We would like to thank the two anonymous reviewers for their suggestions. We also thank the numerous undergraduates who have helped with constructing pots, microcosms, and slotted containers and who have assisted with maintaining and harvesting experiments. We also thank North Central College for startup funds (to JW) and current facilities, as well as Ashley Wojciechowski for obtaining a North Central College Richter Grant supporting an experiment using these methods. Part of this work was funded by a National Science Foundation Doctoral Dissertation Improvement Grant (DEB-1401677).

&#160;1. Construction and assembly of rotatable cores
<ol>
	<li>Modify commercial tubular seedling containers (subsequently called &#8216;containers&#8217;; Table of Materials) to have 19 mm wide x 48 cm length openings.
	<ol>
		<li>Using a drill-press with a 19 mm hole saw without a central, pilot twist drill, cut two holes, one above the other, in the sides of a container (2.5 cm diameter x 12.1 cm length) so that the holes are about 1 cm apart. Hold the container against a fence on the drill press and have a stop with a short dowel that will fit inside the container to help hold it in place while drilling. Use a container with flexible plastic to prevent cracking.</li>
		<li>Cut the remaining thin piece of plastic between the holes with scissors, a wire cutter, or tin snips (for rigid plastic use a sabre-saw) to make one elongated opening about 2 cm wide and 5 cm long.</li>
		<li>Repeat steps 1.1.1 &#8211; 1.1.2 on the opposite side of the container.</li>
	</ol>
	</li>
	<li>Cover the slots with nylon mesh and/or hydrophobic membrane&#160;(Figure 1A).
	<ol>
		<li>Cut nylon mesh with 40 &#956;m pores into 9.5 cm x 8.5 cm pieces. Cut as many pieces as there are containers.</li>
		<li>Glue the nylon mesh externally onto the containers to cover both openings with some slight overlap in the fabric using high-strength, industrial hot glue.</li>
		<li>If the prevention of water movement is needed, such as when using water-soluble nutrients or stable isotopes, cover the nylon mesh layer with a hydrophobic membrane31,32 (Table of Materials) that allows AM fungus hyphae to pass, but only the movement of water vapor and not liquid water.</li>
		<li>Place the hot glue around the openings on the container and along the long edges of the nylon mesh. Roll the container onto the fabric to avoid burning fingers. Add a&#160;layer of glue along the fabric edge where the mesh edges overlap. Press the edge onto some cardboard to firmly seal it. Always roll consistently in one direction that will be the same direction of the rotation of the finished containers within pots or microcosms so that the overlapped mesh edge will not be pushed to potentially dig into the substrate.</li>
		<li>Once the glue has cooled, tape the top and bottom ends of the fabric to the container to prevent loose edges and ripping using a flexible tape, such as electrician&#39;s tape.</li>
	</ol>
	</li>
	<li>Using the same tape as in step 1.2.5, cover the small holes on the sides of the conical end (not the hole at the tip of the bottom) of each container to prevent root growth out of the container into the rest of the pot/microcosm.</li>
	<li>To prevent soil loss while providing drainage, place a glass marble into the bottom of each container.</li>
	<li>For a control treatment that does not involve any potential for a CMN to form between plants, use solid, unmodified containers (Figure 1A).</li>
</ol>
2. Assembly of pots or microcosms to fit the conical ends of the containers
<ol>
	<li>To ensure containers stand upright in a fixed position and have proper drainage, flip a pot over so that the bottom is facing up. Cut around the bottom of the pot, leaving a small lip for support, using a sabre-saw.</li>
	<li>Preparation of polystyrene foam
	<ol>
		<li>Cut polystyrene foam, about 36 mm thick, to the same diameter as the bottom of the pot using a bandsaw with a circle-cutting jig.</li>
		<li>Drill holes into the foam using a drill press and 19 mm hole saw (without a central twist drill) in the pattern in which the containers will be positioned.</li>
