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 to remove the plastics after a sufficient period of tree establishment (approximately 3 years) and properly dispose of or recycle the plastic debris.
Another possible solution to the plastics issue is to utilize biodegradable materials for the tethers (wooden or bamboo poles), pots (peat pots), and peat bags (burlap or a similar material) with the intent that the containment material would maintain integrity and contain the soil and peat until the tree sapling was established in place. Since the purpose of this study was to demonstrate the proof-of-concept and to keep the cost reasonable, the original containers of the trees and peat were modified for use in this study. This allowed for the implementation of a basic study in which the container materials were likely to maintain integrity for the duration of tree establishment.
We have observed that some of the bags began to split apart after about 30 months following deployment. Removal of the plastics should take place when this is first observed to avoid plastic waste floating away. Preliminary observations following the removal of the plastics from some of the pop-ups after about 32 months indicate that the roots of many of the saplings and of the other plants colonizing the peat bags can maintain the consolidation of the peat and thus the elevation provided solely by the peat and pot soil at deployment (Figure 4).
We demonstrated a promising technique for planting trees in regions of limited access and variable hydrology at a retail cost of less than 20 USD (sapling = 3-4 USD, peat bag = 11 USD, other materials = 2-3 USD) per pop-up. This technique will work best in peat or organic soil-based regions with a potential to restore forested areas. We developed the technique with the goal of restoring degraded tree islands in the Everglades but it could readily be adapted to other environments with similar restoration goals.

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&#39;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. aurea, and A. rubrum. For each species, 5 replicates of 4 treatments and 2 sets of controls were planted for a total of 35 individuals of each specie. Most of the saplings grown in the study survived and thrived, with growth dependent upon the treatment4. Survival rate was 80%, with 18 F. aurea and 3 A. glabra found dead at the end of the 3 year study. Results indicate that the simplest and most effective configuration has a single sapling and provides the higher profile of 0.46 m (greater height, less flooding), with one fertilizer spike, and without additional flotation. Since the daily growth rate in double planted bags was less than the control, this treatment should not be used4. The reduction in growth by saplings receiving two fertilizer spikes as opposed to a single spike may be attributed to over-fertilization. Over-fertilization has been suggested as causing poor growth by altering soil chemistry and the soil microbial community, as well as leading to poor root establishment and reduced water absorption12.
Sapling basal diameter data for the high-profile peat bag orientation yielded a statistically significant treatment effect as well as a statistically significant difference between the low fertilizer treatment data compared to the controls and compared to the high fertilizer treatment4. The surface of the soil in the pots and the punctures in the top surface of the peat bags provided sites for the colonization of herbaceous plants and ferns over the time of the study4 (Figure 4). In addition, willow trees (Salix caroliniana) invaded several of the pop-ups as well.
The saplings responded to the treatments generally as predicted. The fertilized trees generally were the most successful (and the one spike treatment was even better), followed by the controls (single planting), the flotation treatment, and double planting (no statistically significant effects)4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56636/56636fig1.jpg" /> 
Figure 1: Photograph of removal of the sapling pot bottom. The sapling roots and soil are removed from the plastic pot and a large portion (or the complete bottom) of the pot bottom is removed with a box cutter. The pot is then replaced over the roots and soil. All photographs are courtesy of the South Florida Water Management District. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56636/56636fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56636/56636fig2.jpg" /> 
Figure 2: Insertion of bottomless sapling pot into the peat bags, with cut triangles shown on the sides. The photograph shows a bag with two pots inserted although the preferred method was determined to be one pot per bag. The bag shown also is in the low-profile configuration, which is not the preferred orientation. For the preferred final orientation, refer to Figure 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56636/56636fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56636/56636fig3.jpg" /> 
Figure 3: Photographs of an A.glabra sapling with the bottomless pot duct-taped to the peat bag soon after deployment. Complete pop-up shown floating in place on the left; closer view shown on the right. (Labels) A: peat bag; B: potted sapling; C: duct tape around pot to hold in place; D: PVC pipe tethers; E: holes made by inserting a metal rod; F: fertilizer spikes (the two-spike treatment is shown, however, adding only one spike is recommended). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56636/56636fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56636/56636fig4.jpg" /> 
Figure 4: Photograph of a pop-up following pot and bag removal. Peat from the bag and the pot soil are now held together by the roots which have also grown down into the substrate. Note the fern and other plants that have colonized the mound of peat remaining from the pop-up. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56636/56636fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Environment

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

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

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. ...

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal behavior questions. The protocol described here provides a simple, reproducible, and controlled method to manipulate DO to study behavioral response in aquatic organisms resulting from hypoxic and anoxic conditions. While performing degasification of water with nitrogen is commonly used in laboratory settings, no explicit method for ecological (aquatic) application exists in the literature, and this protocol is the first to describe a protocol to degasify water to observe organismal response. This technique and protocol were developed for direct application for aquatic macroinvertebrates; however, small fish, amphibians, and other aquatic vertebrates could be easily substituted. It allows for easy manipulation of DO levels ranging from 2 mg/L to 11 mg/L with stability for up to a 5 min animal-observation period. Beyond a 5 min observation period water temperatures began to rise, and at 10 min DO levels became too unstable to maintain. The protocol is scalable to the study organism, reproducible, and reliable, allowing for rapid implementation into introductory teaching labs and high-level research applications. The expected results of this technique should relate dissolved oxygen changes to behavioral responses of organisms.

This article describes a simple and reproducible protocol to manipulate dissolved oxygen conditions in a laboratory setting for animal behavior studies. This protocol may be used in both teaching and research laboratory settings to evaluate organismal response of macroinvertebrates, fishes, or amphibians to changes in dissolved oxygen concentration.

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Forced Flowering in Mandarin Trees under Phytotron Conditions

Phytotron has been widely used to assess the effect of numerous parameters on the development of many species. However, less information is available on how to achieve fast profuse flowering in young fruit trees with this plant growth chamber. This study aimed to outline the design and performance of a fast clear methodology to force flowering in young mandarin trees (cv. Nova and cv. Clemenules) and to analyze the influence of induction intensity on inflorescence type. The combination of a short water stress period with simulated spring conditions (day 13 h, 22 &#176;C, night 11 h, 12 &#176;C) in the phytotron allowed flowers to be obtained only after 68-72 days from the time the experiment began. Low-temperature requirements were adequately replaced with water stress. Floral response was proportional to water stress (measured as the number of fallen leaves): the greater the induction, the larger the quantity of flowers. Floral induction intensity also influenced inflorescence type and dates for flowering. Details on artificial lighting (lumens), photoperiod, temperatures, plant size and age, induction strategy and days for each stage are provided. Obtaining flowers from fruit trees at any time, and also several times a year, can have many advantages for researchers. With the methodology proposed herein, three, or even four, flowering periods can be forced each year, and researchers should be able to decide when, and they will know, the duration of the entire process. The methodology can be useful for: flower production and in vitro pollen germination assays; experiments with pests that affect early fruit development stages; studies on fruit physiological alterations. All this can help plant breeders to shorten times to obtain male and female gametes to perform forced-crosses.

Here, we present a protocol to force flowering in mandarin trees under phytotron conditions. Water stress, high illuminance and a simulated spring photoperiod allowed viable flowers to be obtained in a short period of time. This methodology allows researchers to have several flowering periods in 1 year.

Phytotron has been widely used to assess the effect of numerous parameters on the development of many species. However, less information is available on ...