We are very grateful to Frank Yates for his precious advice. This work was supported by Sup&#39;Biotech.

1. Inocula preparation
<ol>
	<li>Prior to the experiment, lay 5 &#181;L of Trichoderma spp. SBT10-2018 spores stored at 4 &#176;C on potato dextrose agar medium (PDA) and incubate for 4 days at 30&#176;C with regular light exposure to promote conidia formation42 (Figure 1, panel A). 
	NOTE: Trichoderma spp. SBT10-2018 has been isolated from wood and is used as the model in this study for its rapid growth and ease of spore recovery. This strain is preserved by our laboratory.</li>
	<li>Recover conidia (Figure 1, panel A)
	<ol>
		<li>Lay 3 mL of 0.05% Tween-20 on the Trichoderma mycelium.</li>
		<li>Use a rake to release conidia from conidiophores; avoid pressing down on the mycelium to prevent hyphae from being torn away.</li>
		<li>Recover the solution rapidly with a micropipette to avoid it being absorbed by the agar medium and transfer into a 15 mL tube.</li>
		<li>Count the total number of spores using a hemocytometer and prepare a solution containing 3 x 106 spores/mL. 
		NOTE: This step must be performed carefully to prevent hyphae from being extracted. Spore preparation is then checked under microscope. Eventually, for strains presenting highly aerial and fluffy mycelium, a step of filtration using 40 &#181;M strainer filter can be added to eliminate residual mycelium fragment.</li>
	</ol>
	</li>
</ol>
2. Fungal plates preparation (Figure 1, panel B)
<ol>
	<li>Deposit 100 &#181;L of 3 x 106 spores/mL with a micropipette on a 9 cm diameter Petri dish containing PDA medium to obtain 4,800 spores/cm2 corresponding to 925 spores/5 mm diameter-agar plug.</li>
	<li>Add 10 g of 2 mm diameter glass beads with a sterile spatula and perform forward and backward movements parallel and perpendicular to the operator&#39;s arm to evenly distribute the spores on the surface of the agar.</li>
	<li>Rotate the plate by 90&#176; and repeat the rotating movements (as in section 2.2); repeat these steps until the plate has been rotated completely.</li>
	<li>Use the plate immediately to set up experiments requiring spores or incubate the plates at 30 &#176;C for 17 h or 24 h when early hyphae or mycelium, respectively, are needed. 
	NOTE: To compare antifungal activity measured after mycelium plug-transfer and mycelium disk-transfer, use sterile tweezers and place sterile 5 mm cellulose disks randomly onto the surface of the agar plate after spore spreading.</li>
</ol>
3. Antifungal compounds preparation
<ol>
	<li>Plant-derived product preparation: garlic-powder preparation
	<ol>
		<li>Peel the cloves of fresh garlic and cut the cloves into 2-3 mm wide slices using a scalpel blade.</li>
		<li>Air-dry the slices for 2 days at 40 &#176;C.</li>
		<li>Grind the slices for 3 x 15 seconds using a knife mill to obtain a fine powder.</li>
		<li>Store the garlic powder at 4 &#176;C in 50 mL tubes before use. 
		NOTE: As garlic is not autoclaved (to prevent the degradation of temperature-sensitive antifungal compounds) clean the grinder, the scalpel, and the air-dryer with 70% ethanol before use.</li>
	</ol>
	</li>
	<li>Essential oil preparation
	<ol>
		<li>Prepare 0.5%, 1%, 2.5%, 5% and 20% Thymus vulgaris essential oil solutions in 0.5% Tween-80.</li>
		<li>Mix well to form an emulsion before adding it into the PDA medium (see section 4.2).</li>
	</ol>
	</li>
	<li>Carbendazim preparation
	<ol>
		<li>Weigh carbendazim to prepare a 200 mg/L ethanol solution (carbendazim is poorly soluble in water).</li>
		<li>Store the solution at room temperature before adding it into the PDA medium (see section 4.2). 
		CAUTION: Carbendazim presents a health and environmental hazard. Wear gloves and mask when handling this product. Store it in a ventilated space.</li>
	</ol>
	</li>
</ol>
4. Contact-inhibition assay
<ol>
	<li>Preparation of agar plates containing garlic powder
	<ol>
		<li>Prepare and autoclave PDA medium.</li>
		<li>Weigh the desired garlic powder quantity into a 50 mL tube using a sterile spatula, to obtain concentrations generally ranging from 0.25 mg/mL to 16 mg/mL.</li>
		<li>Add 10 mL of PDA after having checked the temperature of the medium on the inside of the wrist. The temperature must be as low as possible to prevent degradation of sensitive molecules. Ideally, this temperature should be 45 &#176;C.</li>
