The authors acknowledge a joint seed grant to Seyfferth and Tappero to support collaboration between the University of Delaware and Brookhaven National Laboratory. Parts of this research used the XFM (4-BM) Beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704.

1. Preparation of slam-freezing equipment
<ol>
	<li>Place two copper blocks (~5 cm x 5 cm x 15 cm) horizontally inside of a clean cooler capable of holding liquid nitrogen and pour enough liquid nitrogen to submerge the blocks. Once the bubbling subsides, place two spacers on top of one copper block at each end. 
	NOTE: The spacer height determines the height of the sample to be frozen; this example uses a 2 cm spacer to create cubes approximately 3 cm x 3 cm x 2 cm. The volume of the liquid nitrogen will depend o...

This paper describes a protocol to obtain preserved bulk soil + rhizospheres of wetland plant roots using a slam-freezing technique that can be used for elemental imaging and/or chemical speciation mapping.
There are several benefits of this method over existing methods. First, this method allows the simultaneous investigation of roots and the surrounding rhizospheres. Methods currently exist to preserve and chemically image roots out of their soil environment by washing away the soil and pres...

Roots and their rhizospheres are dynamic, heterogeneous, and critically important for understanding how plants obtain mineral nutrients and contaminants1,2,3. Roots are the primary pathway by which nutrients (e.g., phosphorus) and contaminants (e.g., arsenic) move from soil to plants and thus understanding this process has implications for food quantity and quality, ecosystem functioning, and phytoremediation. However, roots are dynamic in space and time growing in response to nutrient acquisition needs and they often vary in function, diameter, and structure (e.g., lateral roots, adventitious roots, root hairs)2. Heterogeneity of root systems can be studied on spatial scales from cellular to ecosystem-level and on temporal scales from hourly to decadal. Thus, the dynamic and heterogeneous nature of roots and their surrounding soil, or rhizosphere, poses challenges for capturing rhizosphere chemistry over time. Despite this challenge, it is imperative to study roots in their soil environment to characterize this critical plant-soil relationship.
The rhizosphere chemistry of wetland plants is particularly challenging to investigate because of steep oxygen gradients that exist from bulk soil to the roots, which change in space and time. Because roots need oxygen to respire, wetland plants have adapted to the low oxygen conditions of wetland soils by creating aerenchyma4, 5. Aerenchyma are hollowed cortical tissues that extend from shoots to roots, allowing the diffusion of air through the plant into the roots. However, some of this air leaks into the rhizosphere in less suberized parts of the roots particularly near lateral root junctions, less mature root tips and elongation zones6,7,8,9. This radial oxygen loss creates an oxidized zone in the rhizosphere of wetland plants that affects rhizosphere (bio-geo)chemistry and is distinct from the reduced bulk soil10,11,12. To understand the fate and transport of nutrients and contaminants in wetland rhizospheres and roots, it is critical to preserve the chemically reduced bulk soil, the oxidized rhizosphere, and roots of wetland plants for analysis. However, because the bulk soil contains reduced soil constituents that are oxygen-sensitive, root and soil preservation methods must preserve root structures and minimize oxygen-sensitive reactions.
Methods exist to fix plant tissues and preserve the ultrastructure for imaging, but those methods cannot be applied to chemically preserve roots growing in wetland soil. For investigations where only the elemental distribution within plant cells is desired, plants are typically grown hydroponically and roots can be easily removed from solution, fixed under high-pressure freezing and freeze substitution and sectioned for a variety of imaging applications including high-resolution secondary ion mass spectrometry (nanoSIMS), electron microscopy, and synchrotron X-ray fluorescence (S-XRF) analysis13,14,15. To investigate Fe plaque on the outside of wetland roots, these hydroponic studies must artificially induce Fe plaque formation in solution16, which does not accurately represent the heterogeneity of the distribution and mineral composition of Fe plaque formation and associated elements in situ17,18,19,20. Methods exist to preserve wetland soil and associated microorganisms with freeze-coring21, but it is difficult to obtain roots with this technique. Current methods to visualize roots growing in soil and their rhizospheric chemistry consist of two primary measurement types: elemental fluxes and total elemental concentration (and speciation). The former is typically measured using diffusive gradients in thin films (DGT)22,23,24, in which soil is placed into rhizoboxes to support plant growth in a laboratory setting and labile elements in the soil diffuse through a gel into a binding layer. This binding layer can then be imaged to quantify the labile elements of interest. This technique can successfully illustrate relationships between roots and the rhizosphere24,25,26,27, but artefacts from root-bounding may exist by growing plants in rhizoboxes, and information on the root interior is not captured with DGT. The latter involves sampling of the roots and rhizosphere, preserving the sample, and directly analyzing elemental distribution on a sample section. For this environmental sampling of wetland plant roots and their surrounding rhizosphere, careful sample handling is required to avoid artefacts from sample preparation.
Here a protocol is described that effectively preserves root structures and rhizosphere chemistry of wetland plants by slam-freezing and freeze drying. Flash-freezing can drastically slow down transformations of oxygen sensitive solutes but may damage roots and may cause mobilization when samples dry out. However, slam-freezing where the sample is frozen between copper blocks pre-cooled with liquid nitrogen minimizes root damage and sample distortion28. The preserved samples are then embedded in an epoxy resin that preserves As speciation20, 29 and can be cut and polished for imaging of roots within their rhizosphere soil. The samples in this report were analyzed by S-XRF chemical speciation imaging after thin sectioning. However, other imaging techniques could also be used, including laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS), particle induced x-ray emission (PIXE), secondary ion mass spectrometry (SIMS), and laser induced breakdown spectroscopy (LIBS) imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Copper blocks</td>
			<td>McMaster Carr</td>
			<td>89275K42</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond blade</td>
			<td>Buehler</td>
			<td>15 LC, 102 mm x 0.3 mm</td>
			<td>operation speed: 225 rpm</td>
		</tr>
		<tr>
			<td>Epoxy forms</td>
			<td>Struers</td>
			<td>40300085</td>
			<td>FixiForm</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Epotek</td>
			<td>301-2FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Loctite</td>
			<td>404</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin sectioning machine</td>
			<td>Buehler</td>
			<td>PetroThin</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet saw</td>
			<td>Buehler</td>
			<td>IsoMet 1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to preserve wetland roots and rhizospheres for elemental imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

