The author thanks the Patent Office of University of Padova, the TeSAF Department and Vitzani srl for their financial and technical support that allowed the development of the instrument, Dr. Jonathan Cocking (UK) for his kind cooperation and linguistic revision, the Municipality of Ponte San Nicol&#242; PD for kind hospitality during the video shooting, and the anonymous reviewers for their purposeful, detailed suggestions.

<ol>
	<li>Perform treatments preferably between bud break and late summer, in sunny and breezy days, according to general rules in tree endotherapy17.</li>
	<li>Wear gloves, protective glasses and any other safety device according to regulations in force.</li>
	<li>Get familiar with the instrument, disassembling and reassembling it. Its main components are reported in Figure 2.</li>
	<li>Get familiar with the method, described in the following steps and in Video 2. Start practicing infusion technique using water in an un-pruned tree with smooth bark, fully developed canopy and very broad leaves (i.e. Platanus spp., Quercus spp.).</li>
	<li>Use only toxic liquids (insecticides, fungicides, desiccants, etc.) authorized for tree injection according to local or national rules, and diluted according to the producer information (see label).</li>
	<li>Measure the circumference (crf = diam x 3.14) of the tree at breast height and calculate the quantity of solution according to the technical information provided by the liquid&#39;s producer (i.e. if cfr is 190 cm and dosage is 0.7 ml /cm, a total amount of 133 ml of diluted liquid will be injected).</li>
	<li>Calculate the number of ports to be made according to the diameter (diam) or circumference (crf) at breast height, envisaging 1 port/25-30 cm crf and rounding the result to the following unit (i.e. 190 cm crf = 6.3 = 7 ports).</li>
	<li>Recalculate the distance between ports according to the final number of ports (i.e. 190 cm/7 ports = 27 cm)</li>
	<li>Calculate the final volume of liquid/port (i.e. 133 cc in total/7 ports = 19 ml/port).</li>
	<li>When using pre-filled capsules, calculate the number of ports according to the manufacturer' instructions.</li>
	<li>Select equidistant insertion sites (i.e. 7) along the circumference in the first 150 cm from the ground, preferring lightly convex, smooth locations above root flares.</li>
	<li>Avoid any part with anomalies able to interfere with sap dynamics, above or below the insertion site (i.e. knots, wood decay, pruning cuts).</li>
	<li>If necessary, superficially smooth the bark with a knife to allow the external gasket to make a perfect seal with the bark. If the site is too rough or too curved, move a few cm to one side.</li>
	<li>Choose a blade with a length compatible with both bark thickness and tree diameter and screw it to the body. In broadleaves, at least 2 cm must enter the woody tissues; in conifers and palms, longer blades are preferable to overcome the resin vessels or because of the monocotyledonae anatomy, respectively.</li>
	<li>Fill a disposable pharmaceutical syringe (the cheapest and most effective solution) of suitable volume with the solution required for a single insertion (i.e. 17.14 ml), keeping the plunger in for at least 3 cm deep (i.e. a 20 ml syringe).</li>
	<li>As an alternative to the syringe, use any container for tree injections (i.e. drip bags, refillable syringes, pre-filled capsules, external tanks) fitting with the conical hole (female hypodermic needle standard) or the 1/8" thread located on the tool&#39;s arm.</li>
	<li>Hold the body firmly with a hand, directing the blade to the tree&#39;s center. The blade&#39;s edge must be directed parallel to the fibers (i.e. vertically).</li>
	<li>With the other hand strike the sliding hammer on the body until the external latex gasket is completely squeezed.</li>
	<li>Insert the syringe in the conical opening and gently draw out the plunger: air that entered the instrument during the percussion will flow out through the liquid. The resistance of the plunger indicates the perfect insertion of the blade. Note: with different couplings air cannot be drawn out and treatment will be slower.</li>
	<li>Wait for total uptake.</li>
	<li>If infusion is quick (i.e. 10 ml/1 min), to decrease the total number of ports refill the container before it&#39;s empty and then recalculate the number of ports. Note: to ensure a good distribution of the liquid to the canopy, at least one port /40 cm along the circumference is suggested.</li>
	<li>If infusion takes too long (more than 1 ml/min; i.e. on cloudy days or in conifers and palms) turn to light injection mode, re-inserting the plunger and applying a low pressure with the thumb, or connecting the tool to a pressurized tank (ca. 1.5 bar; Figures 3a and b).</li>
	<li>If injection doesn&#39;t happen, postpone the treatment until suitable physiological and climatic conditions prevail.</li>
	<li>When the container is empty, wait for the remaining liquid to exit the blade (8-10 sec), remove the syringe and extract the blade by striking the hammer in the opposite direction.</li>
	<li>Move to the next port.</li>
	<li>Use an inert wax or gum if protection of the wound is desirable or compulsory. Note: pruning or grafting gums containing pesticides can be phytotoxic for the cambium, slowing down the wound closure.</li>
	<li>In case of plants affected by transmittable diseases, disinfect both the blade and the body after treatment. Where locally acceptable, use alcohol or hydrogen peroxide. In case of heat disinfection, remove both the external and two internal rubber gaskets and treat them separately, or substitute them.</li>
</ol>

