in the USA for several thousand years. However, a novel genotype introduced from Europe began displacing the native genotype in early 20th century and rapidly spread across the continent. This alien Phragmites alters the soil, produces copious seeds (that can remain dormant for several years), and can propagate vegetatively producing dense, nearly monoculture stands. As Phragmites invades an area, plant species diversity declines and critical habitat for fish, reptiles, amphibians, and birds is lost. Presently, Phragmites has colonized many marshes throughout the eastern United States, is rapidly invading the few remaining marshes in the Great Lakes, and has spread to the Pacific coast, where it is again displacing local vegetation.
Conventional wisdom holds that introduced plants are largely unaffected by predators, pathogens, and other factors that would naturally regulate their density (i.e., the enemies release hypothesis). Native plants, having this density-dependent regulation, are at a consequent competitive disadvantage. Hence, invasive plants are thought to displace native plants simply because the introduced plants have fewer enemies. However, the problem may be more complex.
What has not been previously considered is that the introduced plants may have microbial helpers. Research has shown that native plants in the western USA could not grow in areas that had been invaded by Eurasian plant Halogeton unless the soil had been autoclaved or treated with methylbromide to kill the microbes associated with the invading Halogeton. It is well known that all plants form associations with microbes, and the colonizing success of plants depends upon not only their genotype but also on forming the right associations with the microbial community. We believe that these associations provide potential control targets in dealing with invasive plants. These targets have not previously been examined as potential control measures. If the associations between invasive Phragmites and its microbes can be disrupted, the competitive advantage of Phragmites may be reduced and native plant assemblages can be maintained when invasive Phragmites is present.
A second innovative control method potentially lies in silencing the genes that control the species specific competitive abilities of Phragmites. USGS will work with Wayne State University scientists to develop a biological control regimen involving a species-specific reduction of Phragmites’ invasive and competitive abilities. Scientists at Wayne State University have developed a gene-silencing vector that has been successfully applied to a number of dicot plant species. This vector acts to suppress the activity of specific genes by targeting the initial products of the gene for degradation. As long as the exact gene sequence is known, any gene may theoretically be chosen for treatment. As a proof of principal, it has been demonstrated that genes involved in photosynthesis, flower organ formation, and organ number can be silenced in spinach, tobacco, and tomato. Wayne State scientists have demonstrated that the vector can be applied either through biolistic or Agrobacterium applications. The vector cannot spread either systemically through the plant or be transferred incidentally to other individuals and thus should be safe to apply in an open area. The proposed project will develop this vector or a similar vector to be applied to Phragmites. Target genes involved in flower, and thus seed and pollen production, and in rhizome development will be the primary focus. If these genes were successfully silenced, colonization of new areas could be reduced, the spread of established colonies through aggressive vegetative expansion could be minimized, and native plant species could be allowed to compete and eventually replace Phragmites without causing ancillary ecological disturbances.
The two-pronged approach of this project (i.e., first targeting the organisms that may help Phragmites spread and second using a molecular genetic approach to silence the genes in Phragmites that allow it to reproduce and grow) will involve researchers from the USGS – Great Lakes Science Center, Wayne State University, University of Washington, USGS - Western Fisheries Research Center, and State University of New York – Brockport. Phase one of this work will be initiated as a FY10 GLRI project coordinated by the USGS – Great Lakes Science Center.
This project addresses mission, vision, and multiple priorities identified in the USGS Strategic Plan and the USGS – Great Lakes Science Center Strategic Plan. The USGS - Great Lakes Science Center has the mission, staff, facilities, and equipment to lead the cutting-edge research involved in this basin-wide effort. Approximately 70% of the funding will go to Wayne State University for experimental design, laboratory testing, data analysis, and reporting; 26% will go to the USGS – Western Fisheries Research Center for greenhouse and laboratory experiments and data analysis; 3% of the annual funding will go to SUNY – Brockport for assistance with field work and data analysis; 1% of the annual funding will go to the USGS – Great Lakes Science Center for project management. Subawards are not expected.
This project would address a basin-wide issue by collecting samples from Saginaw Bay of Lake Huron, Lake St. Clair, and western Lake Erie and developing products applicable basin wide.
How can our knowledge of endophytes and gene silencing potentially result in control of invasive plants?
- Determine if there is a delay in rapid Phragmites expansion after initial colonization due to the fungi and plant evolving a relationship that permits the fungus to invade the plant’s ovary and integuments without harming the remainder of the ovule and subsequent seed.
- Determine if invasive plants have broadly adapted endophytes, or if the plants are able to acquire or replace endophytes more easily than non-invasive or native plants.
- Test whether infecting plants with antagonistic endophytes or pathogens of the endophyte limit the ability of the plants to grow, survive, and reproduce.
- Determine if chemically inhibiting the growth of the endophyte, inside the plant, reduces the plant’s vigor or reproductive potential.
