Name: investigation of plant interactions across common mycorrhizal networks using rotated cores
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Description: Watch this Scientific Journal Video about Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores at JoVE.com

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
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	<li>Modify commercial tubular seedling containers (subsequently called &#8216;containers&#8217;; Table of Materials) to have 19 mm wide x 48 cm length openings.
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		<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 a...

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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.

<table border="0" cellpadding="0" cellspacing="0" style="width:704px;" width="705">
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<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 ...

Most plants in nature are likely interconnected by symbiotic root-inhabiting fungi called mycorrhizal fungi. This method explicitly examines how common mycorrhizal networks influence plant interactions and the subsequent ecological consequences. With this method, we can explicitly examine the role of common mycorrhizal networks while allowing all plants to be colonized by fungi, as opposed to excluding fungi, which results in poorly growing plants.To begin modifying commercial tubular seedling containers with flexible plastic, use a drill press and hold the container against a fence on the drill press, and have a stop with a short dowel that fits inside the container to help hold it in place while drilling. Then, drill two holes, one above the other, in the sides of the container, so that the holes are about one centimeter apart. Cut the remaining thin piece of plastic between the holes to make one elongated opening, about two centimeters wide, and five centimeters long.Repeat cutting these elongated openings on the opposite side of the container. To cover the slots with nylon mesh, cut 40 micrometer pore mesh into as many 9.5 centimeter by 8.5 centimeter pieces as there are containers. Then, using high-strength industrial hot glue, fasten the nylon mesh externally onto the containers to cover both openings, with some slight overlap in the fabric.Apply hot glue around the openings on the container, and along the long edges on the nylon mesh, and roll the container onto the nylon mesh, and hydrophobic membrane, when used, to avoid burning your fingers. Add a layer of glue along the fabric edge where the mesh edges overlap, and press the edge onto some cardboard to firmly seal it. Once the glue has cooled, use flexible tape to attach the top and bottom ends of the fabric to the container, to prevent loose edges and ripping.Using the same tape, cover the small holes on the sides of the conical end of each container to prevent root growth out of the container into the rest of the pot. Place a glass marble into the bottom of each container to prevent soil loss while providing drainage, and then assemble pots as described in the manuscript. Use solid, unmodified containers for a control treatment that does not involve any potential for a CMN to form between plants.Add AM fungus inoculum to the soil, by uniformly mixing 1.2 centimeter-long chopped root pieces thoroughly with the soil. Select a desired soil mixed with an infertile silica sand or glass beads to decrease the concentration of mineral nutrients available to plants. Position the filled containers in previously-constructed drilled foam in the pots.To make nutrient-poor silica sand mixture of adequate drainage, mix medium particle size sand with small particle size sand in a cement mixer. Fill the interstitial space with this mixture using a funnel to assist in filling small spaces. Next, transplant pretreatment, or so-called nurse plants, of the desired species into each container to sustain AM fungi, which will spread among the containers and establish common mycorrhizal networks.Grow the plants for two to three months at approximately 24 degrees Celsius in a greenhouse to allow for CMN establishment. Sew experimental plants by seeding into containers. Wait until all containers have germinated seedlings before removing pretreatment nurse plants, by clipping their shoots.To establish CMN treatments, leave some containers not moved for the duration of the experiment. To physically sever hyphae extending among the modified containers, rotate some of the containers weekly, making sure to do it through one full rotation, to avoid unintentionally altering above-ground interactions. Immediately after rotation, heavily water all pots to reestablish contact between the interstitial substrate and the sides of containers.Soaking all pots with water after severing common mycorrhizal networks is critical to reestablish contact between the container and the interstitial substrate, therefore preventing unintended aeration of the containers. Fertilize neighboring plants with 0.5%nitrogen 15 enriched potassium nitrate, and ammonium chloride. Fertilize the target individual with a nitrogen 14 fertilizer of equal concentration.Re-randomize the positions of the pots over the course of the experiment, at least monthly. Measure growth weekly by measuring longest leaf length, to monitor when growth begins to slow, making sure to harvest before the plants become root bound. To harvest, clip all aboveground tissue, and place individual plants into envelopes labeled with their treatment, pot, and position.Then dry above-ground tissues at 60 degrees Celsius in a forced air drying oven, to constant weight, and then measure the dry weight of each plant tissue. Allow the soil to dry two to three days before removing the containers, and harvesting the roots. After harvesting, gently brush off as much soil as possible from the root systems.Wash the roots on a sieve with 250 micron pore size, to avoid root loss. After allowing the roots to air dry, weigh the whole root system. Clip the root system randomly, and place the fragments in 50%ethanol.Re-weigh the remaining root system, and store it in a labeled paper envelope to dry at 60 degrees Celsius for dry weight assessment. When growing Andropogon gerardii Vitman in a target plant experiment, severing or preventing CMNs diminished target's aboveground dry weight, suggesting that intact CMNs promoted plant growth. Plants with severed CMNs and no CMNs responded similarly to their treatments.Competition, in which the growth of one individual suppresses the growth of another nearby individual, was detected in the intact CMN treatment, but not in the severed or no CMN treatments. Thorough comparisons of different mineral nutrient leaf tissue concentrations, versus plant size-only concentrations of manganese, were positively associated with target plant aboveground dry weights, over all treatments. Target plant tissue was assessed for nitrogen 15 in leaf tissues versus plant size, after adding nitrogen 15 label only to neighbors'containers.Target plants with intact CMNs had higher nitrogen 15 concentrations, compared to both other treatments. Intact CMNs had a strongly positive, significantly different slope of nitrogen amount, from that of the severed CMNs treatment, suggesting that large plants obtained more nitrogen from CMNs reaching into neighboring containers, than small target individuals. In a field experiment using rotated cores made of PVC pipe, extraradical mycelium beyond pipes had little effect on growth of soapberry during the 13-month experiment.However, severing it by rotation of pipes reduced foliar nitrogen, phosphorus, and copper concentrations, substantially shown by the chlorotic plants. Rotate the containers in the same direction that the fabric is glued onto each container to avoid tearing of the fabric, and rotate 360 degrees to reduce aboveground changes to the plants. This procedure can be used in the field with modified PVC pipes filled with sterile soil to examine the role of extraradical mycelium in root colonization, and its influence on nutrient uptake.This technique allows for comparison of plants with common mycorrhizal networks, to mycorrhizal plants that are not interconnected by networks, instead of to small plants without mycorrhizas, as is most often done.

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