<ol>
	<li>Kielbaso, J. J., Ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc. of the Symposium on Systemic Chemical Treatments in Tree Culture. , (1978).</li><li>Miller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Symposium on Systemic Chemical Treatments in Tree Culture. , (1991).</li><li>Chen, R. F., Wang, H. H., Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+and+metabolism+of+injected+glyphosate+in+lead+tree+(Leucaena+leucocephala).">Translocation and metabolism of injected glyphosate in lead tree (Leucaena leucocephala).</a> Weed Science. 57, 229-234 (2009).</li><li>Gentile, S., Valentino, D., Tamietti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+potassium+phosphyte+in+the+control+of+Chestnut+ink+disease.">Effectiveness of potassium phosphyte in the control of Chestnut ink disease.</a> Acta Hort. 866, 417-424 (2010).</li><li>Poltronieri, Y., Martinez, H. E. P., Cecon, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+zinc+and+its+forms+of+supply+on+production+and+quality+of+coffee+beans.">Effect of zinc and its forms of supply on production and quality of coffee beans.</a> J. Sci. Food Agric. 91, 2431-2436 (2011).</li><li>Zuo, Y., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+and+crop+management+strategies+to+prevent+iron+deficiency+in+crops.">Soil and crop management strategies to prevent iron deficiency in crops.</a> Plant and Soil. 339, 83-95 (2011).</li><li>Da Vinci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Codex Atlanticus. , 12-76 (1478-1519).</li><li>Helton, A. W., Rohrbach, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+twelve+fungicidal+compounds+injected+into+trees+of+Prunus+domestica.">Translocation of twelve fungicidal compounds injected into trees of Prunus domestica.</a> Phytopathology. 56, 933-939 (1966).</li><li>Jones, T. W., Gregory, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+apparatus+for+pressure+injection+of+solutions+into+trees.">An apparatus for pressure injection of solutions into trees.</a> USDA Forest Service Reserch Paper. 233, 1-9 (1971).</li><li>Reil, W. O., Beutel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pressure+machine+for+injecting+trees.">A pressure machine for injecting trees.</a> Calif. Agric. 30, 4-5 (1976).</li><li>Perry, T. O., Santamour, F. S., Stipes, R. J., Shear, T., Shigo, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+alternatives+to+tree+injection.">Exploring alternatives to tree injection.</a> J. Arb. 17, 217-226 (1991).</li><li>Shigo, A. L., Campana, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discolored+and+decayed+wood+associated+with+injection+wounds+in+American+elm.">Discolored and decayed wood associated with injection wounds in American elm.</a> J. Arb. 3, 230-235 (1977).</li><li>Neely, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+closure+rates+on+trees.">Wound closure rates on trees.</a> J. Arb. 14, 250-254 (1988).</li><li>Shang, Q., Liao, K., Liu, H., Zhao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+Structure+of+Needle+Head+and+Seal+Mechanism+of+Tree+Trunk+Injection.">Study on Structure of Needle Head and Seal Mechanism of Tree Trunk Injection.</a> , (2011).</li><li>Montecchio, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BITE:+a+low+impact+tool+for+xylematic+injections.">BITE: a low impact tool for xylematic injections.</a> , (2012).</li><li>Antonini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BITE,+un+nuovo+strumento+per+i+trattamenti+endoterapici+agli+alberi.">BITE, un nuovo strumento per i trattamenti endoterapici agli alberi.</a> Giardini e ambiente. 3, 70-73 (2013).</li><li>Chaney, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+related+to+chemical+movement+in+trees.">Anatomy and physiology related to chemical movement in trees.</a> J. Arb. 12, 85-91 (1988).</li><li>Cocking, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+on+the+22nd+AGM+of+the+European+Arboricultural+Council.">Report on the 22nd AGM of the European Arboricultural Council.</a> The ARB Magazine. 158, 27 (2012).</li><li>Strazzabosco, L., Klaudatos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separa+non+buca.">Separa non buca.</a> Acer. 2, 33-37 (2013).</li><li>Shigo, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern Arboriculture. , (1991).</li></ol>

Patent references of the described instrument are PD2011A000245, EP2012/063680, WIPO WO/2013/010909.

