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 ...

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