		<li>For a target plant experiment, drill a central hole with equally spaced holes for neighboring individuals surrounding it. For a pot with a 15.5 cm diameter, space six holes 12 mm apart around the circumference of an 11 cm diameter circle (Figure 1B).</li>
		<li>Lay out the holes hexagonally or in a square array (Figure 1C, D) for a microcosm experiment.</li>
	</ol>
	</li>
</ol>
3. Filling of the containers and pots with soil and sand mixtures
<ol>
	<li>Select a desired soil mixture and add AM fungus field-collected or pot-cultured inoculum to the soil by uniformly mixing chopped root pieces (1 &#8211; 2 cm long) thoroughly with the soil. Mix the desired soil with an infertile silica sand or glass beads to decrease the concentration of mineral nutrients available to plants.</li>
	<li>Position the filled containers in the drilled foam or microcosm bottom and fill the interstitial space with an infertile substrate.</li>
	<li>Fill the interstitial space between containers with nutrient-poor silica sand mixture using a funnel to assist in filling small spaces. To ensure adequate drainage and mimic the texture of the soil, mix medium particle-size sand, such as 6-20 grade, with small particle-size sand, such as 30-65 grade, in a cement mixer.</li>
</ol>
4. Establishment of CMNs throughout pots/microcosms
<ol>
	<li>Plant pretreatment &#8216;nurse&#8217; plants of the desired species into each container to sustain AM fungi so that they can spread among the containers and establish CMNs.</li>
	<li>When all containers have established seedlings, remove shoots by clipping so that only one individual remains in each container.</li>
	<li>Allow 2-3 months for plant growth and CMN establishment.</li>
</ol>
5. Establishment of experimental plants and treatments
<ol>
	<li>Sow experimental plants by seeding or transplanting into containers. If seeding, wait until all containers have a germinated seedling&#160;before removing pre-treatment nurse plants by clipping their shoots. If transplanting, clip all pre-treatment plants before transplanting experimental seedlings to prevent unintended competitive effects.</li>
	<li>Establish CMN treatments by either leaving the containers not moved for the duration of the experiment (intact CMNs) or rotating them weekly to physically sever hyphae extending among the modified containers (severed CMNs; Figure 1A). When severing CMNs, rotate each container through one full rotation to avoid unintentionally altering aboveground interactions, particularly for heliotropic plants.</li>
	<li>Heavily water all pots or microcosms immediately after rotation to reestablish contact between the interstitial substrate and the sides of containers.</li>
</ol>
6. Tracing of mineral nutrient movement across CMNs
<ol>
	<li>Fertilize neighboring plants with 0.5% 15N enriched KNO3 and NH4Cl.</li>
	<li>Fertilize the target individual with a 14N fertilizer of equal concentration.</li>
</ol>
7. Monitoring and maintenance of experiment
<ol>
	<li>Regularly (at least monthly) re-randomize the positions of the pots or microcosms over the course of the experiment.</li>
	<li>Weekly measure growth, such as height or longest leaf length (for grasses) to monitor when growth begins to slow, because it is important to harvest before the plants become root-bound.</li>
</ol>
8. Harvest of the experiment
<ol>
	<li>Clip all aboveground tissue and place individual plants into labeled envelopes that identify their treatment, pot or microcosm, and position.</li>
	<li>Dry aboveground tissues at 60 &#176;C to constant weight. Measure the dry weight of each plant tissue.</li>
	<li>Allow the soil to dry before extracting the containers and harvesting the roots.</li>
	<li>Delicately brush off as much soil as possible from the root systems and wash them in a pan of water or under a gentle stream of water on a sieve of 250 micron pore size.</li>
	<li>Allow the roots to air-dry and weigh the whole root system.</li>