In spite of the acknowledged environmental advantages of tree endotherapy, up to now the main factor limiting a wider spread of this method has been related to the negative side effects of the drill holes used in traditional pressure methods (i.e. delayed wound closure, parasitic infections through the hole, production of inactive discolored wood).
In contrast with other methods, the described one was envisaged to work in compliance with host physiology, considering the delivery rate ...

In recent times, trunk endotherapy has progressively replaced traditional air spray methods in woody plants1-6, but this is not a recent idea. In the 15th century Leonardo Da Vinci described in detail how he was able to intoxicate apples by injecting arsenic solution into the trunk of an apple tree through deep holes made with a gimlet7. Little has changed since then: chemicals easily available in nature have gradually been replaced with more efficient synthetic active ingredients (insecticides, fungicides, bactericides, fertilizers, plant growth regulators and desiccants). Atmospheric pressure injections evolved into high pressure, and hand-driven gimlets have been replaced by modern battery drills8-10. Unfortunately, even the sharpest drill bit tears and overheats the cambial tissue responsible for the hole closure. Consequently, wound closure is delayed and large sections of the adjacent woody tissues lose their functionality (&#34;discolored wood&#34;) from the injection site to several feet above and below11. Furthermore, unplugged holes can easily be colonized by bacteria and fungi attracted by the bleeding sap and leading to long-term internal decay, with a consequential loss of wood strength and stability12,13.
Realizing that 1) a group of longitudinal fibers separates according to a lenticular biconvex geometry (Figure 1a), and 2) sap movements into vessels fulfill the Bernoulli&#39;s principle on fluid dynamics, in 2011 the University of Padova designed a new14 drill-free endotherapic instrument with an essential lenticular, biconvex and hollow blade that enters the wood separating its fibers. In this way, inner xylematic vessels are reached with minimal friction (Figure 1b), and the temporary reduction of their section increases sap velocity, accelerating the natural uptake of an external liquid (Figure 1c, Video 1)15,16.

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			<td class="xl63" height="21" style="height:15.75pt;width:162pt" width="216">BITE</td>
			<td class="xl63" style="width:290pt" width="387">Vitzani srl, Perarolo di Cadore, Italy</td>
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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.

Due to its small dimension and unique shape, the lenticular blade does not remove cambial and woody tissues, and when compared with traditional drill holes, the wound is visibly smaller (Figure 4a). Usually, the edges of the hole are slowly compartmentalized by cambial tissues (Figure 4b1), often because of overheating during drilling. Conversely, after treating the same tree with a lenticular blade, the woody fibers revert to their previous shape, and the cambium starts to produce meris...

Watch this Scientific Journal Video about A Venturi Effect Can Help Cure Our Trees at JoVE.com

A Venturi Effect Can Help Cure Our Trees

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. ...

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17.7K Views.  Unversity of Padova. 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.

Video: A Venturi Effect Can Help Cure Our Trees

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the two-spotted spider mite (TSSM), Tetranychus urticae Koch, on greenhouse grown cotton. TSSM is a polyphagous pest which occurs on a variety of field and horticultural crops. It often becomes an early season pest of cotton in damaging proportions as opposed to being a late season innocuous pest in the mid-southern United States. Evaluation of acaricides is important for maintaining the efficacy of and preventing resistance to the currently available arsenal of chemicals and newly developed control agents. Enumeration of spider mites for efficacy evaluations is laborious and time consuming. Therefore, subjective visual damage rating is commonly used to assess density of spider mites. The NDVI (Normalized Difference Vegetation Index) is the most widely used statistic to describe the spectral reflectance characteristics of vegetation canopy to assess plant stress and health consequent to spider mite infestations. Results demonstrated that a multispectral optical sensor is an effective tool in distinguishing varying levels of infestation caused by T. urticae on early season cotton. This remote sensing technique may be used in lieu of a visual rating to evaluate insecticide treatments.

This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the ...

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 ...

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

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.

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 ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

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 ...