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Chemical Hormesis on Plant Pathogenic Fungi and Oomycetes Sumit Pradhan,1 Francisco J. Flores,2 Hassan Melouk,1 Nathan R. Walker,1 Julio E. Molineros,1 and Carla D. Garzon*,1 1Department

of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078 United States 2Department of Live Sciences and Agriculture, Universidad de las Fuerzas Armadas-ESPE, Sangolquí, Ecuador *E-mail: [email protected].

Hormetic effects of fungicides on fungi and oomycetes include moderate increase in growth rate, secondary metabolite production, and disease severity by plant pathogens. Hormesis has been documented in plant pathogens for exposure to low-doses of fungicides with both high and low fungicide resistance risk. Exposure to sub-inhibitory doses of fungicides can occur from improper use (e.g., over use of an active ingredient, poor application technique, or dilution of fungicide solutions in recirculation systems) or from natural degradation of the active ingredient, which may also lead to selection of fungicide resistant strains. One of the main challenges of chemical hormesis research is reproducibility. Systematic and meticulous screening and selection of target organisms, optimal mycelial age, chemical stressors and appropriate endpoints are required elements in experimental design for hormesis studies. Furthermore, multiple replicates and assay repetitions are necessary to ensure reproducibility and accuracy. Attempts to elucidate the mechanisms behind chemical hormesis have been fruitless so far. However, recent research suggests that increased mutation rates may result from exposure to sublethal doses of fungicides in fungi. Awareness of the risks associated to hormetic stimulation of fungi and oomycetes © 2017 American Chemical Society Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

among scientists, educators, growers and the general public is necessary to prevent aggravated damages and crop losses that may result from accidental stimulation of pathogens.

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Introduction As stated in other chapters in this volume, hormesis is the toxicological phenomenon where biological systems exhibit opposed responses to high and low doses of a stressor, characterized by low-dose stimulation and high-dose inhibition (1). Hormetic responses are consistent and independent of biological system, stressor, endpoint or mechanisms studied. Calabrese (1) highlighted that one of the most distinctive features of hormetic dose responses is the display of modest stimulatory responses at doses that are below and contiguous with the no observed adverse effect level (NOAEL) of the threshold dose-response model. Such modest stimulatory responses represent percentages rather than fold increments, often peaking at ranges between 10 to 60%, even though stressor doses may vary 10 to 20 fold, and in exceptional cases even 1000 fold (1–4). Recent studies on the hormetic effects of fungicides on fungi and oomycetes are consistent with the expectations derived from numerous case studies in so many other biological systems. Accidental exposures to subinhibitory doses of fungicides often derive from over use of fungicide active ingredients in disease management plans without proper fungicide rotation plans, leading to selection of fungicide resistant fungal strains. Most reports of fungicide hormesis are related to fungicides with high risk of resistance development, such as phenylamides, quinone inside inhibitors (QiI), and methyl benzimidazole carbamates (MBC) (4–8). However, hormetic responses to subtoxic doses of propamocarb, a low to medium risk fungicide, have also been observed (4). Furthermore, non-resistant isolates also display hormetic responses to subinhibitory doses of fungicides (4), but their hormetic dose ranges may be several degrees of magnitude lower than those of resistant isolates. Poor application technique, dilution of fungicide solutions in recirculation systems, natural degradation processes, among others, may also result in accidental exposure of fungal plant pathogens to subinhibitory doses of fungicides (Figure 1). Phytopathological literature reviews reveal numerous examples of plant pathogens stimulated by subtoxic doses of fungicides (4, 9–18). Most of them were accidental discoveries that were described as unexpected outcomes in fungicide efficacy studies and often reported as a curiosities. Unfortunately, much of this evidence has been overlooked and often explained as experimental error, deemed not relevant to the main objectives of research, or as anecdotal.

