Multi-site fungicides suppress banana Panama disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4

Abstract

Global banana production is currently challenged by Panama disease, caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (FocTR4). There are no effective fungicide-based strategies to control this soil-borne pathogen. This could be due to insensitivity of the pathogen to fungicides and/or soil application per se. Here, we test the effect of 12 single-site and 9 multi-site fungicides against FocTR4 and Foc Race1 (FocR1) in quantitative colony growth, and cell survival assays in purified FocTR4 macroconidia, microconidia and chlamydospores. We demonstrate that these FocTR4 morphotypes all cause Panama disease in bananas. These experiments reveal innate resistance of FocTR4 to all single-site fungicides, with neither azoles, nor succinate dehydrogenase inhibitors (SDHIs), strobilurins or benzimidazoles killing these spore forms. We show in fungicide-treated hyphae that this innate resistance occurs in a subpopulation of "persister" cells and is not genetically inherited. FocTR4 persisters respond to 3 μg ml-1 azoles or 1000 μg ml-1 strobilurins or SDHIs by strong up-regulation of genes encoding target enzymes (up to 660-fold), genes for putative efflux pumps and transporters (up to 230-fold) and xenobiotic detoxification enzymes (up to 200-fold). Comparison of gene expression in FocTR4 and Zymoseptoria tritici, grown under identical conditions, reveals that this response is only observed in FocTR4. In contrast, FocTR4 shows little innate resistance to most multi-site fungicides. However, quantitative virulence assays, in soil-grown bananas, reveals that only captan (20 μg ml-1) and all lipophilic cations (200 μg ml-1) suppress Panama disease effectively. These fungicides could help protect bananas from future yield losses by FocTR4. Author summary Bananas are amongst the most popular fruits eaten world-wide, yet their production is seriously challenged by the fungus Fusarium oxysporum f. sp. cubense, Tropical Race 4 (FocTR4). Hitherto, no effective strategy to control this devastating disease has been described. Indeed, even fungicides, which are generally considered to be our "front-line weapon" against plant pathogenic fungi, are deemed ineffective against FocTR4. Here, we analyse the use of 12 single-site and 9 multiple-site fungicides against FocTR4 and FocR1 (Fusarium oxysporum f. sp. cubense, Race 1) and compare these findings with data raised from the wheat pathogen Zymoseptoria tritici. We perform quantitative growth and cell survival assays, using FocTR4 hyphae and the 3 spore types (macroconidia, microconidia, chlamydospores). We show that all FocTR4 morphotypes are highly tolerant of the most widely-used single-site fungicides (azoles, succinate dehydrogenase inhibitors, strobilurins). Analysis of gene expression in surviving "persister" hyphae suggests that they cope with single-target fungicides by multiple mechanisms. This includes increased production of fungicide target enzymes, efflux proteins and detoxification enzymes. Only multi-site fungicides, which have multiple ways to affect the pathogen cell, proved to be effective in killing FocTR4. However, quantitative assessment of disease symptom development in fungicide-treated bananas revealed that only captan and three lipophilic cations have the potential to control Panama disease.