We thank Case Western Reserve University&#8217;s Squire Valleevue and Valley Ridge Farms, including A. Locci, C. Bond, and A. Alldridge, for help establishing the common garden. J. Hooks, L. Huffman, L. Gonzales, S. C. Leahy, B. Ochocki, A. Ubiles, C. Yu, X. Zhao, and N. M. Zimmerman provided field assistance. A.J.B. and J.H.B. were funded by startup funds from CWRU to J.H.B. G.A.D. was supported by a Summer Programs in Undergraduate Research grant from CWRU funded by the Howard Hughes Medical Institute. This work was also supported by National Science Foundation funding to J.H.B. (DEB 1250170).

1. Collect Field Soils to Produce Heterogeneous and Homogeneous Soil Treatments
<ol>
	<li>Identify two cooccurring plant species in similar habitats and soil types for study (e.g. Figure 1a). Determine amount of soil needed based on soil patch size to use in the experiment (e.g. one-third to one-half of the focal species' primary root zone, or volume encompassed by the root ball) and the number of patches needed for the experiment (see Protocol 2 and 3, below).
	<ol>
		<li>For Rumex spp. (Figure 1a), use soil patches of 10 cm x 10 cm x 18 cm (length x width x depth) for approximate root zone of 10 cm radius from base of plant to 18 cm depth (5,655 cm3 soil from this defined zone of root influence for one individual plant yields 3 soil patches of 1,800 cm3).</li>
	</ol>
	</li>
	<li>Survey field site to locate adult individuals of the two focal species. Avoid individuals whose root zone likely overlaps with the other focal species (e.g. Rumex individuals located within 0.25 m of the other species). Mark the number of individuals needed to obtain the required amount of soil with pin flags.
	<ol>
		<li>Mark 20 randomly chosen individuals of each Rumex spp. for 20 experimental units (2 species x 2 soil treatments x 5 replicates) that each require 3 soil patches from each focal species (as in Protocol 2, below).</li>
	</ol>
	</li>
	<li>Sterilize soil collecting equipment (shovels, gloves, transporting containers) with an approximately 1:10 mixture of bleach (5-10% NaClO) and water to remove all soil particles. Dry equipment before use to avoid bleach effects on soil biota. Label each piece of equipment with laboratory tape to indicate the focal species whose soil it will collect or transport to avoid cross-contamination (e.g. label one shovel for species A and a second shovel for species B).</li>
	<li>Check weather and soil conditions before beginning soil collection for recent precipitation events. Avoid collecting soils that are too wet (muddy or fully saturated) or collecting soil during a precipitation event to minimize soil compaction. Avoid collecting soils that are too dry (very hard and difficult to insert a shovel into) to aid in separating soil from roots.</li>
	<li>Collect field soil from marked individuals of the two focal species in replicate batches to avoid pseudoreplication (e.g. a single soil sample containing a rare pathogen being subsequently mixed across experimental replicates).
	<ol>
		<li>Dig up the complete root zone of an individual of species A (as defined in step 1.1) with the appropriately-labeled sterile shovel, remove visible coarse roots from the soil with the appropriately-labeled sterile gloves, and place soil in the appropriately-labeled sterile transport container (e.g. bucket). For an experiment with 4 experimental units in each block and 1 individual plant's root zone providing enough soil for 1 experimental unit (as in Section 1.2.1), repeat this procedure for 3 additional individuals of species A.</li>
		<li>Repeat Section 1.5.1 for species B.</li>
		<li>Transport the replicate soil collection from both species to the experimental site. Place soil in experimental units for block 1 of the experiment (as in Protocol 2 or 3, below), where a block contains an experimental unit from each treatment combination (i.e. each species x each soil treatment).</li>
		<li>Repeat Section 1.5.3.</li>
		<li>Repeat Sections 1.5.1-1.5.4 for the remaining blocks in the experiment. Collect soil from groups of individuals at least 1 m distant from previous collecting locations for each replicate collection to minimize pseudoreplication.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51580/51580fig1highres.jpg" width="500" /> 
Figure 1. Example field experimental design manipulating plant-induced soil heterogeneity. (a) Soils are collected from the zone of root influence of conspecifics (&#945;) and heterospecifics (&#946;) in the field, following standard protocols for studying the effects of plant-soil feedbacks9. (b) Experimental treatments with heterogeneous soils composed of soils from plant A (&#945; soils) and soils from plant B ('&#946;' soils) are arranged in a grid, and homogenized soil treatments created with an equal mixture of soils from these two origins. In this example, grids of field soils are inserted into large-diameter pots sunken into the ground and the area around each grid is filled with coarse, sterilized sand. This figure has been modified from Brandt et al.10
2. An Example Field Experiment, Creating Grids of Heterogeneous and Homogeneous Soil to Measure Plant Population Responses
<ol>
	<li>Use soils from a replicate field soil collection (Protocol 1) to produce a heterogeneous treatment (i.e. alternating grid cells contain soil collected from species A or species B) and a homogeneous treatment (i.e. each grid cell contains a 1:1 mixture of soil from species A and B) (Figure 1b) for block 1 of the experiment in the following steps.
