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 Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact.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

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....

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	/