This research was funded and conducted under the Everglades Systems Assessment Section of the South Florida Water Management District.

1. Preparation and Transportation to Planting Location
<ol>
	<li>Obtain 2 or 3 gal (7 or 11 L) saplings of locally grown native trees (in plastic pots), commercial peat bags (measuring 0.3 x 0.46 x 0.6 m3), tree and shrub fertilizer spikes, duct tape, polyvinyl chloride (PVC) pipe (2 to 3 m length, 0.8 in (2.1 cm) diameter, schedule 40), box cutter, trash bags, and a metal rod or rebar (1 to 2 m in length, 1.3 cm diameter) to puncture the peat bags. Potential tree species applicable to the Everglades are Annona glabra, Ficus aurea, and Acer rubrum. Wearing work gloves is highly recommended.</li>
	<li>Determine the best location to deploy the pop-ups, typically in a high area of the ghost tree island with at least 0.3 m of peat remaining above the bedrock where inundation would be minimized during the wet season.</li>
	<li>Transport saplings, commercial peat bags (one per sapling), tree and shrub fertilizer spikes (one per sapling), duct tape (number of rolls as appropriate), PVC pipe (two per sapling), and other tools and materials to the planting location (typically by airboat).</li>
</ol>
2. Pop-up Assembly
NOTE: Assemble the potted sapling and pop-up (steps 2.1-2.6) in the boat prior to placing it in the water over the tree island (step 3)
<ol>
	<li>Slide the plastic pot off the sapling roots and pot soil, and cut a large hole in the pot bottom, or remove the entire bottom of each sapling pot using a box cutter or scissors (Figure 1).</li>
	<li>Slide the pot with the open bottom back onto the sapling roots and pot soil for insertion into the peat bag.</li>
	<li>Make a 20 to 30 cm X-shaped cut (slightly larger than the pot diameter) in the peat bag at the center of what will be the upper surface, using a box cutter.</li>
	<li>Remove about 2,000 to 5,000 cm3 (about one-half the volume of the pot) of the contents of the peat bag from the X-shaped opening and place in a trash bag for removal.</li>
	<li>Set the bottomless pot with the sapling within the X-shaped opening/depression created by removing the peat from the peat bag. The bottomless pot should be about halfway down in the X-shaped opening, with the points (triangles) pointing up against the sides of the pot (Figure 2). The saplings are left in the pots to provide additional elevation and physical support.</li>
	<li>Secure the pot in place with several windings of duct tape, around the pot and covering the bag triangles. This will affix the bag triangles to the pot (Figure 3) and hold the pot in place during the next 2-3 years of growth. This creates the pop-up.</li>
</ol>
3. Pop-up Deployment
<ol>
	<li>Set the pop-up in the water. Enter the water and move the floating pop-up to the planting site.</li>
	<li>Tether the pop-up in place with two approximately 2 to 3 m long lengths of PVC pipe inserted through the bag on either side of the sapling, into approximately one-third to one-half m of the substrate (soil beneath the bag) (Figure 3).</li>
	<li>Puncture the bags on the top, bottom, and sides by driving the metal rod (rebar) completely through the bag, creating openings (at least four in the top/bottom and four in each side) to allow water to enter and to allow the roots to emerge (Figure 3). The pop-ups will float for about 6 months.</li>
	<li>Insert one tree fertilizer spike entirely into the top of the bag in one of the locations shown in Figure 3.</li>
</ol>
4. Follow-up for Maintenance, Measurements, and Plastics Removal
<ol>
	<li>Return to the site as frequently as deemed necessary, particularly following severe storm events and note the condition and survival, and measure the tree height and basal diameter of each remaining tree on an annual basis.</li>
	<li>After 3 years, return to the site with box cutters/scissors and trash bags to remove the pots and plastic from the peat bag and recycle them. The pot and plastic from the bag (top and sides) are readily removed without disturbing the sapling; the plastic beneath the peat bag may not be accessible (Figure 4).</li>
	<li>Return to the site on an annual basis to evaluate survival and growth.</li>
</ol>