	<li>Clip the root system haphazardly and store the root fragments in 50% ethanol. After they are&#160;stained33, use these fragments for quantification of root colonization using the gridline intersection method34.</li>
	<li>Re-weigh the remaining root system and store it in a labeled paper envelope to dry at 60 &#176;C for assessment of dry weight. Use the following equation to calculate the weight of the entire root system:</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/59338/59338eq1v2.jpg" style="opacity: 0.9;" />
9. Mineral nutrient and stable isotope analyses
<ol>
	<li>Group the seedlings by biomass into &#8220;deciles&#8221; or 10 groups, &#8220;octiles&#8221; or 8 groups, &#8220;quartiles&#8221; or four groups, etc. after rank-ordering them by weight if the tissue quantity is too low for minimum requirements for digestion to determine mineral nutrient concentrations.</li>
	<li>Send foliar samples to a contracted lab for mineral nutrient and stable isotope analyses (Table of Materials).
	<ol>
		<li>Describe isotopic abundance using the following customary expression: 
		<img alt="Equation 1" src="/files/ftp_upload/59338/59338eq2v2.jpg" /> 
		where R represents the 15N/14N ratio of a sample or of the standard which is atmospheric N.</li>
		<li>Use the non-modified, solid container treatment to serve as a control for background 15N ratios in the following mass balance equation when quantifying the amount of 15N taken up by a target plant in severed or intact CMN treatments: 
		<img alt="Equation 1" src="/files/ftp_upload/59338/59338eq3v2.jpg" style="opacity: 0.9;" /> 
		where &#948;15N represents the isotopic abundance of targets, neighbors, and target plants in the no CMN treatment, and x represents (as a decimal fraction) the percent nitrogen obtained by the target plant from neighbor containers to which label was added. Values for &#948;15NNeighbors are obtained for each target plant&#8217;s composited neighbors.</li>
	</ol>
	</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbuscular+common+mycorrhizal+networks+mediate+intra-and+interspecific+interactions+of+two+prairie+grasses.">Arbuscular common mycorrhizal networks mediate intra-and interspecific interactions of two prairie grasses.</a> Mycorrhiza. , 1-13 (2017).</li><li>Weiner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+competition+in+plant+populations.">Asymmetric competition in plant populations.</a> Tree. 5 (11), 360-364 (1990).</li><li>Damgaard, C., Weiner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Describing+inequality+in+plant+size+or+fecundity.">Describing inequality in plant size or fecundity.</a> Ecology. 81 (4), 1139-1142 (2000).</li><li>Johnson, C. R., Joiner, J. N., Crews, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+N,+K,+and+Mg+on+growth+and+leaf+nutrient+composition+of+3+container+grown+woody+ornamentals+inoculated+with+mycorrhizae.">Effects of N, K, and Mg on growth and leaf nutrient composition of 3 container grown woody ornamentals inoculated with mycorrhizae.</a> Journal of the American Society for Horticultural Science. 105 (2), 286-288 (1980).</li><li>Estrada-Luna, A. A., Davies, F. T., Egilla, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycorrhizal+fungi+enhancement+of+growth+and+gas+exchange+of+micropropagated+guava+plantlets+(Psidium+guajava+L.)+during+ex+vitro+acclimatization+and+plant+establishment.">Mycorrhizal fungi enhancement of growth and gas exchange of micropropagated guava plantlets (Psidium guajava L.) during ex vitro acclimatization and plant establishment.</a> Mycorrhiza. 10 (1), 1-8 (2000).</li><li>Hurlbert, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudoreplication+and+the+design+of+ecological+field+experiments.">Pseudoreplication and the design of ecological field experiments.</a> Ecological Monographs. 54 (2), 187-211 (1984).</li><li>Arg&#252;ello, A., 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=Options+of+partners+improve+carbon+for+phosphorus+trade+in+the+arbuscular+mycorrhizal+mutualism.">Options of partners improve carbon for phosphorus trade in the arbuscular mycorrhizal mutualism.</a> Ecology Letters. 19 (6), 648-656 (2016).</li></ol>