	<ol>
		<li>Sterilize soil handling equipment (trowels, gloves, plastic grid) with an approximately 1:10 mixture of bleach (5-10% NaClO) and water to remove all soil particles. Dry equipment before use to avoid bleach effects on soil biota. Label each piece of equipment with laboratory tape to indicate the focal species whose soil it will handle to avoid cross-contamination (e.g. label one trowel for species A and a second trowel for species B) and the plastic grid to indicate whether it will be used for the heterogeneous or homogeneous soil treatment.</li>
		<li>Place removable plastic grid into a pot sunken into the ground [or other plot or container large enough to accommodate the grid] to be filled with field collected soil (e.g. 1,800 cm3/grid cell as in step 1.1.1). Use a custom-constructed grid of the desired dimensions, e.g. a 2 x 3 grid with 6 cells of the size determined in step 1.1 (Figure 1b).
		<ol>
			<li>For the heterogeneous treatment of alternating soil patch types, place spadefuls of soil from species A into grid cells for &#945; soil and soils from species B into cells for &#946; soil (Figure 1b).</li>
			<li>For the homogeneous treatment, place alternating spadefuls of soils a and b into each grid cell, taking care to evenly distribute the two soil types and avoid the formation of layers of each soil type (Figure 1b). For the example soil patch of 1,800 cm3 (Section 1.1.1), place 900 cm3 of each soil type in each grid cell.</li>
			<li>Be sure to produce equal amounts of disturbance (i.e. break up soil clods to the same degree) when creating heterogeneous and homogeneous soil treatments to avoid confounding soil disturbance with soil heterogeneity.</li>
		</ol>
		</li>
		<li>If any empty area remains around the outside of the grid within the large pot used (as in Figure 1b), fill this area with sterile sand. Sterilize sand using any standard method (e.g. autoclaving, gamma-irradiation).</li>
		<li>Lift the plastic grid vertically out of the pot, leaving the soil patches intact. This allows roots from different individuals to interact in the soil and each plant to experience multiple patches, which is essential for allowing plant roots to grow into multiple soil patches (i.e. grid cells).</li>
		<li>Repeat Sections 2.1.2-2.1.4 for a second pair of experimental units to produce a complete experimental block (i.e. a grid of heterogeneous soil into which to plant each focal species and a grid of homogeneous soil into which to plant each focal species, for a total of 4 experimental units).</li>
	</ol>
	</li>
	<li>Repeat step 2.1 for remaining blocks in the experiment with the remaining soil replicate collections (Protocol 1). Randomize blocks, and plots within blocks, throughout the experimental area, using landscape fabric around the plots if desired, to keep shading from outside vegetation at a minimum (not shown).</li>
	<li>Plant seeds of focal species into each grid cell of each experimental pot (2 focal species x 2 soil treatments). Use pooled seed to avoid confounding plant genotypes or seed-surface microbial communities with the soil treatments. For example, plant seeds of each Rumex spp. into the pot for that species (Figure 1a) individually glued to plastic toothpicks with water-soluble glue in 12 planting positions (2 positions per grid cell) to clearly identify the planted individuals.</li>
	<li>Measure population responses of the experimental units, such as germination and survival, through a regular census of individual marked planting locations of seeds (e.g. using toothpicks as in step 2.3). Minimize disturbance of the soil for any response measured to avoid mixing soil among patches.