<ol>
	<li>Sklar, F. H., van der Valk, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tree Islands of the Everglades. , (2002).</li><li>Wetzel, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+tree+islands+in+the+Florida+Everglades:+nutrient+redistribution+is+the+key.">Maintaining tree islands in the Florida Everglades: nutrient redistribution is the key.</a> Front Ecol Environ. 3 (7), 370-376 (2005).</li><li>Aich, S., Ewe, S. M. L., Gu, B., Dreschel, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+peat+loss+from+an+Everglades+tree+island,+Florida,+USA.">An evaluation of peat loss from an Everglades tree island, Florida, USA.</a> Mires and Peat. 14 (2014), 1-15 (2014).</li><li>Dreschel, T. W., Cline, E. A., Hill, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everglades+tree+island+restoration:+testing+a+simple+tree+planting+technique+patterned+after+a+natural+process.">Everglades tree island restoration: testing a simple tree planting technique patterned after a natural process.</a> Restor Ecol. , (2017).</li><li>Stanturf, J. A., Conner, W. H., Gardiner, E. S., Schweitzer, C. J., Ezell, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognizing+and+Overcoming+Difficult+Site+Conditions+for+Afforestation+of+Bottomland+Hardwoods.">Recognizing and Overcoming Difficult Site Conditions for Afforestation of Bottomland Hardwoods.</a> Ecol Restor. 22 (3), 183-193 (2004).</li><li>Sharitz, R., Barton, C. D., DeSteven, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tree+Plantings+in+Depression+Wetland+Restorations+Show+Mixed+Success+(South+Carolina).">Tree Plantings in Depression Wetland Restorations Show Mixed Success (South Carolina).</a> Ecol Restor. 24 (2), 114-115 (2006).</li><li>Brandt, L. A., Portier, K. M., Kitchens, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+Change+in+Tree+Islands+in+Arthur+R.+Marshall+Loxahatchee+National+Wildlife+Refuge+from+1950-1991.">Patterns of Change in Tree Islands in Arthur R. Marshall Loxahatchee National Wildlife Refuge from 1950-1991.</a> Wetlands. 20 (1), 1-14 (2000).</li><li>Armentano, T. V., Jones, D. T., Ross, M. S., Gamble, B. W., Sklar, F. H., van der Valk, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetation+pattern+and+process+in+tree+islands+of+the+southern+Everglades+and+adjacent+areas.">Vegetation pattern and process in tree islands of the southern Everglades and adjacent areas.</a> Tree Islands of the Everglades. , 225-281 (2002).</li><li>Jones, D. T., Sah, J. P., Ross, M. S., Oberbauer, S. F., Hwang, B., Jayachandran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+physiological+responses+of+twelve+tree+species+common+in+Everglades+tree+islands+to+simulated+hydrologic+regimes.">Growth and physiological responses of twelve tree species common in Everglades tree islands to simulated hydrologic regimes.</a> Wetlands. 26 (3), 830-844 (2006).</li><li>Stoffella, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+growth+responses+of+eight+Everglades+tree+species+along+an+experimental+hydrological+gradient+on+two+tree+island+types.">Survival and growth responses of eight Everglades tree species along an experimental hydrological gradient on two tree island types.</a> Appl Veg Sci. 13 (4), 439-449 (2010).</li><li>Aich, S., Dreschel, T. W., Cline, E., Sklar, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+geographic+information+system+(GIS)+to+document+research+in+an+Everglades+physical+Model.">The development of a geographic information system (GIS) to document research in an Everglades physical Model.</a> J Environ Sci Eng. 5 (2011), 289-302 (2011).</li><li>Weinbaum, S. A., Johnson, R. S., DeJong, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+Consequences+of+Overfertilization+in+Orchards.">Causes and Consequences of Overfertilization in Orchards.</a> HortTechnology. 2 (1), 112-121 (1992).</li></ol>

This work was performed under funding and oversight of the South Florida Water Management District, an agency of the state of Florida. Florida enjoys a broad public records law. This document is subject to that law and considered to be part of the public record.

This proposed approach to replanting tree islands allows the increase in elevation and deployment of trees from an airboat without the building of roads required to bring fill to each site. This minimizes the cost and disturbance of implementing tree island plantings.
An issue with this technique is the aesthetic and environmental problems associated with putting a large quantity of PVC pipe, plastic bags, and plastic pots on a marsh feature that was once a tree island. The current approach is...

