Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

Summary

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.

Abstract

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 Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact.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.

Introduction

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 storage^2,3, 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 accumulated^5,6, novel alternatives to old prophylactic strategies are being developed for pre- and post-harvest treatments^7,8,9. 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 damage^11,12. 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 vivo^2,9. 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 inhibition^17,18,19. 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 compounds^13,20,21. 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 mycelium^17,20,22 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, peptides^23,24, and metabolites having wide chemical diversity and belonging to different classes of molecules such as polyphenols, terpenes, alcaloïds²⁵, 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 culture^{29,30,31,32,33}. 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 method^20,24. 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.

Protocol

1. Inocula preparation

1. Prior to the experiment, lay 5 µL of Trichoderma spp. SBT10-2018 spores stored at 4 °C on potato dextrose agar medium (PDA) and incubate for 4 days at 30°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.
2. Recover conidia (Figure 1, panel A)
   1. Lay 3 mL of 0.05% Tween-20 on the Trichoderma mycelium.
   2. Use a rake to release conidia from conidiophores; avoid pressing down on the mycelium to prevent hyphae from being torn away.
   3. Recover the solution rapidly with a micropipette to avoid it being absorbed by the agar medium and transfer into a 15 mL tube.
   4. 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 µM strainer filter can be added to eliminate residual mycelium fragment.

2. Fungal plates preparation (Figure 1, panel B)

1. Deposit 100 µL 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.
2. 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's arm to evenly distribute the spores on the surface of the agar.
3. Rotate the plate by 90° and repeat the rotating movements (as in section 2.2); repeat these steps until the plate has been rotated completely.
4. Use the plate immediately to set up experiments requiring spores or incubate the plates at 30 °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.

3. Antifungal compounds preparation

1. Plant-derived product preparation: garlic-powder preparation
   1. Peel the cloves of fresh garlic and cut the cloves into 2-3 mm wide slices using a scalpel blade.
   2. Air-dry the slices for 2 days at 40 °C.
   3. Grind the slices for 3 x 15 seconds using a knife mill to obtain a fine powder.
   4. Store the garlic powder at 4 °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.
2. Essential oil preparation
   1. Prepare 0.5%, 1%, 2.5%, 5% and 20% Thymus vulgaris essential oil solutions in 0.5% Tween-80.
   2. Mix well to form an emulsion before adding it into the PDA medium (see section 4.2).
3. Carbendazim preparation
   1. Weigh carbendazim to prepare a 200 mg/L ethanol solution (carbendazim is poorly soluble in water).
   2. 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.

4. Contact-inhibition assay

1. Preparation of agar plates containing garlic powder
   1. Prepare and autoclave PDA medium.
   2. 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.
   3. 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 °C.
   4. 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).
   5. With the Petri dish placed at room temperature, wait until the agar solidifies.
2. Preparation of agar plates containing essential oil or carbendazim
   1. Introduce 10 mL of PDA into a 50 mL tube. Check the temperature as for section 4.1.3.
   2. Add 100 µL 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).
   3. 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).
   4. Homogenize carefully by turning the tube upside down, quickly pour 10 mL into a 5 cm diameter Petri dish (Figure 1, panel C).
   5. With the Petri dish placed at room temperature, wait until the agar solidifies.
3. Contact inhibition assay (Figure 1)
   1. 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).
   2. 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).
   3. 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).
   4. Incubate the plates containing spores for 48 h at 30 °C, 31 h for the plates containing early hyphae and, 24 h for the plates covered with mycelium (panel C).
   5. Measure the diameter of radial growth and calculate the percent of fungal-growth inhibition over control using the formula (panel D)
      ​% 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

5. Vapor-Phase inhibition assay

1. Preparation of agar plates containing garlic powder
   1. Proceed as in section 3.1.
2. Preparation of agar plate containing essential oil or carbendazim
   1. Proceed as in section 3.2 and 3.3.
3. Preparation of fungal plates
   1. Proceed as in section 2.
4. Vapor-phase antifungal inhibition assay (Figure 1)
   1. 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).
   2. 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).
   3. 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).
   4. Form plugs with a 5 mm diameter sterile stainless-steel tube randomly into the fungal plates as in section 4.3.2 (panel B).
   5. 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).
   6. Incubate at 30 °C as in section 4.3.4 (panel C).
   7. Measure the diameter of radial growth and calculate the percent of fungal growth inhibition using the formula in section 4.3.5 (panel D).

Results

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 plants^39,40. Thymus vulgaris essential oil has been largely described for its antibacterial and antifungal activity and is used as natural f...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
Autoclave-vacuclav 24B+	Melag
Carbendazim	Sigma	378674-100G
Distilled water
Eppendorf tubes	Sarstedt	72.706	1.5 mL
Falcons tubes	Sarstedt	547254	50 mL
Five millimeters diameter stainless steel tube	retail store		/
Food dehydrator	Sancusto		six trays
Garlic powder	Organic shop
Glass beads	CLOUP	65020	2 mm
Hemocytometer counting cell	Jeulin	713442	/
Incubator	Memmert	UM400	30 °C
Knife mill	Bosch	TSM6A013B
Manual cell counter	Labbox	HCNT-001-001	/
Measuring ruler	retail store
Microbiological safety cabinets	FASTER		FASTER BHA36, TYPE II, Cat 2
Micropipette	Mettler-Toledo	17014407	100 - 1000 µL
Micropipette	Mettler-Toledo	17014411	20 - 200 µL
Micropipette	Mettler-Toledo	17014412	2 - 20 µL
Petri dish	Sarstedt	82-1194500	55 mm
Petri dish	Sarstedt	82-1473	90 mm
Pipette Controllers-EASY 60	Labbox	EASY-P60-001	/
Potato Dextrose Agar	Sigma	70139-500G
Precision scale-RADWAG	Grosseron	B126698	AS220.R2-ML 220g/0.1mg
Rake	Sarstedt	86-1569001	/
Reverse microscope AE31E trinocular	Grosseron	M097917	/
Sterile graduated pipette	Sarstedt	1254001	10 mL
Thymus essential oil	Drugstore		Essential oil 100%
Tips 1000 µL	Sarstedt	70.762010
Tips 20 µL	Sarstedt	70.760012
Tips 200 µL	Sarstedt	70.760002
Tooth pick	retail store
Trichoderma spp strain			Strain of LRPIA laboratory
Tween-20	Sigma	P1379-250ML
Tween-80	Sigma	P1754-1L
Tweezers	Labbox	FORS-001-002	/