	<ol>
		<li>Determine the appropriate census intervals based on the expected rate of population responses for the focal species. For example, conduct weekly censuses for Rumex spp. that can germinate quickly.</li>
		<li>Continue the experiment for an appropriate duration based on the life history of the focal species. For example, continue the experiment for at least 2 years for short-lived perennial Rumex spp. to obtain data on all life stages.</li>
	</ol>
	</li>
</ol>
3. An Example Greenhouse Experiment, with Heterogeneous and Homogenized Soil in Pots to Measure Individual Plant Responses
<ol>
	<li>Use soils from a replicate field soil collection (Protocol 1) to produce a heterogeneous treatment (i.e. each half of a pot filled with soil collected from species A or species B) and a homogeneous treatment (i.e. pot contains a 1:1 mixture of soil from species A and B) (Figure 2a) for block 1 of the experiment in the following steps.
	<ol>
		<li>In the transport container (e.g. bucket) and before potting, mix the field collected soil from each species with sterile sand to produce a 1:1 mixture. Do this to mitigate soil compaction and improve drainage in the pots, which is especially helpful for washing roots, facilitating the separation of roots from soil. Sterilize sand using any standard method (e.g. autoclaving, gamma-irradiation).</li>
		<li>Be sure to produce equal amounts of disturbance (i.e. break up soil clods to the same degree) when mixing each batch of field soil with sand.</li>
	</ol>
	</li>
	<li>Sterilize soil handling equipment (trowels, gloves, plastic sheet) with an approximately 1:10 mixture of bleach (5-10% NaClO) and water to remove all soil particles. Dry equipment before use to avoid bleach effects on soil biota. Label each piece of equipment with laboratory tape to indicate the focal species whose soil it will handle to avoid cross-contamination (e.g. label one trowel for species A and a second trowel for species B) and each side of a stiff plastic sheet to indicate which soil type will be potted on each side of it in the heterogeneous soil treatment.</li>
	<li>Create a heterogeneous soil treatment using the field soil-sand mixtures from the two focal species (from Section 3.1.1).
	<ol>
		<li>Place the stiff plastic sheet in the center of a pot to divide it in half (Figure 2a). Use a pot sized such that half of it is equivalent in volume to the appropriate soil patch size (step 1.1). For example, use a pot with a 15 cm diameter and 18 cm depth for the Rumex spp. in Figure 1a (total volume of 3,181 cm3).</li>
		<li>Have two researchers simultaneously add the field collected soil from each species to the appropriate side of the pot (e.g. &#945; soil from species A) with appropriately-labeled trowels, to keep the sides even as the pot is filled.</li>
		<li>Remove the plastic divider by lifting it vertically out of the soil, leaving the soil patches intact. This allows the plants to experience both patches of soil within a pot, essential for the plants to experience heterogeneity.</li>
		<li>Repeat Sections 3.3.1-3.3.3 for 3 additional experimental units to produce a complete block of the heterogeneous soil treatment (2 species x 2 soil patch types in the heterogeneous treatment; Figure 2).</li>
	</ol>
	</li>
	<li>Create a homogeneous soil treatment using the field soil-sand mixtures from the two focal species (from Section 3.1.1).
	<ol>
		<li>Repeat Section 3.3.1. Have two researchers simultaneously add both &#945; and &#946; soils to both sides of the pot with appropriately-labeled trowels, evenly distributing both soil types within each patch and avoiding the creation of layers. Repeat Section 3.3.3.</li>
		<li>Repeat Section 3.4.1 for a second experimental unit to produce a complete block of the homogeneous soil treatment (one pot for each of the 2 focal species).</li>
	</ol>
	</li>
	<li>Repeat steps 3.2-3.4 for remaining blocks in the experiment with the remaining soil replicate collections (Protocol 1). Randomize blocks, and plots within blocks, across the greenhouse bench.</li>
</ol>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51580/51580fig2highres.jpg" width="500" /> 
Figure 2. Example greenhouse experimental design manipulating plant-induced soil heterogeneity. (a) Soils collected from the zone of root influence of species A (&#945; soils) and species B (&#946; soils) in the field are placed in each half of a pot (heterogeneous treatment) or mixed throughout the pot (homogeneous treatment). (b) Plants of species A are then planted into the experiment in each soil patch type in the heterogeneous treatment and on one side of the homogeneous treatment. Here, only one species (A) is shown planted into this design. A fully reciprocal design would include plants of the second focal species (B) planted into each soil treatment and patch type within the heterogeneous treatment.