Natural recolonization of degraded Everglade tree islands by native tree species is possible through seed dispersal if there are nearby islands with a diversity of tree species1,2. According to Sklar and van der Valk1 restoring severely subsided tree islands may be possible, &#34;by raising the elevation of their heads, although such efforts would be expensive and labor intensive.&#34; Currently, there are very few native (desirable) trees present near the degraded (also known as &#34;ghost&#34;) tree islands in Everglades Water Conservation Area (WCA)-2A (one of five WCAs created to store water for and prevent flooding of surrounding urban and agricultural areas) to provide the seeds needed for recolonizing the degraded tree islands3.
To raise the elevation of a subsided tree island for reforestation, appropriate fill would have to be transported to the island. This would require building roads for trucks or creating waterways for barges, leading to severe damage to the Everglades at a great financial cost. A simple, low cost technique is needed to repopulate degraded tree islands with native tree species that will provide elevation (reduce hydroperiod) and improve survival under the variable hydrology endemic to the Everglades4.
The size of these pop-ups allows the ease of transportation to the planting site. This provides an advantage compared to conventional filling of areas and planting of trees which may require roads be built for transporting. This technique thus reduces the ecological impact of tree planting activity. Additionally, these pop-ups have the ability to float for the initial six months following deployment, to allow establishment of the sapling. Under conventional tree planting scenarios, the trees may experience increased flooding stress during establishment if water levels rise.
A number of designs were tested and it was determined that greater pop-up height (bag orientation), one tree fertilizer spike to add nutrients, and planting the pop-up with a single sapling achieved the best configuration for deployment upon an Everglade degraded tree island4. Although other researchers describe the planting of trees for restoring forested wetland areas5,6, to our knowledge, none of the planting techniques described added elevation to reduce hydroperiod.
This procedure mimics the formation of floating Everglades tree islands that arise from peat that breaks away from the bedrock and floats to the surface. Upland plants can establish to form a &#34;pop-up&#34; or floating tree island which eventually moves and settles onto the peat surface and becomes what is termed a &#34;battery&#34; tree island1,7.
Everglades tree island tree species have varying tolerances to flooding for growth and survival8,9,10. In a controlled study, Jones et al. demonstrated the importance of the hydroperiod to the survival of Everglades tree island species9. Stoffella et al. found that relative elevation (to the water surface) had varying and profound effects on planted native tree species survival and growth, due to the specific tolerance of each species to flooding10. Tree species can be selected for the specific conditions present. The tree planting technique described here can provide additional elevation and consequently, tree seed sources in formerly forested regions that have lost elevation and are now subject to an inundation environment that is harmful to native tree species.
Formerly forested regions that experience extended hydroperiods might benefit from this method to reintroduce woody species to these areas. This technique provides elevation, thus reducing the hydroperiod. In addition, the trees will build soil (root and leaf biomass) and provide a seed source for continued colonization of the area.

<table><tbody><tr><td>One or Two Gallon Native Tree Saplings in plastic pots.</td><td>Local Native Plant Nurseries</td><td>N/A</td><td>Obtain native tree saplings grown from locally obtained seed</td></tr><tr><td>Commercial Peat Bags. 5 cubic ft.</td><td>Lambert</td><td>Lambert Canadian Sphagnum Peat Moss (6649801115)</td><td>Product description: Lambert peat moss is an excellent soil amendment because of its capacity to retain water, aerate the soil and it is completely natural. All of Lambert&#039;s peat moss packages carry the OMRI (Organic Material Review Institute) logo, which indicates that they are approved for use in organic production. Size: 5.5 cu.ft.</td></tr><tr><td>Vigoro Tree Spikes (Tree and Shrub Fertilizer Spikes, 15-Count)</td><td>Vigoro, Swiss Farms Products Inc.</td><td>Model # 154205</td><td>GUARANTEED ANALYSIS (%) TOTAL IN PRODUCT (ppm): Total Nitrogen (N) 12.0000; Available Phosphoric Acid (P2O5) 5.0000; Soluble Potash (K2O) 7.0000</td></tr><tr><td>PVC Pipe: 1/2 in schedule 40</td><td>JM Eagle</td><td>Model # 67447</td><td>Any manufacturer or supplier will do.</td></tr><tr><td>Duct Tape, 1-7/8 in x 12 yds</td><td>MSC Industrial Supply</td><td>Gorilla Heavy-Duty Duct Tape, Model # 60012</td><td>Any manufacturer or supplier will do.</td></tr><tr><td>Metal Rod (Rebar 1.3 cm diameter, 1.2 m long)</td><td>Weyerhaeuser</td><td>Model # 35616</td><td>Any manufacturer or supplier will do.</td></tr><tr><td>Box Cutter</td><td>Stanley</td><td>Model # STHT10273</td><td>Any manufacturer or supplier will do.</td></tr><tr><td>Work Gloves</td><td>Firm Grip</td><td>Model # 3101-96</td><td>Any manufacturer or supplier will do.</td></tr><tr><td>Trash Bags (42 gallon)</td><td>HUSKY</td><td>Model # HK42WC050B</td><td>Any manufacturer or supplier will do.</td></tr></tbody></table>

a simple planting technique for re-establishing trees where frequent inundation occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

The study using this technique4 was conducted within a 0.32 km2 Everglades physical model11, on a ridge to simulate testing on a ghost tree island. During this study, two bag orientations for height (low: 0.3 m, high: 0.46 m), with one versus two saplings per bag growing, were tested as well as the use of tree fertilizer spikes to promote survival and growth. Three&#160;species of saplings were used: A. glabra, F. aur...