		<li>Homogenize carefully by turning the tube upside down to evenly distribute the powder into the PDA medium. Quickly pour 10 mL into a 5 cm diameter Petri dish (Figure 1, panel C).</li>
		<li>With the Petri dish placed at room temperature, wait until the agar solidifies.</li>
	</ol>
	</li>
	<li>Preparation of agar plates containing essential oil or carbendazim
	<ol>
		<li>Introduce 10 mL of PDA into a 50 mL tube. Check the temperature as for section 4.1.3.</li>
		<li>Add 100 &#181;L of the different solutions of Thymus vulgaris essential oil in PDA to obtain 0.005%, 0.01%, 0.025%, 0.05% and 0.2% solutions (see section 3.2.1).</li>
		<li>Add the required volume of carbendazim from the 200 mg/L solution to obtain solutions ranging from 0.0625-2 mg/L (see section 3.3.1).</li>
		<li>Homogenize carefully by turning the tube upside down, quickly pour 10 mL into a 5 cm diameter Petri dish (Figure 1, panel C).</li>
		<li>With the Petri dish placed at room temperature, wait until the agar solidifies.</li>
	</ol>
	</li>
	<li>Contact inhibition assay (Figure 1)
	<ol>
		<li>With a 5 mm diameter sterile stainless-steel tube, plot a circle in the center of Petri dishes containing either PDA or PDA including antifungal compounds. Dispose of the agar cylinder using a sterile toothpick (panel C).</li>
		<li>With a 5 mm diameter sterile stainless-steel tube, plot circles randomly into the fungal plates from section 2. Plot between 15-20 circles per plate (panel B).</li>
		<li>Carefully withdraw the agar-cylinders covered by spores, early hyphae, or mycelium with a sterile toothpick and place the plugs into the empty space of Petri dishes containing either PDA or PDA including antifungal compounds (panel C).</li>
		<li>Incubate the plates containing spores&#160;for 48 h at 30 &#176;C, 31 h for the plates containing early hyphae and, 24 h for the plates covered with mycelium (panel C).</li>
		<li>Measure the diameter of radial growth and calculate the percent of fungal-growth inhibition over control using the formula (panel D) 
		&#8203;%&#160;fungal growth inhibition = (C - A/C)* 100 
		where C is the diameter of radial growth in PDA medium and A the diameter of radial growth in PDA medium containing the antifungal compounds. 
		NOTE: To compare antifungal activity measured after mycelium plug-transfer and mycelium disk-transfer, using sterile tweezers, transfer one 5 mm diameter disk previously deposited onto the surface of the fungal plates (section 2 note) at the center of Petri dishes containing either PDA or PDA containing antifungal compounds and proceed exactly as for agar-plug transfer</li>
	</ol>
	</li>
</ol>
5. Vapor-Phase inhibition assay
<ol>
	<li>Preparation of agar plates containing garlic powder
	<ol>
		<li>Proceed as in section 3.1.</li>
	</ol>
	</li>
	<li>Preparation of agar plate containing essential oil or carbendazim
	<ol>
		<li>Proceed as in section 3.2 and 3.3.</li>
	</ol>
	</li>
	<li>Preparation of fungal plates
	<ol>
		<li>Proceed as in section 2.</li>
	</ol>
	</li>
	<li>Vapor-phase antifungal inhibition assay (Figure 1)
	<ol>
		<li>Pour 10 mL of PDA medium into the lid of the 5 cm diameter Petri dishes containing either 10 mL PDA medium or 10 mL of PDA medium containing antifungal compounds into the bottom of the dishes. Wait until complete solidification of the agar at room temperature (panel C).</li>
		<li>Use a 50 mL centrifugal tube as a calibration tool to obtain a circle of PDA in the center of the lid; remove the PDA around the circle with a sterile spatula (panel C).</li>
		<li>Plot a circle in the center of the PDA medium placed into the lid with a 5 mm diameter sterile stainless-steel tube. Discard the agar-cylinder with a sterile toothpick (panel C).</li>
		<li>Form plugs with a 5 mm diameter sterile stainless-steel tube randomly into the fungal plates as in section 4.3.2&#160;(panel B).</li>
		<li>Using a sterile toothpick, carefully transfer the plugs covered either with spores, early hyphae, or mycelium from fungal plates into the lids of assay plates (panel C).</li>
		<li>Incubate at 30 &#176;C as in section 4.3.4 (panel C).</li>
		<li>Measure the diameter of radial growth and calculate the percent of fungal growth inhibition using the formula in section 4.3.5&#160;(panel D).</li>
	</ol>
	</li>
</ol>

