Efficacy of Plant-Derived Fungicides at Inhibiting Batrachochytrium salamandrivorans Growth

Abstract

The emerging fungal amphibian pathogen, Batrachochytrium salamandrivorans (Bsal), is currently spreading across Europe and given its estimated invasion potential, has the capacity to decimate salamander populations worldwide. Fungicides are a promising in situ management strategy for Bsal due to their ability to treat the environment and infected individuals. However, antifungal drugs or pesticides could adversely affect the environment and non-target hosts, thus identifying safe, effective candidate fungicides for in situ treatment is needed. Here, we estimated the inhibitory fungicidal efficacy of five plant-derived fungicides (thymol, curcumin, allicin, 6-gingerol, and Pond Pimafix^®) and one chemical fungicide (Virkon^® Aquatic) against Bsal zoospores in vitro. We used a broth microdilution method in 48-well plates to test the efficacy of six concentrations per fungicide on Bsal zoospore viability. Following plate incubation, we performed cell viability assays and agar plate growth trials to estimate the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of each fungicide. All six fungicides exhibited inhibitory and fungicidal effects against Bsal growth, with estimated MIC concentrations ranging from 60 to 0.156 μg/mL for the different compounds. Allicin showed the greatest efficacy (i.e., lowest MIC and MFC) against Bsal zoospores followed by curcumin, Pond Pimafix^®, thymol, 6-gingerol, and Virkon^® Aquatic, respectively. Our results provide evidence that plant-derived fungicides are effective at inhibiting and killing Bsal zoospores in vitro and may be useful for in situ treatment. Additional studies are needed to estimate the efficacy of these fungicides at inactivating Bsal in the environment and treating Bsal-infected amphibians.

Keywords: Batrachochytrium salamandrivorans; amphibian; antifungal; minimum fungicidal concentration; minimum inhibitory concentration; plant-derived; salamander; zoospore.

Figure 1

Bsal zoospore viability (% of the positive control) of the six tested concentrations and minimum inhibitory concentration [MIC] of (A) 6-gingerol [25 μg/mL], (B) curcumin [5 μg/mL], (C) thymol [25 μg/mL], (D) allicin [0.156 μg/mL], (E) Pond Pimafix^® [31.25 μg/mL], and (F) Virkon^® Aquatic [60 μg/mL]. An asterisk (*) is above the MIC for each fungicide. Each box plot represents Bsal zoospore viability relative to the positive control (Bsal zoospores in half-strength TGhL broth) for each concentration tested per fungicide (n = 45 wells per concentration across all plates). Midlines in each boxplot denote the median and the upper and lower sections of each box represent the first and third quartiles. Colored points extending beyond the boxplot represent outliers. As mentioned in the main text, “%” values can be negative based on the standard equation that we used if observed values are less than the negative control.