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Multi-site fungicides suppress banana Panama disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 [1]

['Stuart Cannon', 'Biosciences', 'University Of Exeter', 'Exeter', 'United Kingdom', 'Institute Of Biomedical', 'Clinical Science', 'William Kay', 'Department Of Plant Sciences', 'University Of Oxford']

Date: 2022-12 

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.

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.

Competing interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: Aspects of the research described herein are covered by patent application GB2202216.4 (Fusarium treatment in soil with MALCs) and patent WO2020201698A1 (Antifungal Compositions).

Funding: This work was supported by the Biotechnology and Biological Sciences Research Council ( https://www.ukri.org/councils/bbsrc/ ; grants: BB/N020847/1, led by Dr. Dan Bebber with SJG and GS, and in association Prof. Gert Kema, Wageningen, The Netherlands; BB/P018335/1, led by GS with SJG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The authors confirm that all relevant data are included in the paper or in the Supporting Information file. Additional information is available from the authors upon request. Raw sequencing data are available from the NCBI Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA803733 ). Numerical data and statistical analysis are provided in the Supplementary Information file S1 Data .

Copyright: © 2022 Cannon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we address these claims and develop robust and quantitative assays to evaluate 21 fungicides from 7 classes on (i) in vitro FocTR4 and FocR1 colony formation and, for comparison, the fungicide-sensitive wheat pathogen Zymoseptoria tritici, (ii) survival of FocTR4 macrospores, microspores, chlamydospores in liquid culture. This gave a subset of fungicides to test for their potential to protect against Panama disease caused by these pathogens. We then focussed our study on the current threat to Cavendish banana production by FocTR4, and compared the transcriptional responses of FocTR4 and Z. tritici to the single-site targeting azoles, SDHIs and strobilurins. This analysis provided better insight into the molecular drivers of tolerance against these fungicide classes. This systematic approach enabled us to (i) describe likely reasons for the failure of major fungicide classes to control FocTR4 (ii) identify that captan and mono-alkyl lipophilic cations (MALC S ) suppress Panama disease.

Different strategies to control FocTR4 include (i) crop husbandry, including removing infected tissues [ 9 ], (ii) biological control agents [ 10 ], (iii) efforts to generate resistant varieties [ 5 , 11 ] (iv) biosecurity protocols to reduce spread [ 10 ]. However, none of these have been particularly effective [ 1 ]. The best way to control fungi on crops is with fungicides [ 12 ]. Moreover, disinfectants are used to clean contaminated agricultural equipment [ 13 , 14 , 15 , 16 ]. Studies on the effect of various fungicides on FocTR4 in vitro [ 13 , 17 , 18 ] or in vivo, by corm injection [ 19 ], bare root immersion and/or soil drenches [ 13 , 17 , 18 , 20 ] provided contradictory results. Consequently, it is thought that "chemical measures are of limited or questionable efficacy" [ 1 , 9 ], or "Fusarium wilt cannot be controlled by fungicides" (ProMusa, https://www.promusa.org/Fungicides +used+in+banana+plantations). It is currently not known whether the insensitivity of Fusarium is related to the application of fungicides to soil or to the physiological response of the pathogen itself. Indeed, most highly effective and widely used fungicides, such as azoles, succinate dehydrogenases inhibitors (SDHIs) and strobilurins, target a single enzyme catalysing an essential process. These single-site fungicides can be overcome by point mutations in their respective target gene or by increased expression of mutated isoforms of the target enzyme [ 21 , 22 ]. Such increased transcription can be a consequence of mutations in transcription factors and thus is genetically inherited to the next generation of pathogen cells [ 23 , 24 ]. This risk of resistance development can be minimised by using multi-site inhibitors, which have multiple modes of action, thereby avoiding target-site resistance-related mechanisms [ 25 ].

FocTR4 (also named Fusarium odoratissimum [ 6 ]) produces 3 asexual spore forms, macroconidia, microconidia and chlamydospores [ 6 ]. Studies in Foc sub-tropical Race 4 suggest that these spore forms participate in the infection of bananas [ 7 ]. The thick-walled, melanised chlamydospores tolerate adverse environmental conditions and persist in soil debris for decades [ 3 ]. They spread as silent passengers on non-host species or are carried in soil, on footwear or on farm equipment [ 1 ]. Chlamydospores germinate under favourable conditions [ 8 ], penetrate root tissues, invade the cortex and enter the endodermis, where they colonise and block xylem vessels, inciting host tyloses formation [ 3 ]. The conidial forms are thought to play important roles during colonization [ 6 ]. Infection results in wilted, yellowed leaves, vasculature browning, corm necrosis and plant death [ 1 ].