The approach presented here allows for the evaluation of antifungal properties of minimally processed plant-derived products. In this protocol, homogenous distribution of spores on the agar surface is achieved using 2 mm glass beads. This step requires handling skills to properly distribute the beads and to obtain reproducible results, ultimately allowing the comparison of antifungal effects at different stages of fungal growth. We found that 5 mm glass beads or excessive rotation while homogenizing during spreading can ...

Global losses of fruits and vegetables can reach up to 50% of production1 and result mostly from food decay caused by fungi spoilage in field or during post-harvest storage2,3, despite the extensive employment of synthetic fungicides since the middle of the twentieth century4. The use of these substances is being reconsidered since it represents serious environmental and health hazards. As the harmful consequences of their use are showing up throughout ecosystems and evidence of potential health impacts has accumulated5,6, novel alternatives to old prophylactic strategies are being developed for pre- and post-harvest treatments7,8,9. Hence the challenge we face is two-fold. Novel fungicidal strategies must, firstly, maintain the levels of efficacy of food protection against phytopathogens and concomitantly, secondly, contribute to dramatically reducing the environmental footprint of agricultural practices. To fulfill this ambitious goal, strategies inspired by the natural defenses evolved in plants are being proposed as more than 1000 plants species have been highlighted for their antimicrobial properties8. For instance, plants which have developed natural fungicides to fight phytopathogens are a novel resource in exploring the development of new biocontrol products2. Essential oils are flagship molecules of this type. For example, Origanum essential oil protects tomato plants against gray mold in greenhouses 10 and Solidago canadensis L. and cassia essential oils have been shown to preserve post-harvested strawberries from gray mold damage11,12. These examples illustrate that biocontrol and notably plant-derived products represent a solution that combines biological efficacy and environmental sustainability.
Thus, plants are an important resource of molecules of potential interest for the crop-protection industry. However only a handful of plant products have been proposed to be used as biocontrol products even though they are generally recognized as safe, non-phytotoxic and eco-friendly2. Some difficulties in the transposition from the lab to the field have been observed, such as efficacy decreasing once applied in vivo2,9. Thus, it becomes important to improve the ability of lab tests to better predict field efficacy. In this context, antifungal testing methods for plant-derived products are necessary both to evaluate their antifungal efficacy and to define their optimal conditions for use. Specifically, biocontrol products are generally less efficient than chemical fungicides, so a better understanding of their mode of action is important for proposing suitable formulations, to identify the mode of application in fields, and to define which developmental stage of the pathogen is vulnerable to the candidate bioproduct.
Current approaches addressing antibacterial and antifungal activities include diffusion methods such as agar-disk diffusion, dilution, bioautography and flow cytometry13. Most of these techniques, and more specifically, the standard antifungal susceptibility testing - agar-disk diffusion and dilution assays - are well-adapted for evaluating the antimicrobial activity of soluble compounds on bacterial and fungal spores in liquid suspensions14. However, these methods are generally not suitable for testing solid compounds such as dried plant powder or to quantify antifungal activity during mycelium growth as they require spore dilution or spore spreading on agar plates and/or dilution of antifungal compounds13. In the food-poisoned method, agar plates containing the antifungal agent are inoculated with a 5-7 mm diameter disk sampled from a 7-day old fungi culture without considering the precise quantity of starting mycelium. After incubation, the antifungal activity is determined as a percent of radial-growth inhibition17,18,19. With this approach we can evaluate the antifungal activity on mycelial growth. By contrast, the agar-dilution method is performed to determine the antifungal activity on spores directly inoculated on the surface of the agar plate containing the antifungal compounds13,20,21. These two approaches give complementary results on antifungal activity. However these are two independent techniques used in parallel that do not provide accurate side-by-side comparison of the relative efficacy of antifungal compounds on spores and mycelium17,20,22 as the quantity of starting fungal material differs in the two approaches. Moreover, the antifungal activity of a plant-derived product often results from the combination of antifungal molecules synthesized by plants to face pathogens. These molecules encompass proteins, peptides23,24, and metabolites having wide chemical diversity and belonging to different classes of molecules such as polyphenols, terpenes, alcalo&#239;ds25, glucosinolates8, and organosulfur compounds26. Some of these molecules are volatile or become volatile during pathogen attack27. These agents are most often poorly water soluble and high vapor-pressure compounds that have to be recovered through water distillation as essential oils, some of whose antimicrobial activities have been well established28. Vapor-phase mediated susceptibility assays have been