This method allows for preservation of roots and chemical species in the roots and rhizosphere of wetland plants and into the bulk soil. In this work, the method was used to evaluate As speciation and co-localization with Fe and Mn oxides and plant nutrients in the rhizosphere of rice (Oryza sativa L.). Rice was grown at the RICE Facility at the University of Delaware where 30 rice paddy mesocosms (2 m x 2 m, 49 plants each) are used to grow rice under various soil and water management conditions with the goal o...

Watch this Scientific Journal Video about A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging at JoVE.com

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

This method provides researchers with a new way to prepare aquatic plant roots, the surrounding rhizosphere and bulk soil for elemental imaging techniques. The main advantage of this method is that it's relatively quick and affordable while also accurately preserving the distribution and speciation of elements of interest. This technique can be used to answer questions regarding many elements and plant systems from nutrients in agricultural systems to fate and transport of contaminants in fettermiation systems.Be sure to work quickly while also be mindful of the desired area or areas of the root system for sampling. And take multiple samples per root system. To prepare the slam freezing equipment for an experiment, place two, five by five by 15 centimeter copper blocks horizontally into a clean cooler capable of holding liquid nitrogen.And wearing the appropriate PPE, pour enough liquid nitrogen into the cooler to submerge the blocks. Once the bubbling subsides, place one spacer on each end of one copper block. Then, using tongs and cryogenic gloves, stand the other copper blog on its end to make retrieval easier when the sample is in place.To collect the sample, either use a potted plant or use a shovel to begin extracting the desired plant and rhizosphere from the wet soil, taking care of that the dug hole is much larger than the desired root volume. Use a steel blade to cut away any excess soil, taking care not to disturb the soil within the desired area. When the desired areas reach, cut an approximately three by three by two centimeter root cube, and immediately placed the cube between the two spacers on the horizontal copper block.For a slam freezing of the sample, used the cryogenic gloves to place the vertical copper block onto the spacers for about five minutes. When the bubbling subsides transfer the slam frozen rhizosphere cube sample into a piece of pre-labeled aluminum foil square. To freeze dry the soil cubes, when the freeze dryer has reached the proper vacuum pressure and temperature, place one frozen rhizosphere cube sample for clean and acid washed 50 milliliter tube or directly into the freeze dryer and use a clean disposable wipe to prevent dust from entering the vacuum pump.Place the samples into the freeze dryer vessel for at least a few days until dry. When the tissues have been freeze dried, use a steel blade to dried soil cubes to an appropriate size for the analysis before placing the cubes into labeled forms inside a vacuum desiccator. After preparing epoxy according to manufacturer's instructions, use a dropper to slowly add epoxy to the form on one side of the soil, until the epoxy entirely covers the sample.The soil will darken in color as the epoxy wets the soil. Once the forms are filled with epoxy, close the desiccator and turn on the vacuum. Check the level of a epoxy every 30 to 90 minutes for the first one to four hours, adding additional epoxy if necessary.Once the epoxy has hardened, remove the sample. After embedding, use a diamond blade precision wet saw to cut the sample. Cut the sample in a different direction if no roots are obtained in the previous cut.After trimming, manually sand the cut side of each sample with progressively finer sandpaper for 30 seconds per grit size. In this figure, several root diameters can be observed within the soil matrix as transverse sections. The roots may demonstrate varying levels of quality.For example, in this image, a well-preserved root, a root distorted by the freeze drying process, and a root that was pulled out during the thin sectioning process can be observed. Analysis of this root transverse section with a lateral root in longitudinal section by synchrotron x-ray fluorescence imaging as demonstrated allows the detection of iron, manganese, and arsenic. The presence of iron within the soil and surrounding the root in the iron plaque is also visible in the light micrograph images.Manganese is uniquely present within the cortex of the lateral root, but also co-localizes with iron in some areas within the iron plaque as a green-blue hue. Arsenic is mostly found within the vasculature of the lateral root merging into the vasculature of the primary root. Chemical speciation imaging reveals a variability in the localization of the arsenic species.As observed in this tri-color plot, arsenite and arsenite glutathione are closely associated in the vasculature, while arsenate primarily localizes within the exterior of the root, associated with the iron plaque. Prior to sample collection, determine the desired part of the root system for elemental imaging. This will dictate the location orientation of the root key removed.Following this protocol, samples can be imaged at micron scale or coarser using a variety of techniques, depending on the specific research questions, elements of the interest, or instrumentation available.

a-method-to-preserve-wetland-roots-rhizospheres-for-elemental

Research

JoVE Journal

Environment

3.2K Views.  University of Delaware. This method provides researchers with a new way to prepare aquatic plant roots, the surrounding rhizosphere and bulk soil for elemental imaging techniques. The main advantage of this method is that it's relatively quick and affordable while also accurately preserving the distribution and speciation of elements of interest. This technique can be used to answer questions regarding many elements and plant systems from nutrients in agricultural systems to fate and transport of contaminants in fet...

Video: A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

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, ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...