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.

investigation-plant-interactions-across-common-mycorrhizal-networks

Research

JoVE Journal

Environment

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

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 ...

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and ...

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 ...

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

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 ...

Advanced Workflow for Taking High-Quality Increment Cores - New Techniques and Devices

In dendroecological research, precise dating of each single growth ring is a basic requirement for all studies, focusing on ring-width variations only, chemical or isotope analyses, or wood anatomical studies. Independent of the sampling strategy for a certain study (e.g., climatology, geomorphology), the way samples are taken is crucial for their successful preparation and analyses.
Until recently, it was sufficient to use a (more or less) sharp increment corer to obtain core samples that could be sanded for further analyses. Since wood anatomical characteristics can be applied to long time series, the need to obtain high-quality increment cores has taken on a new meaning. Essentially, the corer needs to be sharp(ened) when used. When coring a tree by hand, there are some problems in handling the corer, resulting in the hidden occurrence of micro cracks along the entire core: When starting to drill by hand, the drill bit is strongly pressed against the bark and the outermost ring until the thread has fully entered the trunk. At the same time, the drill bit is moved up and down as well as sideward. Then, the corer is drilled all the way into the trunk; however, it is necessary to stop after each turn, change the grip, and turn again. All these movements, as well as the start/stop-coring, puts mechanical stress on the core. The resulting micro cracks make it impossible to create continuous micro sections, as they fall apart along all these cracks.
We present a protocol to overcome these obstacles by applying a new technique using a cordless drill to minimize these problems when coring a tree, as well as its effect on the preparation of long micro sections. This protocol includes the preparation of long micro sections, as well as a procedure to sharpen corers in the field.

Here, we present a protocol on how to avoid micro cracks in increment cores by applying a cordless drill with a torque multiplier to minimize problems when coring trees, as well as its effect on preparing long micro sections. This protocol also includes a procedure to sharpen corers in the field.

In dendroecological research, precise dating of each single growth ring is a basic requirement for all studies, focusing on ring-width variations only, ...