developed to measure the antimicrobial activity of volatile compounds following evaporation and migration via the vapor phase29. These methods are based on the introduction of antifungal compounds at a distance from the microbial culture29,30,31,32,33. In the commonly used vapor-phase agar assay, essential oils are deposited on a paper disk and placed in the center of the cover of the Petri dish at distance from the bacterial or fungal spore suspension, which is spread on agar medium. The diameter of the zone of growth inhibition is then measured in the same way as for the agar-disk diffusion method20,24. Other approaches have been developed to provide quantitative measurement of the vapor-phase antifungal susceptibility of essential oils, derived from the broth-dilution method from which an inhibitory vapor-phase mediated antimicrobial activity was calculated32, or derived from agar-disk diffusion assays31. These methods are generally specific to vapor-phase activity studies andnot appropriate to contact-inhibition assays. This precludes the determination of the relative contribution of volatile and non-volatile agents to the antifungal activity of a complex bioactive mixture.
The quantitative method we have developed aims to measure the antifungal effect of dried-plant powder on controlled quantities of spores and grown mycelium deposited on the surface of an agar medium to reproduce the aerial growth of phytopathogens during infection of plants15 as well as an interconnected mycelial network16. The approach is a modified experimental setup based on the agar-dilution and food-poisoned methods that also allows, in the same experimental setup, side-by-side quantification of the contribution of both volatile and non-volatile antifungal metabolites. In this study, the method has been benchmarked against the activity of three well-characterized antifungal preparations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclave-vacuclav 24B+</td>
			<td>Melag</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbendazim</td>
			<td>Sigma&#160;</td>
			<td>378674-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Sarstedt</td>
			<td>72.706</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Falcons tubes</td>
			<td>Sarstedt</td>
			<td>547254</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Five millimeters diameter stainless steel tube</td>
			<td>retail store</td>
			<td></td>
			<td>/</td>
		</tr>
		<tr>
			<td>Food dehydrator</td>
			<td>Sancusto</td>
			<td></td>
			<td>six trays</td>
		</tr>
		<tr>
			<td>Garlic powder</td>
			<td>Organic shop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>CLOUP</td>
			<td>65020</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/61798/61798eq1.png" /> 2 mm</td>
		</tr>
		<tr>
			<td>Hemocytometer counting cell</td>
			<td>Jeulin</td>
			<td>713442</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert</td>
			<td>&#160;UM400</td>
			<td>30 &#176;C</td>
		</tr>
		<tr>
			<td>Knife mill</td>
			<td>Bosch</td>
			<td>TSM6A013B</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual cell counter</td>
			<td>Labbox</td>
			<td>HCNT-001-001</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Measuring ruler</td>
			<td>retail store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological safety cabinets</td>
			<td>FASTER</td>
			<td></td>
			<td>FASTER BHA36, TYPE II, Cat 2</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Mettler-Toledo</td>
			<td>17014407</td>
			<td>100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Mettler-Toledo</td>
			<td>17014411</td>
			<td>20 - 200 &#181;L</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Mettler-Toledo</td>
			<td>17014412</td>
			<td>2 - 20 &#181;L</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82-1194500</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/61798/61798eq1.png" /> 55 mm</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82-1473&#160;</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/61798/61798eq1.png" /> 90 mm</td>
		</tr>
		<tr>
			<td>Pipette Controllers-EASY 60</td>
			<td>Labbox</td>
			<td>EASY-P60-001</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Potato Dextrose Agar</td>
			<td>Sigma&#160;</td>
			<td>70139-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision scale-RADWAG</td>
			<td>Grosseron</td>
			<td>B126698</td>
			<td>AS220.R2-ML 220g/0.1mg&#160;</td>
		</tr>
		<tr>
			<td>Rake</td>
			<td>Sarstedt</td>
			<td>86-1569001</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Reverse microscope AE31E trinocular</td>
			<td>Grosseron</td>
			<td>M097917</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Sterile graduated pipette</td>
			<td>Sarstedt</td>
			<td>1254001</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Thymus essential oil</td>
			<td>Drugstore</td>
			<td></td>
			<td>Essential oil 100%</td>
		</tr>
		<tr>
			<td>Tips 1000 &#181;L&#160;</td>
			<td>Sarstedt</td>
			<td>70.762010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 20 &#181;L&#160;</td>
			<td>Sarstedt</td>
			<td>70.760012</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 200 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.760002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth pick</td>
			<td>retail store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trichoderma spp strain</td>
			<td></td>
			<td></td>
			<td>Strain of LRPIA laboratory</td>
		</tr>
		<tr>
			<td>Tween-20&#160;</td>
			<td>Sigma&#160;</td>
			<td>P1379-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sigma&#160;</td>
			<td>P1754-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Labbox</td>
			<td>FORS-001-002</td>
			<td>/</td>
		</tr>
	</tbody>
</table>

