This work was jointly supported by the National Natural Science Foundation of China (41877139), the Major Projects of the National Natural Science Foundation of China (41991335), the National Key Research and Development Program of China (2016YFD0800204), the Natural Science Foundation of Jiangsu Province (No. BK20161616), the &#34;135&#34; Plan, and the Frontiers Program of the Chinese Academy of Sciences (ISSASIP1615).

The aim of this protocol is to provide a general pipeline, from a hydroponic culture experiment to metabolomic analysis, quantifying the effect of DEHP on alfalfa root exudates.
1. Hydroponic culture experiment
NOTE: This protocol presents an example of an alfalfa hydroponic culture experiment designed to obtain alfalfa (Medicago sativa) seedlings under the stress of different concentrations of DEHP. Three treatments were set up: the control without any additions, and the nutrient solution spiked with 1 mg kg-1 and 10 mg kg-1 of di(DEHP. The concentrations of DEHP were set according to the real content of DEHP in soil23. Each treatment had six replicates.
<ol>
	<li>Sterilize alfalfa seeds with 0.1% sodium hypochlorite for 10 min and 75% ethyl alcohol for 30 min.
	<ol>
		<li>Rinse the sterilized seeds several times with distilled water and then germinate on moist filter paper in a sterile Petri dish at 30 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Transfer 20 uniform, germinated, big-plump seeds onto an engraftment basket in a culture bottle filled with nutrient solution, composed of (in &#181;M): Ca(NO3)2, 3,500; NH4H2PO4, 1,000; KNO3, 6,000; MgSO4, 2,000; Na2Fe-ethylenediaminetetraacetic acid (EDTA), 75; H3BO3, 46; MnSO4, 9.1; ZnSO4, 0.8; CuSO4, 0.3; and (NH4 )6Mo7O24, 0.02. Adjust the solution pH to 7.0 using 0.1 M KOH. Renew all solutions weekly.</li>
	<li>Place all the culture bottles in a controlled growth chamber with a light intensity of 150-180 &#181;mol m-2 s-1 with a photoperiod of 16 h each day, at 27 &#176;C and 20 &#176;C representing day (16 h) and night (8 h), respectively.</li>
	<li>Transfer 15 uniform alfalfa seedlings to a new glass bottle for culture experiments under 1 mg kg-1 and 10 mg kg-1 DEHP stress after 2 weeks. Wrap the glass bottles with aluminum foil and parafilm to prevent photolysis and volatilization of the DEHP. To apply the same conditions, also wrap the control bottles with aluminum foil and parafilm. Supplement the nutrient solution daily to maintain the liquid level.</li>
	<li>Randomly place and rotate the bottles every 2 days to ensure consistent growth conditions for the alfalfa seedlings.</li>
	<li>After 7 days of cultivation, remove the alfalfa seedlings from the bottles and wash with ultrapure water several times, preparing for the collection of root exudates.</li>
</ol>
2. Collection, extraction, and metabolomic analysis of root exudates
NOTE: This protocol is divided into three parts: a collection experiment, an extraction experiment, and metabolomic analysis of the root exudates. The goal of the collection experiment is to transfer the metabolites secreted in plant samples to the solution system for subsequent extraction.
<ol>
	<li>Collection experiment
	<ol>
		<li>Transfer 10 uniform alfalfa seedlings to centrifuge tubes filled with 50 mL of sterilized deionized water. Submerge the roots in water to collect root exudates for 6 h; keep the tubes upright. Perform at least six replicates for each treatment.</li>
		<li>Wrap the centrifuge tubes with aluminum foil to protect the roots from light.</li>
		<li>Remove the plants and freeze-dry the collected liquid for metabolite profiling.</li>
	</ol>
	</li>
	<li>Extraction experiment
	<ol>
		<li>Add 1.8 mL of extraction solution (methanol:H2O = 3:1, V/V) to the tubes and vortex for 30 s.</li>
		<li>Apply ultrasound waves to the tubes for 10 min in an ice water bath.</li>
		<li>Centrifuge the samples at 4 &#176;C and 11,000 &#215; g for 15 min.</li>