The world’s banana supply was previously challenged by Fusarium oxysporum f. sp. cubense (Foc) as, in the 1950s, Race 1 (FocR1) decimated the commercially-dominant Gros Michel variety [ 1 , 3 ]. This threat was overcome by introduction of a resistant Cavendish variety [ 1 ]. However, with the appearance of Tropical Race 4 (FocTR4), identified in Taiwan in 1967, the world’s banana supply faced renewed jeopardy [ 2 ]. This aggressive strain has spread across the continents, reaching South America in 2019 [ 4 ]. The threat of Panama disease is of high significance, as Cavendish bananas currently account for ~40% of world production and >90% of all exports [ 5 ].

Bananas are amongst the most popular fruits eaten world-wide. Indeed, bananas (including plantains) are the 4 th most important global staple food crop, produced in over 150 countries at >114 million metric tons per annum (2019, http://www.fao.org/economic/est/est-commodities/oil crops/bananas). Bananas provide staple local food for almost half a billion people, whilst exports support economic stability [ 1 ]. They are thus an essential calorie crop and a revenue-generating trade commodity [ 2 ].

Results

FocTR4 forms fungicide-tolerant "persister" hyphae In the presence of several azoles, strobilurins and SDHIs, colony growth of FocTR4 and FocR1 was strongly inhibited at low concentrations. Paradoxically, sparse growth occurred at higher concentrations (Fig 3, e.g. epoxiconazole, pyraclostrobin; see S2 Fig, phase 1 and phase 2). This response indicates a majority of fungicide-sensitive cells and a subpopulation of fungicide-tolerant “persisters”. Microscopic investigation revealed that persisters grown on media-containing agar plates represented a mixture of “yeast-like”cells (Fig 2G and 2H, closed arrowheads) as well as thicker, swollen and irregular hyphae (Fig 2G and 2H, open arrowheads). To ensure optimal persister growth conditions, we incubated liquid media cultures of microconidia for 9 days in 3 μg ml-1 epoxiconazole, 500 μg ml-1 fluxapyroxad or 500 μg ml-1 azoxystrobin. Again, FocTR4 survived this treatment (S3A Fig) and formed hyphae (S3B Fig), whereas IPO323 cells died (S3A Fig).

Azole, SDHI and strobilurin FocTR4 fungicide-tolerance is not based on single-target site mutations FocTR4 persisters survive high concentration of the global market-leader fungicides, notably azoles, SDHIs and strobilurins [25]. These are single-site fungicides, binding directly to essential pathogen enzymes. Amino acid residue exchanges in the fungicide-interacting region can confer resistance [33,34,35,36]. BLAST searches using published nuclear and mitochondrial FocTR4 genomes [37,38] for putative target genes revealed 3 isoforms of the azole-target lanosterol 14α-demethylase (Foc_Erg11/1, Foc_Erg11/2 and Foc_Erg11/3), homologues of the SDHI-targeted subunits of succinate dehydrogenase (Foc_Sdh2, Foc_Sdh3/1, FocSdh3/2, Foc_Sdh4) and the mitochondrial respiration complex III protein cytochrome b (Foc_Cytb, see S3 Table for UniProt IDs and BLAST error probabilities). We sequenced genomic DNA of epoxiconazole-, fluxapyroxad- or azoxystrobin-treated persisters and searched for mutations in their promoters (1500 bp upstream of translation initiation codon ATG) and in their coding sequences. This revealed no variations between untreated cells and fungicide-grown persisters. However, mutations in unknown transcription factors can confer fungicide resistance (e.g. [24]). We therefore tested if the innate resistance of the newly-generated FocTR4 persister cells is genetically inherited. Persisters were replated onto agar, containing 3 μg ml-1 epoxiconazole, 500 μg ml-1 fluxapyroxad or 500 μg ml-1 azoxystrobin and their growth was compared with fungicide naive cells. We found that fungicide sensitivity of persister cells was the same as untreated wild-type cells (Fig 2I) and conclude that the ability of FocTR4 to cope with high fungicide loads of azoles, SDHIs and strobilurins is not genetically inherited.