Watch this Scientific Journal Video about A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs at JoVE.com

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

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6.1K Views.  South Florida Water Management District. This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions. 

Video: A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

A Venturi Effect Can Help Cure Our Trees

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. According to these factors and their dynamics, small amounts of sap-compatible liquids (i.e. pesticides) can be injected into the xylem system, reaching their target from inside. This endotherapic method, called &#34;trunk injection&#34; or &#34;trunk infusion&#34; (depending on whether the user supplies an external pressure or not), confines the applied chemicals only within the target tree, thereby making it particularly useful in urban situations. The main factors limiting wider use of the traditional drilling methods are related to negative side effects of the holes that must be drilled around the trunk circumference in order to gain access to the xylem vessels beneath the bark.
The University of Padova (Italy) recently developed a manual, drill-free instrument with a small, perforated blade that enters the trunk by separating the woody fibers with minimal friction. Furthermore, the lenticular shaped blade reduces the vessels&#39; cross section, increasing sap velocity and allowing the natural uptake of an external liquid up to the leaves, when transpiration rate is substantial. Ports partially close soon after the removal of the blade due to the natural elasticity and turgidity of the plant tissues, and the cambial activity completes the healing process in few weeks.

Compared to the more traditional hole-based methods, most of which require the tree to be drilled, tools with lenticular blades transform the basics of endotherapy easing the closure of the wound and allowing the natural uptake of the solutions.

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. ...

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

A Method for Quantifying Foliage-Dwelling Arthropods

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density requires a method with high detection probability and a known sampling area. While most described methods provide a qualitative or semi-quantitative estimate adequate for describing species presence, richness, and diversity, few provide an adequately consistent detection probability and known or consistent sampling areas to provide an index or estimate with adequate precision to detect differences in abundance across environmental, spatial, or temporal variables. We describe how to quantify leaf-dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate and quantify them. As we demonstrate, this method can be used at a landscape scale to quantify leaf-dwelling arthropods with adequate precision to test for and describe how spatial, temporal, environmental, and ecological variables influence arthropod richness and abundance. This method allowed us to detect differences in density, richness, and diversity of leaf-dwelling arthropods among 5 genera of trees commonly found in southeastern deciduous forests.

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density ...

Using Tree-Rings to Reconstruct Fire History Information from Forested Areas

Annual tree-ring patterns are a source of ecological and environmental information including the history of fires in forested areas. Tree-ring based fire histories include three fundamental phases: field collection, laboratory methods (preparation and dating), and data analysis. Here we provide step-by-step instructions and issues to consider, including the process for selecting the study area, sampling sites, plus how and which fire-scarred trees to sample. In addition, we describe fire-scar sample preparation and dating which are done in the laboratory. Finally, we describe basic analysis and relevant results, including examples from studies that have reconstructed fire history patterns. These studies allow us to understand the historical fire frequency, changes in those frequencies related to anthropogenic factors, and analyzes of how climate influences fire occurrence over time. The description of these methods and techniques should provide a greater understanding of fire history studies that will benefit researchers, educators, technicians, and students interested in this field. These detailed methods will allow new researchers to this field, a resource to start their own work and achieve greater success. This resource will provide a greater integration of tree-ring aspects within other studies and lead to a better understanding of natural processes with forested ecosystems.

This work explains the most appropriate techniques and methods for conducting a fire history study from beginning site selection to final analysis of fire-climate relationship.

Annual tree-ring patterns are a source of ecological and environmental information including the history of fires in forested areas. Tree-ring based fire ...