<ol start="6">
	<li>Plant seedlings of focal species A and B into each soil patch type in a factorial design (2 species x 3 soil patch types [&#945; soil, &#946; soil, and a homogeneous mixture of &#945; and &#946; soil]; Figure 2b).</li>
	<li>Measure any individual plant responses that indicate plant performance, such as plant size, biomass production, or functional traits. Appropriate traits to measure will depend on the focal species and the scientific questions of interest.
	<ol>
		<li>Continue the experiment until the plants appear large relative to soil patches (e.g. approximately 1.5x the width of the soil patch for rosette-forming forbs) to ensure the plant roots are growing into both soil patches. Experimental duration will thus depend on the growth rate of focal species.</li>
		<li>Harvest plants and/or measure responses before plants become pot-bound (i.e. root growth is limited by the confines of the pot and roots circle the bottom of the pot) if realistic responses to the soil environment are desired. For example, roots of pot-bound plants are unlikely to forage throughout the soil column in a similar manner to plants in the field.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Plant-induced soil heterogeneity is highly likely in natural communities because plants have large and often species-specific effects on their soil environment and the subsequent plants that experience that soil (e.g. Petermann&#160;et al.13). However, our understanding of the role of this type of heterogeneity on plant communities is minimal10,14. Here, we present a method for manipulating plant-induced soil heterogeneity, using soil from different origins (i.e. zones of ...

One of the primary goals of community ecology is to explain and predict the processes governing community assembly. However, plant communities are frequently more diverse than predicted by coexistence theory1, and restoration ecologists need to understand coexistence mechanisms to successfully restore diverse native communities2. Environmental heterogeneity is a theoretically important mechanism that can help explain high levels of community diversity, but experimental manipulations of heterogeneity are infrequent3 and focus on abiotic heterogeneity (e.g. reviewed in Lundholm4). Theory that incorporates heterogeneity typically assumes that heterogeneity is extrinsic to the assembling community. Extrinsic heterogeneity is governed by factors such as landscape typology, which are independent of the community composition. Extrinsic heterogeneity can result in coexistence through niche partitioning (reviewed in Melbourne&#160;et al.3, e.g. Pacala and Tilman5&#160;and&#160;Chesson6). However, much of the environmental heterogeneity relevant to plant communities may be intrinsic to the community, developing as the community assembles and depending on the identity of the species in the community. Intrinsic heterogeneity can result from biotic feedbacks, which can lead to coexistence through negative frequency-dependence (e.g. Bever&#160;et al.7). Here, we describe a novel method for manipulating plant-induced soil heterogeneity, a type of soil heterogeneity that is intrinsic to the community and arises from plant-soil feedbacks.
Plant-soil feedbacks (PSF) occur when plants influence the soil structure, chemistry, or biota in a manner that affects subsequent plant performance in that soil, and PSF have large mean effects on plant performance in native plant communities8. Studies of PSF have typically either collected soils from the field or conditioned soils experimentally, then asked how plants perform in conspecific soil relative to heterospecific or sterilized soil9. If plants perform better in conspecific soil relative to reference soils, then PSF are positive, while if plants perform better in reference soils, PSF are negative. Reciprocal negative PSF can lead to frequency-dependent coexistence between species7. While the mean effects of PSF are well-characterized8, the effects of spatial heterogeneity in PSF are poorly understood10.