All three FocTR4 spore forms survive treatment with most fungicides So far, our fungicide assays were performed in nutrient-rich medium, where persister hyphae survived azole, SDHI and strobilurin treatment. To better reflect the nutrient-poor soil environment, fungicide toxicity on chlamydopores, microspores and macrospores was assessed after 10 days in sterile distilled water (SDW), using quantitative LIVE/DEAD staining (Fig 6A shows examples). We also attempted to investigate hyphae in these assays. However, even in the absence of fungicide hyphae perished within 3–10 days in water (~60% dead after 10 days, not shown). Consequently, we only incubated all 3 spore forms with the highly effective fungicides (MIC<10 μg ml-1) at ~5-times of their MIC (carbendazim, 4.5 μg ml-1; thiabendazole, 10 μg ml-1; chlorothalonil, 5 μg ml-1; captan, 10 μg ml-1; mancozeb, 35 μg ml-1) and all less effective chemistries (MIC>10 μg ml-1) at 100 μg ml-1. All single-site fungicides, including azoles, strobilurins, SDHIs and benzimidazoles were largely ineffective in killing all 3 FocTR4 spore types; only tebuconazole and pyraclostrobin caused modest mortality in macro- and microconidia, but not in chlamydospores (Fig 6B–6D). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Fungicide sensitivity of FocTR4 spores in liquid culture. A Examples of LIVE/DEAD dye-stained microconidia and chlamydospores after 10 days in ddH 2 O, supplemented with carbendazim, mancozeb or captan. Living cells exclude the dye and often accumulate it in the cell wall (open arrowheads); dead cells fluoresce red. Scale bars = 10 μm. B Relative number of living microconidia (LIVE/DEAD staining negative) after 10 d-treatment with fungicides; fungicide classes are indicated above parenthesis. C Relative number of living macroconidia (LIVE/DEAD staining negative) after 10 d-treatment with fungicides; fungicide classes are indicated above parenthesis. D Relative number of living chlamydospores (LIVE/DEAD staining negative) after 10 d-treatment with fungicides; fungicide classes are indicated above parenthesis. E Morphology and LIVE/DEAD staining of microconidia, macroconidia and chlamydospores after 10 day-incubation in SDW, supplemented with epoxiconazole, fluxapyroxad and azoxystrobin (all 100 μg ml-1). Note that all spore forms can persist at high concentration of fungicides. Scale bar = 10 μm. Bars shown in (B—D) represent the mean proportion (±SEM) of cells that did not take up LIVE/DEAD staining, thus were considered alive; red dots represent averages of 3 independent experiments. All spore types in (B—D) were incubated for 10 days in SDW at 25°C and under rotation; concentrations used were in category I (= inhibition on agar plates at <10 μg ml-1): thiabendazole, 10 μg ml-1; carbendazim, 4.5 μg ml-1; chlorothalonil, 5 μg ml-1; captan, 10 μg ml-1; mancozeb, 35 μg ml-1; in category II (= inhibition on agar plates at >10 μg ml-1): copper, 100 μg ml-1 (applied as Copper(II) sulfate pentahydrate); LMW chitosan, 100 μg ml-1 (applied as a lactate salt); garlic oil, 100 μg ml-1; CTAB (Cetrimonium bromide), 100 μg ml-1; dodine, 100 μg ml-1; C 18 DMS; 100 μg ml-1; triticonazole, 100 μg ml-1; epoxiconazole, 100 μg ml-1: tebuconazole, 100 μg ml-1; azoxystrobin, 100 μg ml-1; trifloxystrobin, 100 μg ml-1; pyraclostrobin, 100 μg ml-1; bixafen, 100 μg ml-1; fluxapyroxad, 100 μg ml-1; boscalid, 100 μg ml-1; thiophanate methyl, 100 μg ml-1; concentrations in (e) are 100 μg ml-1. https://doi.org/10.1371/journal.ppat.1010860.g006 Interestingly, spores suspended in SDW culture were not killed by the single-site fungicides carbendazim and thiabendazole, or by the multi-site compound chlorothalonil (Fig 6B–6D). As these fungicides effectively inhibit growth on agar plates, we conclude that these compounds are fungistatic and not fungitoxic. Only the multi-site fungicides mancozeb, copper and three MALCs effectively killed all micro-, macroconidia and chlamydospores (Fig 6B–6D). This included CTAB and C 18 DMS, which showed lower effectiveness in agar plates, but greater efficacy in liquid culture. We also noted that several multi-site fungicides were less effective against chlamydospores (Fig 6B–6D, e.g. captan and LMW chitosan). As these durable soil-surviving spores initiate infection [3], a more detailed analysis was performed. This revealed that chlamydospores are enveloped by a fibrillar outer layer and an inner multi-layered wall (S6A Fig). This laminated wall is significantly thicker than in other morphotypes (S6B Fig) and could reduce fungicide efficacy. Finally, we tested if azole-, SDHI- and strobilurin-persisting spores undergo a morphological transition and found that surviving spores after 10 days incubation with 100 μg ml-1 still display their characteristic morphologies (Fig 6E), suggesting that these spores are innately resistant to these fungicides. In summary, our data show that all 12 tested single-site and 1 multi-site fungicide show no, or low, fungitoxicity in spores. This includes the major fungicide classes azoles, SDHIs and strobilurins.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010860

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