The authors have nothing to disclose.

Our results affirm that our rotated core method can sharply focus on the role of CMNs in belowground plant interactions. There are several critical steps in the protocol, however, that if altered, have potential to influence the ability to detect CMN effects. It is critical to fill the interstitial area surrounding containers with a nutrient-poor medium. In our unsuccessful, rotated-core target plant experiment with guava tree seedlings, although there was a marked reduction of target growth in the presence of any number...

Arbuscular mycorrhizal (AM) fungi assisted plants in the colonization of land 460 million years ago1 and today, they are ubiquitous symbionts of most plants2, providing them with vital mineral nutrients for growth. The thin, thread-like hyphae of AM fungi forage for mineral nutrients beyond nutrient depletion zones near roots, often encountering and colonizing root systems of neighboring plants in a &#8220;common mycorrhizal network&#8221; (CMN). Common mycorrhizal networks also may form when fungal germlings join established networks3, or when AM hyphae fuse (anastomose) with conspecific hyphae4,5,6,7. The extent of these extraradical hyphae in the soil is enormous, with extraradical hyphae constituting 20% to 30% of total soil microbial biomass in prairie and pasture soils8 and stretching for 111 m&#183;cm-3 in undisturbed grassland9.
Common mycorrhizal networks partition mineral nutrients among interconnected neighboring plants10,11,12,13. Plants may receive up to 80% of their phosphorus and 25% of their nitrogen requirements from AM fungi, while providing up to 20% of their total fixed carbon to the fungi in return14. Recent in vitro root organ culture work has found that CMNs preferentially exchange mineral nutrients with host roots that provide the most carbon to the fungi11,12. Furthermore, different species of AM fungi may differ in their quality as symbiotic partners, with some fungi exchanging more phosphorus for less carbon than others15. Although root organ cultures are beneficial models for studying the AM symbiosis because they present carefully controlled environments and the ability to directly observe hyphal interconnections, they do not include photosynthesizing shoots&#160;which affect&#160;important physiological processes such as photosynthesis, transpiration, and diurnal changes, as well as constituting carbon and mineral nutrient sinks.
In nature, seedlings most likely tap into already established CMNs. Until recently, however, scientists have only examined the impact of AM fungi on plant nutrition by growing plants with and without AM fungi, often with a single species of AM fungus. Although this work has been tremendously informative to our understanding of arbuscular mycorrhizas, this method has overlooked the potentially crucial role that CMNs may have in interactions among interconnected host plants. In particular, plants that are highly dependent on AM fungi for growth interact minimally without AM fungi16,17, possibly confounding our interpretation of AM fungus-mediated interactions when used as &#8216;controls&#8217; for baseline reference.
We propose a rotated-core approach for investigation of the role of CMNs in plant interactions and population structuring. Our approach mimics components of the AM symbiosis in nature because whole plants join established CMNs and all plants are grown with AM fungi. By removing root interactions, our methodology specifically focuses on interactions mediated by AM fungi while also tracking mineral nutrient movement within CMNs. Our approach builds on previous work that has used rotated cores both in the field and in the greenhouse to understand AM functioning realistically.
The rotated core method has been established in the literature as a method to manipulate extraradical hyphae18,19,20,21, and it has had several reincarnations depending on its purpose over the past two decades. Initially, mesh bags or barriers allowing in-growth of hyphae were used to provide root-free compartments to quantify the amount of arbuscular mycorrhizal hyphae in the soil22,23. Then, cylindrical cores of soil enclosed in rigid water pipes or plastic tubing with slots covered in a nylon mesh penetrable by hyphae, but not roots, were developed. These could easily be rotated to disrupt extraradical mycelia18,24,25. The rotated cores were placed between plants, and soil hyphal lengths per gram of soil18, 13C fluxes to extraradical mycelia24, or phosphorus uptake from plant-free cores were quantified18. Another use of such cores was to grow plants within them in the field to reduce colonization of roots by AM fungi through frequent hyphal disruption as an alternative to sterilization or the application of fungicides, both of which have indirect effects on soil organic matter and other microbes18.
The hyphal mesh barrier approach has been used to investigate nutrient partitioning and plant interactions across CMNs, but in rectangular microcosms rather than with rotated cores. Walder et al.26 investigated interactions between Linum usitatissimum (flax) and Sorghum bicolor (sorghum) by tracing mineral nutrient for carbon exchange using isotopes across CMNs of either of the AM fungi Rhizophagus irregularis or Funneliformis mosseae26. The microcosms in their study comprised plant compartments separated by mesh barriers, hyphal compartments only accessible to mycorrhizal hyphae, and labeled hyphal compartments that contained radioactive and stable isotopes. As controls, the study used treatments without mycorrhizal fungi. Song et al.27 used a similar approach to find that plant signals could be carried only among established CMNs of F. mosseae when one plant was infected by a fungal pathogen. Also, similarly to Walder et al.26, Merrild et al.28 grew plants in individual compartments separated by mesh to investigate plant performance of Solanum lycopersicum (tomato) seedlings linked by CMNs to a large Cucumis sativus (cucumber) plant that represented an abundant carbon source. They also used treatments without mycorrhizal fungi instead of severing CMNs28. In a second, related experiment, carbon for phosphorus exchange was examined using mesh bags labeled with 32P. Microcosms with hyphal mesh barriers and CMN severing as a treatment were used by Janos et al.29, who investigated competitive interactions between seedlings of the savanna tree species Eucalyptus tetrodonta and transplants of the rain forest tree, Litsea glutinosa. In that study, Janos et al.29 lifted compartments containing seedlings a few centimeters, sliding layers of mesh against one another to break hyphal interconnections29.
The final step in the evolution of the rotated core method has been to grow plants inside cores that are within pots or microcosms20,30. Wyss30 used rotated cores to ascertain if extraradical AM mycelium could colonize Pinus elliottii seedlings when spreading from a donor or &#8216;nurse&#8217; AM host plant, Tamarindus indica, and how extraradical mycelium of ectomycorrhizal fungi influences seedling performance. Large commercial tubular seedling containers (Table of Materials) within microcosms were either solid plastic (no CMNs) or slotted and covered with a hydrophobic membrane. Slotted seedling containers were either not rotated (intact CMNs) or rotated to sever established CMNs. Rotated cores with different mesh barrier sizes were used by Babikova et al.20 to investigate belowground signaling through CMNs among Vicia faba (bean) plants. In their study, a central donor plant in 30 cm diameter mesocosms was interconnected either by roots and hyphae (no barrier) or only by CMNs established through a 40 &#956;m mesh. Central plants were severed from interactions with neighboring plants through rotation of the mesh-enclosed cores, or CMNs were prevented by a fine 0.5 &#956;m mesh enclosing the core.
Here, we present a method that combines aspects of prior rotated-core approaches to examine the influence of CMNs on direct plant interactions combined with stable isotope tracing. Our method uses a &#8216;target plant&#8217; approach, in which the central plant of interest is surrounded by neighboring plants. Plants are grown inside rotatable seedling containers that are slotted and covered with nylon silk-screen mesh, hydrophobic membrane, or are non-modified solid plastic. Common mycorrhizal networks are severed once a week or kept intact, and 15N stable isotopes trace the movement of nitrogen from neighbors&#8217; rotated cores to the central target plant. By comparing plant size with mineral nutrient and stable isotope uptake, we assess which plants may benefit or suffer from CMNs in interactions among host plants.