Because PSF occur on the scale of individual plants7 and because plants are often nonrandomly distributed in space and time, PSF are likely to lead to soil heterogeneity, which we call plant-induced soil heterogeneity. Unlike many other forms of heterogeneity (e.g. landscape topology), this heterogeneity is intrinsic to the assembling community and may thus influence community assembly differently than more extrinsic forms of heterogeneity. In order to understand the influence of this form of heterogeneity on plant performance and coexistence, we need experimental methods that manipulate plant-induced soil heterogeneity. Here, we demonstrate such a method, which uses soils conditioned by two species to create a heterogeneous treatment with separate patches from two soil origins and a homogeneous treatment, which is a mixture of the two soil origins. This soil mixing could represent at least two plausible scenarios in the field: (1) disturbance (e.g. rodent, agriculture) which mixes soils of different origins or (2) plants of two species growing in close proximity, such that their zones of root influence intermingle and homogenize.
We present two example experiments that use plant-induced soil heterogeneity to answer key questions at different levels of ecological organization: (1) Do plant populations respond to plant-induced soil heterogeneity? and (2) Do individual plants respond to plant-induced soil heterogeneity? We describe a field experiment using 6 soil patches to address the first question and a greenhouse experiment using 2 soil patches to address the second question. Quantifying both population and individual plant responses to soil heterogeneity is essential to understanding how heterogeneity influences community assembly.

<table border="0" cellpadding="0" cellspacing="0" width="1119">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shovel(s)</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">It is helpful to have at least two shovels, one for each species of soil origin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trowel(s)</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">It is necessary to have at least two trowels of identical size, one for each species of soil origin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gloves</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Gardening gloves can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diluted bleach</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">We use an ~1:10 concentration of household bleach (containing 5-10% NaClO) to water to sterilize all equipment between soil collections.</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Plastic grid(s)</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">CUSTOM. We used plastic sheeting from the construction of greenhouse walls to create the grid used in the field experiment. However any stiff plastic that can be manipulated can be used. It is helpful to have three grids to produce reciprocal heterogeneous treatments and a homogeneous treatment without needing to sterilize between each experimental unit.</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Plastic dividers</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">CUSTOM. We used stiff sheets of plastic, cut to fit the pot minimum width, such that they can slide down to the bottom of the pot for the greenhouse experiment. It is helpful to have at least two dividers, one for heterogeneous and one for homogeneous treatments, if investigators want to randomize the order in which experimental units within a block are filled without needing to sterilize the divider in between each experimental unit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buckets or wheelbarrows</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Any container for transporting soils.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Seeds</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">We collect seeds in the field by hand. Seeds can also be ordered from horticultural suppliers, if appropriate.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic toothpicks</td>
			<td style="width:136px;">Soodhalter Plastics, Inc.</td>
			<td>805KP</td>
			<td style="width:696px;">We plant individual seeds glued on toothpick in the field experiment, to facilitate monitoring germination and survival of individuals.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Water soluble glue</td>
			<td style="width:136px;">Elmer&#39;s</td>
			<td style="width:71px;">Elmer&#39;s Glue-all</td>
			<td style="width:696px;">Any water soluble glue can be used to adhere seeds to plastic toothpicks.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pots</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Pot size will depend on experimental plants used and number of soil patches desired (e.g. 2 or 6).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sand</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Coarse sand may be mixed with field soils to improve drainage in pots.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lab tape&#160;</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Tape may be used to label equipment used in handling soils with the species of origin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pin flags</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Flags can be used to identify individuals in the field, prior to soil collection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Landscape fabric</td>
			<td style="width:136px;">Any</td>
			<td style="width:71px;">NA</td>
			<td style="width:696px;">Landscape fabric can be used in the field to minimize the growth of plants outside experimental plots.</td>
		</tr>
	</tbody>
</table>

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.

Species responded to plant-induced soil heterogeneity in diverse ways at both the population and individual level (Figures 3 and 4), with implications for community assembly. To determine whether plant populations respond to plant-induced soil heterogeneity, a field experiment was established as in Protocol 2 using three congeneric pairs of species. Plant populations were censused weekly for three months and the total proportion of planted seeds that germinated and the total proportion o...

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Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

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 ...

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Video: Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24&#160;hr&#160;after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...