- Determine if altering the soil chemistry changes the symbiotic relationship between host and endophyte into a pathogenic one for native species.
- Evaluate whether the floral organ identity and root meristem identity genes can be identified in Phragmites.
- Test whether the known gene-silencing vector (pWSRi) can be developed to suppress the activity of specific genes (e.g., those responsible for seed production and rhizome growth).
To answer these questions and other questions, we propose to examine multiple populations and conduct manipulative experiments using common reed (Phragmites australis (Cav.) Trin ex Stuedel)).
Phragmites continues to spread throughout the Great Lakes and have negative impacts on coastal resources including critical fish and wildlife habitat and coastal viewscapes. Current control strategies are time, labor, and resource intensive, so innovative methods to control the spread of Phragmites or minimize its invasive properties are needed. This study will test new strategies to reduce the invasive properties of Phragmites and minimize its competitive advantage.
This restoration project is guided by the Great Lakes Science Center Strategic Plan (September 2005). It is consistent with the U.S. Climate Change Science Program strategic goals, the research goals of multiple programs within USGS (i.e., Ecosystems, Invasive Species, Fisheries: Aquatic and Endangered Resources), and initiatives endorsed by the Great Lakes Regional Collaboration. The proposed work also addresses many of the priority issues identified in the Lake Huron, Lake Erie, and other LaMPs including restoring wetland fish habitats, rehabilitating nearshore habitats, reducing impact of invasive species, and protecting island habitats. Priority management areas (e.g., Saginaw Bay, Lake Huron) and Areas of Concern (e.g., Maumee River, Ohio) are included as study sites in this project.
ENDOPHYTES – We will conduct laboratory experiments to investigate the relationships between Phragmites, endophytes, and chemical agents. We will also conduct field experiments in Phragmites invasion zones at three sites in the Great Lakes region: the Crane Creek drowned river mouth wetland within Ottawa National Wildlife Refuge in western Lake Erie that has deep silt sediments, a site at the inner portion of Saginaw Bay of Lake Huron that has thin silt soils overlying sand, and a site in Lake St. Clair with variable soils. Within each invasion zone, we will establish subzones of a native community, a Phragmites community, and a transition zone.
In laboratory studies, we propose to:
- Isolate and identify fungal endophytes from lower plant sections (roots, crown and stem tissue) and seeds of Phragmities collected at several locations around the great lakes and Washington state.
- Grow endophyte colonized (symbiotic) and noncolonized (nonsymbiotic) Phragmities plants and compare growth, development and stress tolerance (drought, salinity, temperature).
- Grow endophyte colonized (symbiotic) and noncolonized (nonsymbiotic) Phragmities plants in nutrient poor and standard soils, and compare nutrient and elemental content of plant tissues.
- Treat Phragmites with divalent metals ions (Cu, Zn, Mn, Fe) in a proprietary carrier coupled with a surfactant in an attempt to either kill the plants directly or interfere with microbial symbiosis. The carrier is not a chelating agent and promotes a strongly reducing environment. The combination of metal and surfactant should kill Phragmites either by the metal binding to electrons in photosynthesis, glycolysis, and the electron transport chain or via the generation of free radicals (cells use metal ions to generate OH- radicals inside of cells).
- Alter the internal balance of Ca to alter or transform the endophyte symbiotic relationship into a pathogenic relationship—which has been shown to work in tomato (Rodriguez, unpublished data).
In field studies, within each subzone, we propose to characterize existing plant communities prior to experimentation by:
- Measuring plant height and clone circumference of Phragmites
- Developing a proxy for estimation of Phragmites biomass
- Measuring Phragmites seed production and collecting seeds for germination studies
- Identifying and quantifying all other plant taxa present
In field studies, within each subzone, we propose to characterize:
- The elemental concentrations of plant macro- and micro-nutrients in both soils/sediments and plants
- Microbial communities in the rhizosphere
- Endophytic communities
In field studies, within each subzone, we propose to conduct reciprocal transplant experiments involving:
- Moving the dominant plants from each subzone into each subzone within a site
- Moving soils/sediments from each zone into other zones and then conducting plant transplants within each sediment type in each subzone in a full factorial design
- Infecting previously sterilized plants from each subzone with each type of endophyte and then transplanting them into each type of sediment in each subzone
Transplant experiments will be allowed to grow for three years. Each year, we will characterize and quantify the total plant community, assess Phragmites survivorship, and measure Phragmites plant height, clone circumference, seed production, and seed germinability. Photosynthesis, transpiration, and stomatal conductance rates will be determined three times, per plant, during each growing season. At the end of three years, all Phragmites plants will be harvested, divided into above- and below-ground portions, dried, and biomass determined (g). In addition, we will determine the elemental concentrations for elements examined in the sediments (N, P, K, Ca, Cu, Fe, Mg, Mn, Na, Mo, Zn). We will also determine the extent to which each part of the plant (root/rhizome, stem, leaf, seeds) was infected with each of the principal endophytes. Laboratory experiments will be scored for survivorship, growth, and biomass.
