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>

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<li>Talibi, I., Boubaker, H., Boudyach, E. H., Ait Ben Aoumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+methods+for+the+control+of+postharvest+citrus+diseases%5BTitle%5D)+AND+%22Journal+of+Applied+Microbiology%22%5BJournal%5D)">Alternative methods for the control of postharvest citrus diseases.</a> Journal of Applied Microbiology. 117 (1), 1-17 (2014).</li>
<li>Arya, R., Sharma, R., Malhotra, M., Kumar, V., Sharma, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biodegradation+Aspects+of+Carbendazim+and+Sulfosulfuron%3A+Trends%2C+Scope+and+Relevance%5BTitle%5D)+AND+%22Current+Medicinal+Chemistry%22%5BJournal%5D)">Biodegradation Aspects of Carbendazim and Sulfosulfuron: Trends, Scope and Relevance.</a> Current Medicinal Chemistry. 22 (9), 1147-1155 (2015).</li>
<li>European Food Safety Authority. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conclusion+on+the+peer+review+of+the+pesticide+risk+assessment+of+the+active+substance+carbendazim%5BTitle%5D)+AND+%22EFSA+Journal%22%5BJournal%5D)">Conclusion on the peer review of the pesticide risk assessment of the active substance carbendazim.</a> EFSA Journal. 8 (5), 1-76 (2010).</li>
<li>Sakkas, H., Papadopoulou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+activity+of+basil%2C+oregano%2C+and+thyme+essential+oils%5BTitle%5D)+AND+%22Journal+of+Microbiology+and+Biotechnology%22%5BJournal%5D)">Antimicrobial activity of basil, oregano, and thyme essential oils.</a> Journal of Microbiology and Biotechnology. 27 (3), 429-438 (2017).</li>
<li>Steyaert, J. M., Weld, R. J., Mendoza-Mendoza, A., Stewart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproduction+without+sex%3A+conidiation+in+the+filamentous+fungus+Trichoderma%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Reproduction without sex: conidiation in the filamentous fungus Trichoderma.</a> Microbiology. 156, Reading, England. Pt 10 2887-2900 (2010).</li>
<li>Leontiev, R., Hohaus, N., Jacob, C., Gruhlke, M. C. H., Slusarenko, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Comparison+of+the+antibacterial+and+antifungal+activities+of+thiosulfinate+analogues+of+allicin%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">A Comparison of the antibacterial and antifungal activities of thiosulfinate analogues of allicin.</a> Scientific Reports. 8 (1), 1-19 (2018).</li>
<li>Scorzoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+standard+methodology+for+determination+of+antifungal+activity+of+natural+products+against+medical+yeasts+Candida+sp+and+Cryptococcus+sp%5BTitle%5D)+AND+%22Brazilian+Journal+of+Microbiology%22%5BJournal%5D)">The use of standard methodology for determination of antifungal activity of natural products against medical yeasts Candida sp and Cryptococcus sp.</a> Brazilian Journal of Microbiology. 38 (3), 391-397 (2007).</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><tbody><tr><td>Autoclave-vacuclav 24B+</td><td>Melag</td><td></td><td></td></tr><tr><td>Carbendazim</td><td>Sigma&amp;nbsp;</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>&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/61798/61798eq1.png&quot;/&gt; 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>&amp;nbsp;UM400</td><td>30 &amp;deg;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 &amp;micro;L</td></tr><tr><td>Micropipette</td><td>Mettler-Toledo</td><td>17014411</td><td>20 - 200 &amp;micro;L</td></tr><tr><td>Micropipette</td><td>Mettler-Toledo</td><td>17014412</td><td>2 - 20 &amp;micro;L</td></tr><tr><td>Petri dish</td><td>Sarstedt</td><td>82-1194500</td><td>&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/61798/61798eq1.png&quot;/&gt; 55 mm</td></tr><tr><td>Petri dish</td><td>Sarstedt</td><td>82-1473&amp;nbsp;</td><td>&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/61798/61798eq1.png&quot;/&gt; 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&amp;nbsp;</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&amp;nbsp;</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 &amp;micro;L&amp;nbsp;</td><td>Sarstedt</td><td>70.762010</td><td></td></tr><tr><td>Tips 20 &amp;micro;L&amp;nbsp;</td><td>Sarstedt</td><td>70.760012</td><td></td></tr><tr><td>Tips 200 &amp;micro;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&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P1379-250ML</td><td></td></tr><tr><td>Tween-80</td><td>Sigma&amp;nbsp;</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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6.3K 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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Immunology and Infection

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biology

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

Biochemistry

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Source: Chandley, P., et al. <a href="https://app.jove.com/v/64425">A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.</a> J. Vis. Exp. (2022).
This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

Test in vitro w celu oceny wpływu przeciwciał na biofilm Candida tropicalis

1. C. tropicalis&#160;biofilm formation

	Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier ...

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

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Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Test in vitro w celu oceny wpływu przeciwciał na biofilm Candida tropicalis