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		<tr height="85">
			<td height="85" style="height:85px;width:159px;">Commercial tubular seedlings container (called &#39;containers&#39; in the manuscript)</td>
			<td style="width:143px;">Stuewe and Sons, Inc</td>
			<td style="width:179px;">Ray Leach Cone-tainer &#8482;</td>
			<td style="width:224px;">RLC3U</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Course glass beads</td>
			<td style="width:143px;">Industrial Supply, Inc.</td>
			<td style="width:179px;">12/20 sieve</td>
			<td style="width:224px;">Size #1</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Course silica sand</td>
			<td style="width:143px;">Florida Silica Sand</td>
			<td style="width:179px;">6/20 50lb bags</td>
			<td style="width:224px;">None</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:159px;">Fine glass beads</td>
			<td style="width:143px;">Black Beauty</td>
			<td style="width:179px;">Black Beauty FINE Crushed Glass Abrasive (50 lbs)</td>
			<td style="width:224px;">BB-Glass-Fine</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Hydrophobic membrane</td>
			<td style="width:143px;">Gore-tex</td>
			<td style="width:179px;">None</td>
			<td style="width:224px;">None</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:159px;">Large commercial tubular seedling containers</td>
			<td style="width:143px;">Stuewe and Sons, Inc.</td>
			<td style="width:179px;">Deepot &#8482;</td>
			<td style="width:224px;">D16L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:159px;">Medium silica sand</td>
			<td style="width:143px;">Florida Silica Sand</td>
			<td style="width:179px;">30/65 50 lb bags</td>
			<td style="width:224px;">None</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Nylon mesh</td>
			<td style="width:143px;">Tube Lite Company, Inc.</td>
			<td style="width:179px;">Silk screen</td>
			<td style="width:224px;">LE7-380-34d PW YEL 60/62 SEFAR LE PECAP POLYESTER</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:159px;">Soil and foliar nutrient analysis facility</td>
			<td style="width:143px;">Kansas State University Soil Testing Lab</td>
			<td style="width:179px;">None</td>
			<td style="width:224px;">None</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Stable isotope core facility</td>
			<td style="width:143px;">University of Miami</td>
			<td style="width:179px;">None</td>
			<td style="width:224px;">None</td>
		</tr>
	</tbody>
</table>