		<li>Carefully transfer 200 &#181;L of supernatant into a 1.5 mL microcentrifuge tube. Take 45 &#181;L of supernatant from each sample and mix it into quality control (QC) samples at a final volume of 270 &#181;L, which is used for calibration of the metabolome data of samples.</li>
		<li>Freeze-dry the extracts in a vacuum concentrator. Continue drying with 5 &#181;L of the internal standard (ribonucleol).</li>
		<li>After evaporation in a vacuum concentrator, add 30 &#181;L of methoxyamination hydrochloride (dissolved in pyridine at a concentration of 20 mg mL-1) to the tubes and incubate the tubes at 80 &#176;C for 30 min. Then, add 40 &#181;L of bis(trimethylsilyl)trifluoroacetamide (BSTFA) reagent (with 1% trimethylchlorosilane [TMC], V/V) to the samples and place the tubes at 70 &#176;C for 1.5 h for derivatization.</li>
		<li>Cool the samples to room temperature and add 5 &#181;L of fatty acid methyl esters (FAMEs) (in chloroform) to the QC samples.</li>
	</ol>
	</li>
	<li>Metabolomic analysis
	<ol>
		<li>Inject 1.0 &#181;L of the derivatized extracts into a gas chromatograph system coupled to a time-of-flight mass spectrometer (GC-TOF-MS) for metabolomic profiling analysis using a splitless mode.
		<ol>
			<li>Use a capillary column (30 m x 250 &#181;m x 0.25 &#181;m) for the separation of root exudates, with helium as a carrier gas at a flow rate of 1.0 mL min-1. Set the injection temperature to 280 &#176;C, and maintain the transfer line temperature and ion source temperature at 280 &#176;C and 250 &#176;C, respectively.</li>
			<li>For separation, use the following oven program: 1 min isothermal heating at 50 &#176;C, a 10 &#176;C/min-1 oven ramp to 310 &#176;C, and a final isothermal heating at 310 &#176;C for 8 min.</li>
			<li>Perform electron collision mode with -70 eV of energy. Obtain mass spectra using full scan monitoring mode with a mass scan range of 50-500 m/z at a rate of 12.5 spectra/s.</li>
		</ol>
		</li>
		<li>Filter individual peaks to remove noise. The deviation value is filtered based on the interquartile range.</li>
		<li>Fill the missing values with half of the minimum values, standardize, and normalize the data.</li>
		<li>Import the final data in .csv format into statistical analysis software for multivariate analysis.</li>
		<li>Look up the metabolites in Kyoto Encyclopedia of Genes and Genomes (KEGG) database (a database resource for understanding high-level functions and utilities of the biological system), and classify the metabolites into different categories, such as carbohydrates, acids, lipids, alcohols, and amines. Use statistical analysis software to construct a pie chart to indicate the percentage of each category in all the root exudates.</li>
		<li>Apply supervised orthogonal projections to latent structures-discriminate analysis (OPLS-DA) to demonstrate the differences among groups.</li>
		<li>Screen significantly changed metabolites as differential metabolites based on a variable importance in projection (VIP) &#62; 1 and p &#60; 0.05 (Student&#39;s t test).</li>
		<li>Use the metabolome data to construct heat maps with the statistical analysis software and use the fold changes under different treatments to construct histograms.</li>
		<li>Look up the differential metabolites in the KEGG database and Pubchem and compile the metabolic pathways containing the differential metabolites. Perform pathway enrichment analysis or topology analysis.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This protocol provides general guidance on how to collect and measure the root exudates of alfalfa under DEHP stress, as well as how to analyze the metabolome data. Close attention needs to be paid to some critical steps in this protocol. In hydroponic culture experiments, alfalfa seedlings were hydroponically cultured in glass bottles filled with nutrient solutions with different concentrations of DEHP. The glass bottles should be protected from light by covering them with aluminum foil throughout the culture period, in...