measuring volatile and non-volatile antifungal activity of biocontrol products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

To evaluate the ability of the quantitative method to discriminate the mode of action of different types of antifungal compounds, we compared the efficacy of three well-known antifungal agents. Carbendazim is a non-volatile synthetic fungicide which has been widely used to control a broad range of fungal diseases in plants39,40. Thymus vulgaris essential oil has been largely described for its antibacterial and antifungal activity and is used as natural f...

Watch this Scientific Journal Video about Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products at JoVE.com

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

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6.1K Views.  Sup’Biotech. We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

Video: Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often used on a limited number of stolons or rhizomes. For this reason, these traits are limited in their use for morphological characterization of plants. The use of digital image analysis software technology may overcome measurement errors due to human mistakes, which tend to increase as the number and size of samples also increase. The protocol can be used for any kind of crop but is particularly suitable for forage or grasses, where plants are small and numerous. Turf samples consist of aboveground biomass and an upper soil layer to the depth of maximum rhizome development, depending on the species of interest. In studies, samples are washed from the soil, and stolons/rhizomes are cleaned by hand before analysis by digital image analysis software. The samples are further dried in a laboratory heating oven to measure dry weight; therefore, for each sample, the resultant data are total length, total dry weight, and average diameter. Scanned images can be corrected before analysis by excluding visible extraneous parts, such as remaining roots or leaves not removed with the cleaning process. Indeed, these fragments normally have much smaller diameters than stolons or rhizomes, so they can be easily excluded from analysis by fixing the minimum diameter below which objects are not considered. Stolon or rhizome density per unit area can then be calculated based on sample size. The advantage of this method is quick and efficient measurement of the length and average diameter of large sample numbers of stolons or rhizomes.

A software-based image analysis system provides an alternative method to study the morphology of stoloniferous and rhizomatous species. This protocol permits measurement of the length and diameter of stolons and rhizomes and can be applied to samples with a large amount of biomass and to a wide variety of species.

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often ...

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 ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...