investigation of plant interactions across common mycorrhizal networks using rotated cores

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The power of their influence is in extraradical mycelia that spread beyond nutrient depletion zones found near roots to ultimately interconnect individuals within a common mycorrhizal network (CMN). Most experiments, however, have investigated the role of AM fungi in plant interactions by growing plants with versus without mycorrhizal fungi, a method that fails to explicitly address the role of CMNs. Here, we propose a method that manipulates CMNs to investigate their role in plant interactions. Our method uses modified containers with conical bottoms with a nylon mesh and/or hydrophobic material covering slotted openings, 15N fertilizer, and a nutrient-poor interstitial sand. CMNs are left either intact between interacting individuals, severed by rotation of containers, or prevented from forming by a solid barrier. Our findings suggest that rotating containers is sufficient to disrupt CMNs and prevent their effects on plant interactions across CMNs. Our approach is advantageous because it mimics aspects of nature, such as seedlings tapping into already established CMNs and the use of a suite of AM fungi that may provide diverse benefits. Although our experiment is limited to investigating plants at the seedling stage, plant interactions across CMNs can be detected using our approach which therefore can be applied to investigate biological questions about the functioning of CMNs in ecosystems.

To determine how CMNs may influence plant performance through nutrient partitioning, we grew Andropogon gerardii Vitman, a dominant prairie grass, in a target plant experiment with 6 equally spaced neighbors and intact, severed, or no CMNs. We found that severing or preventing CMNs diminished targets&#8217; aboveground dry weights (Figure 2), suggesting that intact CMNs promoted plant growth. Plants with severed CMNs and prevented CMNs responded nota...

Watch this Scientific Journal Video about Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores at JoVE.com

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

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11.9K Views.  North Central College. Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Video: Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...

Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility of three-dimensional visualization and virtual sectioning, has allowed novel and increasingly detailed analysis of complex plant organs. Interactions among plants, including between parasitic plants and their hosts, can also be explored. However, sample preparation before scanning becomes crucial due to the interaction between these plants, which often differ in tissue organization and composition. Furthermore, the broad diversity of parasitic flowering plants, ranging from highly reduced vegetative bodies to trees, herbs, and shrubs, must be considered during the sampling, treatment, and preparation of parasite-host material. Here two different approaches are described for introducing contrast solutions into the parasite and/or host plants, focusing on analyzing the haustorium. This organ promotes connection and communication between the two plants. Following a simple approach, details of haustorium tissue organization can be explored three-dimensionally, as shown here for&#160;euphytoid, vine, and mistletoe parasitic species. Selecting specific contrasting agents and application approaches also allow detailed observation of endoparasite spread within the host body and detection of direct vessel-to-vessel connection between parasite and host, as shown here for an obligate root parasite. Thus, the protocol discussed here can be applied to the broad diversity of parasitic flowering plants to advance the understanding of their development, structure, and functioning.

