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.