Di(2-ethylhexyl) phthalate (DEHP) is a synthetic chemical compound that is widely used in various plastics and polymers as a plasticizer to improve their plasticity and strength. In the past few years, an increasing number of studies have suggested that DEHP is an endocrine disruptor and has adverse effect on the respiratory, nervous, and reproductive systems of humans and other animals1,2,3. Considering its health risk, the United States Environmental Protection Agency, European Union, and Environmental Monitoring Center of China have all classified DEHP in the list of priority pollutants. Soil has been considered as an important sink of DEHP in the environment, due to the application of plastic mulching and organic fertilizers, irrigation with wastewater, and sludge farm application4. As expected, DEHP has been ubiquitously detected in farmland soils, the content of which even reaches up to milligrams per kilogram of dried soil in some regions in China5,6. DEHP can enter plants mainly via the roots and undergo biomagnification at different trophic levels in soil ecosystems7. Therefore, significant concern has been raised about DEHP-induced stress in plants over recent decades.
Plants are usually vulnerable to DEHP exposure. DEHP stress has been observed to exert an adverse effect on seed germination and normal metabolism, thereby inhibiting plant growth and development8,9. For example, DEHP can induce oxidative damage to mesophyll cells, decrease the contents of chlorophyll and osmolytes, and elevate antioxidative enzyme activities, eventually resulting in a decline in the yield and quality of edible plants10,11. However, most of the previous studies on the response of plants to DEHP stress have focused on oxidative stress and physiological and biochemical characteristics. The corresponding mechanisms associated with plant metabolism are less-studied. Root exudates is a generic term describing compounds that plant roots secrete and release into the environment. They have been considered as the interaction media between plants and rhizosphere soil, playing an important role in supporting plant growth and development12. It has been well known that root exudates account for approximately 30%-40% of all photosynthetic carbon13. In polluted environments, root exudates are involved in improving the tolerance of plants to the stress of pollutants through metabolism or external exclusion14. As a consequence, a deep understanding of the response of plant root exudates to pollution stress may help reveal the underlying mechanisms associated with cell biochemistry and biological phenomena15.
Metabolomics technology provides an efficient strategy for measuring a large number of small molecule metabolites simultaneously within cells16,17, tissues18, and even exudates of organisms19, including sugars, organic acids, amino acids, and lipids. Compared with traditional or classical chemical analysis methods, the metabolomics approach greatly increases the number of metabolites that can be detected20, which can help identify metabolites in a higher-throughput way and identify key metabolic pathways. Metabolomics has been widely used in the research field of biological response in stress environments, such as heavy metals21, emerging pollutants22, and nanoparticles19. Most of these studies on plants have focused on the metabolic changes in interior plant tissues, whereas few have been reported on the response of root exudates to environmental stress. Therefore, the aim of this study is to introduce general guidelines for the collection of alfalfa root exudates to study the impact of DEHP on metabolite production. The results will provide a method guidance for the follow-up study of plant metabolomics by DEHP.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adonitol</td>
			<td>SIGMA</td>
			<td></td>
			<td>&#8805;99%</td>
		</tr>
		<tr>
			<td>Alfalfa seeds</td>
			<td>Jiangsu Academy of Agricultural Sciences (Nanjing, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>BSA124S-CW</td>
			<td></td>
		</tr>
		<tr>
			<td>BSTFA</td>
			<td>REGIS Technologies</td>
			<td></td>
			<td>with 1% TMCS, v/v</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>Heraeus Fresco17</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column</td>
			<td>Agilent</td>
			<td>DB-5MS (30 m &#215; 250 &#956;m &#215; 0.25 &#956;m)</td>
			<td></td>
		</tr>
		<tr>
			<td>Di(2-ethylhexyl) phthalate</td>
			<td>Dr. Ehrenstorfer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FAMEs</td>
			<td>Dr. Ehrenstorfer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatography(GC)</td>
			<td>Agilent</td>
			<td>7890A</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding instrument</td>
			<td>Shanghai Jingxin Technology Co., Ltd</td>
			<td>JXFSTPRP-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass spectrometer(MS)</td>
			<td>LECO</td>
			<td>PEGASUS HT</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CNW Technologies</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>Methoxyaminatio hydrochloride</td>
			<td>TCI</td>
			<td></td>
			<td>AR</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>Eppendorf Quality</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd</td>
			<td>DHG-9023A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Adamas</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>R software</td>
			<td></td>
			<td></td>
			<td>statistical analysis software (pathway enrichment, topology)</td>
		</tr>
		<tr>
			<td>SIMCA16.0.2&#160;</td>
			<td></td>
			<td></td>
			<td>statistical analysis software (OPLS-DA etc)</td>
		</tr>
		<tr>
			<td>Ultra low temperature freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Forma 900 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound</td>
			<td>Shenzhen Fangao Microelectronics Co., Ltd</td>
			<td>YM-080S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dryer</td>
			<td>Taicang Huamei biochemical instrument factory</td>
			<td>LNG-T98</td>
			<td></td>
		</tr>
	</tbody>
</table>