Micro-CT is a non-destructive tool that can analyze plant structures in three dimensions. The present protocol describes the sample preparation to leverage micro-CT to analyze parasitic plant structure and function. Different species are used to highlight the advantages of this method when coupled with specific preparations.

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility ...

Dissolved Solute Sampling Across an Oxic-Anoxic Soil-Water Interface Using Microdialysis Profilers

Biogeochemical processes shift rapidly in both spatial (millimeter scale) and temporal (hour scale to day scale) dimensions at the oxic-anoxic interface in response to disturbances. Deciphering the rapid biogeochemical changes requires in situ, minimally invasive tools with high spatial and temporal sampling resolution. However, the available passive sampling devices are not very useful in many cases either due to their disposable nature or the complexity and extensive workload for sample preparation.
To address this problem, a microdialysis profiler with 33 individual polyethersulfone nanomembrane tubes (semipermeable, &#60;20 nm pore size) integrated into the one-dimensional skeleton (60 mm) was established to iteratively sample the dissolved compounds in porewater across the soil-water interface at a high resolution of 1.8 mm (outer diameter plus one spacing, i.e., 0.1 mm between probes). The sampling mechanism is based on the principle of concentration gradient diffusion. The automatic loading of degassed water allows minimal disturbance to the chemical species across the oxic-anoxic interface.
This paper describes the procedures of device setup and continuous porewater sampling across the soil-water interface on a daily basis. Concentration-depth profiles were selectively measured before (on Day 6) and after (on Day 7) disturbances induced by irrigation. The results showed that concentration-depth profiles were undergoing rapid changes, especially for redox-sensitive elements (i.e., iron and arsenic). These protocols can help investigate the biogeochemical responses across the soil-water interface under various disturbances caused by physical, chemical, and biological factors. The paper thoroughly discusses the advantages and disadvantages of this method for potential use in the environmental sciences.

A microdialysis profiler is described to sample dissolved porewater solutes across an oxic-anoxic soil-water interface in situ with minimal disturbance. This device is designed to capture rapid changes in concentration-depth profiles induced by disturbances at the soil-water interface and beyond.

Biogeochemical processes shift rapidly in both spatial (millimeter scale) and temporal (hour scale to day scale) dimensions at the oxic-anoxic interface ...

Culturing Arbuscular Mycorrhizal Fungi Using SAP-AS for Enhanced Observation

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.

Author Spotlight: Enhancing AM Fungi Research with SAP-AS &#8212; A Novel Technique for Single-Spore Cultures

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the ...

Protocol for Field to Lab Tree Ring Digitization in Dendroecological Studies

A New Workflow for Sampling and Digitizing Increment Cores

Here we present a new workflow from taking increment cores in the field, storing and transporting them to the lab, to digitizing their tree rings for further analyses for subsequent dendroecological analyses. The procedure involves the use of new sample carriers for increment cores. These new G&#228;rtner Schneider Core (GSC) holders are designed using three-dimensional (3D) modeling software and finally printed with a 3D printer. Using these mounts from the beginning in the field, the cores can directly be cut with a core microtome, and their surface can then be digitized without further rearrangement using a new high-resolution image-capturing system. They are thus available for direct analysis. This system allows digitizing tree rings from cores and disks, and also taking images from long micro sections (up to 40 cm) using transmitted light. This feature is of special interest for dendroecological and geomorphic applications to identify the onset of any disturbance in micro sections cut with a core-microtome.

Author Spotlight: Innovative Device Development for Advancing Dendroecology and Wood Anatomy Research

We present a protocol to use 3D-printed mounts to fix increment cores in the field without the necessity of unpacking and gluing them on wooden mounts. The new GSC holder allows placing the cores in a core microtome to cut their surface and directly transfer them to digital image capturing.

Here we present a new workflow from taking increment cores in the field, storing and transporting them to the lab, to digitizing their tree rings for ...