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

In Situ Soil Moisture Sensors in Undisturbed Soils

Soil moisture directly affects operational hydrology, food security, ecosystem services, and the climate system. However, the adoption of soil moisture data has been slow due to inconsistent data collection, poor standardization, and typically short record duration. Soil moisture, or quantitatively volumetric soil water content (SWC), is measured using buried, in situ sensors that infer SWC from an electromagnetic response. This signal can vary considerably with local site conditions such as clay content and mineralogy, soil salinity or bulk electrical conductivity, and soil temperature; each of these can have varying impacts depending on the sensor technology.
Furthermore, poor soil contact and sensor degradation can affect the quality of these readings over time. Unlike more traditional environmental sensors, there are no accepted standards, maintenance practices, or quality controls for SWC data. As such, SWC is a challenging measurement for many environmental monitoring networks to implement. Here, we attempt to establish a community-based standard of practice for in situ SWC sensors so that future research and applications have consistent guidance on site selection, sensor installation, data interpretation, and long-term maintenance of monitoring stations.
The videography focuses on a multi-agency consensus of best-practices and recommendations for the installation of in situ SWC sensors. This paper presents an overview of this protocol along with the various steps essential for high-quality and long-term SWC data collection. This protocol will be of use to scientists and engineers hoping to deploy a single station or an entire network.

In Situ Soil Moisture Sensors in Undisturbed Soils

The determination of soil water content is a critical mission requirement for many state and federal agencies. This protocol synthesizes multi-agency efforts to measure soil water content using buried in situ sensors.

Soil moisture directly affects operational hydrology, food security, ecosystem services, and the climate system. However, the adoption of soil moisture ...

Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion annually. The global decline of giant kelp forests due to climate-driven ecological stressors underscores the need for innovative restoration strategies. An emerging restoration technique known as &#39;green gravel&#39; aims to seed young kelps over large areas without extensive underwater labor and represents a promising restoration tool due to cost-effectiveness and scalability. This video article illustrates a protocol and tools for culturing giant kelp, Macrocystis pyrifera. It also provides a resource for further studies to address the successes and limitations of this method in field settings. We outline field and laboratory-based methods for collecting reproductive tissue, sporulating, inoculating, rearing, maintaining, and monitoring substrates seeded with early life stages using the &#39;green gravel&#39;&#160;technique. The protocol simplifies and centralizes current restoration practices in this field to support researchers, managers, and stakeholders in meeting kelp conservation objectives.

This protocol describes the field collection and regular laboratory maintenance of substrates seeded with canopy-forming giant kelp for use in restoration trials to address the success and limitations of the 'green gravel' technique in field settings.

Canopy-forming kelps are essential foundation species, supporting biodiversity and providing ecosystem services valued at more than USD$500 billion ...

Responses to Drought and Flooding

Water plays a significant role in the life cycle of plants. However, insufficient or excess of water can be detrimental and pose a serious threat to plants.

Under normal conditions, water taken up by the plant evaporates from leaves and other parts in a process called transpiration. In times of drought stress, water that evaporates by transpiration far exceeds the water absorbed from the soil, causing plants to wilt. The general plant response to drought stress is the synthesis of hormone abscisic acid that keeps stomata closed and reduces transpiration. Additionally, plants may respond to extreme water insufficiency by shedding leaves. This method, however, reduces photosynthesis and consequently hampers plant growth.

Mitigation of drought stress in plants by microbes

Drought stress limits the growth and productivity of plants in arid and semi-arid regions. However, certain microbes present in the vicinity of plants may release physical and chemical signals that induce changes related to plant defense under drought conditions. For example, the soil bacterium Paenibacillus polymyx is reported to induce drought tolerance in Arabidopsis. The most significant effect of this bacteria was observed in the growth of legumes under water stress. Leguminous plants depend on soil rhizobium for nitrogen fixation - but rhizobia are extremely sensitive to drought stress, resulting in very low nitrogen fixation. However, soil mixed with P. polymyx resulted in increased nitrogen fixation by rhizobium and increased growth of the bean plant.

Excess water is equally as disastrous to plants as a lack of water. Too much water can suffocate plants by reducing air spaces in the soil, thereby restricting oxygen needed for cellular respiration. Certain woody plant species respond to flood conditions by developing hypertrophic growth that appears as swelling of tissues at the stem base. This hypertrophic growth may aid in the downward diffusion of oxygen as well as potential venting of toxic compounds (carbon dioxide, methane, and ethanol) formed from anaerobic metabolism. Other adaptive responses to flood stress include the formation of adventitious roots, increases in root porosity via specialized cells called aerenchyma cells, and a suberized exodermis to prevent loss of oxygen.

Plant Responses to Drought and Floods

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Water plays a significant role in the life cycle of plants. However, insufficient or excess of water can be detrimental and pose a serious threat to ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

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