collection of alfalfa root exudates to study the impact of di(2-ethylhexyl) phthalate on metabolite production

Root exudates are the main media of information communication and energy transfer between plant roots and the surrounding environment. The change in secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol aims to introduce general guidelines for the collection of alfalfa root exudates to study the impact of di(2-ethylhexyl) phthalate (DEHP) on metabolite production. First, alfalfa seedlings are grown under DEHP stress in a hydroponic culture experiment. Second, the plants are transferred to centrifuge tubes containing 50 mL of sterilized ultrapure water for 6 h to collect root exudates. The solutions are then freeze-dried in a vacuum freeze dryer. The frozen samples are extracted and derivatized with bis(trimethylsilyl)) trifluoroacetamide (BSTFA) reagent. Subsequently, the derivatized extracts are measured using a gas chromatograph system coupled with a time-of-flight mass spectrometer (GC-TOF-MS). The acquired metabolite data are then analyzed based on bioinformatic methods. Differential metabolites and significantly changed metabolism pathways should be deeply explored to reveal the impact of DEHP on alfalfa in view of root exudates.

In this experiment, alfalfa root exudates were collected, extracted, and analyzed according to the above methods (Figure 1). Three treatment groups were set up: control, low concentration of DEHP (1 mg L&#8722;1), and high concentration of DEHP (10 mg L&#8722;1).
A total of 778 peaks were detected in the chromatograph of the control, of which 314 metabolites could be identified according to the mass spectra. As shown in F...

Watch this Scientific Journal Video about Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production at JoVE.com

Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production

The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

Collection of Alfalfa Root Exudates to Study the Impact of Di(2-ethylhexyl) Phthalate on Metabolite Production

Root exudates are the main media of information communication and energy transfer between plant roots and the surrounding environment. The change in ...

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1.5K Views.  Chinese Academy of Sciences. The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

Video: Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric chemistry and climate. The complex production mechanisms and reaction pathways make this a challenging research topic. To address these issues, an environmental chamber, providing enough residence time and close-to-ambient concentrations of precursors for secondary organic materials, is needed. The Harvard Environmental Chamber (HEC) was built to serve this need, simulating the production of gas and particle phase species from volatile organic compounds (VOCs). The HEC has a volume of 4.7 m3 and a mean residence time of 3.4 h under typical operating conditions. It is operated as a completely mixed flow reactor (CMFR), providing the possibility of indefinite steady-state operation across days for sample collection and data analysis. The operation procedures are described in detail in this article. Several types of instrumentation are used to characterize the produced gas and particles. A High-Resolution Time-of-Fight Aerosol Mass Spectrometer (HR-ToF-AMS) is used to characterize particles. A Proton-Transfer-Reaction Mass Spectrometer (PTR-MS) is used for gaseous analysis. Example results are presented to show the use of the environmental chamber in a wide variety of applications related to the physicochemical properties and reaction mechanisms of organic atmospheric particulate matter.

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric ...

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be critical for microbial energy production and persistence in many subsurface environments, especially those lacking soluble terminal electron acceptors. While EET-capable microbes have been successfully isolated from various environments, the diversity of bacteria capable of EET is still poorly understood, especially in difficult-to-sample, low energy or extreme environments, such as many subsurface ecosystems. Here, we describe an on-site electrochemical system to enrich EET-capable bacteria using an anode as a respiratory terminal electron acceptor. This anode is connected&#160;to a cathode capable of&#160;catalyzing abiotic oxygen reduction. Comparing this approach with electrocultivation methods that use a potentiostat for poising the electrode potential, the two-electrode system does not require an external power source. We present an example of our on-site enrichment utilized in an alkaline pond at the Cedars, a terrestrial serpentinization site in Northern California. Prior attempts to cultivate mineral reducing bacteria were unsuccessful, which is likely due to the low-biomass nature of this site and/or the low relative abundance of metal reducing microbes. Prior to implementing our two-electrode enrichment, we measured the vertical profile of dissolved oxygen concentration. This allowed us to place the carbon felt anode and platinum-electroplated carbon felt cathode at depths that would support anaerobic and aerobic processes, respectively. Following on-site incubation, we further enriched the anodic electrode in the laboratory and confirmed&#160;a distinct microbial community compared to the surface-attached or biofilm communities normally observed at the Cedars. This enrichment subsequently led to the isolation of the first electrogenic microbe from the Cedars. This method of on-site microbial enrichment has the potential to greatly enhance the isolation of EET-capable bacteria from low biomass or difficult to sample habitats.

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

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The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

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An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: &#34;There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region&#34;, where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: &#34;There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region&#34;.

The protocol focuses on the collection of sea turtle samples, describing all the steps from the animal recovery and necropsy to the classification and quantification of ingested marine litter. Moreover, the representative results show how to use the collected data to elaborate the possible thresholds for Good Environmental Status.

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the ...

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...