GENE SILENCING – Aim 1: At the present time, there are 359 nucleotide sequences listed in GenBank for Phragmites australis. The vast majority of these sequences are chloroplast sequences used in phylogenetic analyses, microsatellite sequences, or highly conserved ribosomal protein sequences. There are no entries for floral or root developmental genes that may be used for species-specific RNAi knockdowns. To effect seed-set, we will focus on the floral organ identity genes involved in stamen and carpel formation, specifically the AGAMOUS homologue. To effect root growth, we will focus on homologues of SCARECROW, SHORT ROOT, and/or PLETHORA. To downregulate energy acquisition, we will focus on rbcS and MG chelatase.
Degenerate primers will be designed based on published sequences from within the Poaceae. Specifically for AGAMOUS, the following sequences will be used: Zea ZAG1(NM_001111851.1), ZAG2(NM_001111908.1), Oryza Os01g0886200 (NP 001045028.1). For the SCARECROW homologue, we will use the following: Zea scl1 (541704),scl23 (542138), Oryza OJ1003C07.9, Ozsa7075. For SHORT ROOT, we will use Oryza 4343769, 4343753. For Mg Chelatase, we will use: Zea oy1 (732841), Oryza 4332690, 4344148, 4332690. rbcS is available for multiple species and will be downloaded. Putative Phragmites homologues will be cloned initially into pGEM Teasy after amplification with an error reducing polymerase (eg. Phusion or Advantage Taq) and will be sequenced. Standard gene phylogenetic analyses will be used to confirm homology.
Aim 2: pWSRi vectors have been developed into forms that can be applied biolistically or via Agrobacterium (pWSRiA). We will test the efficacy of these vectors in Phragmites using the genes cloned in Aim 1. Phenotypic markers and mRNA levels will be tested. Experimental Design: The genes cloned in Aim 1 will be subcloned into the pWSRi vectors. Our experience indicates that fragments of approximately 200-250 bp work well in knockdown experiments. We will identify regions that are specifically highly variable among species to generate species-specific effects in Phragmites. Multiple regions will be used per gene if possible. The pWSRi constructs will be applied to plants via a gene gun. Alternatively, Agrobacterium strains will be transformed with the pWSRiA vectors and applied to seedling leaves using syringes without needles. Plants will initially be tested for the rbcS and chelatase genes, as these will be the easiest to detect phenotypic effects. Affected tissue will then be harvested to extract total RNA. Actual knockdown of the specific mRNAs will be determined using qRT-PCR. We will use the Ambion 18S-competimers as the internal control. Alternatively, a housekeeping gene such as G6pdh may be used. Effects on root growth and flowering will be more time-consuming. The root growth will be determined in plants grown in agar or vermiculite. Root mass will be observed and then weighed when visible effects are detected. Flowering effects will be detected visually. Similar confirmation using qRT-PCR will be used.
Results of these experiments will be used to guide future proposals focused on potential implementation strategies for using successful techniques to control the invasive Phragmites haplotype M. Those experiments would include evaluation of collateral impacts on native species.
After one year, this project is expected to 1) identify whether Phragmites has endophytes; if endophytes are detected, identify how they differ depending upon location, habitat and native vs introduced Phragmites, 2) characterize how Phragmites grows in the absence of endophytes, and 3) test how endophytes could be killed using a specifically designed solutions. The gene silencing work is expected to 1) identify floral or root developmental genes in Phragmites, 2) describe the utility of the pWSRi vectors on Phragmites, and 3) evaluate the treatment on flowering and root growth in greenhouse experiments.
The first year of this project is expected to produce professional presentations and information products (e.g., USGS factsheet, web page) for managers and the public. Initial results are expected to be available by the Summer of 2010. Results of this project will be used to guide future proposals focused on potential implementation strategies for using successful techniques to control the invasive Phragmites haplotype M. Those experiments would include evaluation of collateral impacts on native species.
Point of Contact:
Project Lead: Kurt P. Kowalski
Lead Scientist: D. Carl Freeman
D. Carl Freeman
Dept. of Biological Sciences,
Wayne State University,
Detroit, MI 48202
Regina S. Redman
Dept. of Biology AND College of Forest Resources,
University of Washington,
Seattle, WA 98195
Russell J. Rodriguez
Dept. of Biology AND College of Forest Resources,
University of Washington AND USGS-Western Fisheries Research Center,
Seattle, WA 98115
Edmond Van Hees
Dept. of Geology,
Wayne State University,
Detroit, MI 48202
Edward M. Golenberg
Dept. of Biological Sciences,
Wayne State University,
Detroit, MI 48202
Douglas A. Wilcox
Dept. of Environmental Science and Biology,
Brockport, NY 14420
USGS Great Lakes Science Center,
Ann Arbor, MI 48105