A Historical Perspective The earliest documented study of biphasic dose responses in fungi was reported by Hugo Schulz in 1888 (19). Schultz detected increased efficiency of yeast fermentation in presence of low doses of multiple chemicals, followed 122 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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by inhibition at higher doses. Although the unique biological responses were soon after referred to as the Arndt-Schultz law, because of their overlapping similarities with the observations of the homeopathic physician Rudolph Arndt, the low-dose stimulation concept was undermined at the time due to a lack of understanding of the mechanisms underlying such effects, and because of its needless association with homeopathy, a discipline that was widely criticized and deemed a pseudoscience. Several years later, Branham realized Schulz’s findings had been overlooked in spite of their validity. Stimulated by this realization, Branham studied the sub-lethal dose effects on CO2 production by yeasts of numerous chemical stressors, including mercuric chloride, mercurochrome, metaphen, hexylresorcinol, chloramine-T, iodine, and sodium hypochlorite (20). Exposure to sublethal doses of those chemicals resulted in an outburst of gas production during different phases of fermentation (20). Southam and Ehrlich (21) observed growth stimulation of Fomes officinalis, when multiple strains of the wood decaying fungus were grown on media amended with different concentrations of red-cedar extracts. Southam and Ehrlich used the term “hormesis” (derived from Greek word “hormôn” meaning to stimulate or induce) to describe their observations. After the exciting observations by Southam and Ehrlich, research on chemical hormesis of fungi was halted for decades. Nonetheless, numerous accounts of stimulation of fungi and oomycetes (fungus-like organisms of the Kingdom Stramenopila) by exposure to subtoxic doses of antifungal chemicals can be found in the literature (9–16).

Figure 1. How subinhibitory pesticide doses occur in the environment. 123 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Hormesis in Plant Pathogenic Oomycetes Although not defined as hormetic responses in the original articles, several studies have reported biphasic dose responses in oomycetes exposed to subinhibitory doses of antifungal chemicals. Fenn and Coffey (10) mentioned growth stimulation of up to 28% and 91% over the control at low doses of H3PO3 for Pythium ultimum and Pythium myriotylum, respectively. Likewise, Kato et al. (11) reported the stimulation of Phytophthora undulata linear growth by low doses of hymexazol. Evidence of growth stimulation of Phytopthora infestans by metalaxyl, a commonly used fungicide to control oomycetes, was demonstrated by Zhang et al. (13). In that study, three metalaxyl resistant isolates displayed vigorous growth stimulation with increased aerial biomass when grown on a media amended with metalaxyl (20 µl/ml). An interesting observation was that one of the three isolates grew more in metalaxyl amended media containing fewer nutrients. Based on these observations, metalaxyl was inferred to be beneficial to resistant isolates of Phytopthora infestans under low nutrition conditions. Moorman and Kim (16) observed increased radial growth of some strains of Pythium aphanidermatum, Pythium irregulare (recently re-named Globisporangium irregulare) and P. ultimum (G. ultimum) that were resistant to both propamocarb and mefenoxam. Stimulation of isolates of the three species was observed on media amended with propamocarb (1 µg/ml). Furthermore, a resistant isolate of P. aphanidermatum was stimulated by an unusually high dose of propamocarb (1000 µg/ml). In recent years, a renewed interest on fungicide hormesis emerged in response to casual grower reports of increased disease incidence in ornamental crops after fungicide applications (22). Garzon et al. (5) reported stimulatory effects of low doses of mefenoxam on radial growth of mycelium of mefenoxam and propamocarb resistant isolates of Pythium aphanidermatum and P. cryptoirregulare (currently Globisporangium cryptoirregulare), and increased severity of Pythium damping-off of geranium seedlings. Modest average mycelial radial growth stimulation of up to 10% was observed in vitro, with significant increments in damping-off of geranium seedlings of up to 61 % (Figure 2). Since in vitro growth stimulation is consistent but often modest in chemical hormesis assays, it can be challenging to obtain reproducible stimulation at specific fungicide doses, which might not be detected using traditional, and inappropriate statistical analyses (i.e. ANOVA). In an attempt to improve the accuracy of hormetic estimate, Flores and Garzon (4) reported standardized protocols in vitro for detection and assessment of such effects using radial growth as an endpoint. A statistical analysis capable of differentiating small responses to stimuli from background noise was used. The Brain-Cousens non-linear regression model (23) describes dose-response relationships where stimulation at low doses is followed by inhibition at high doses. Using the standardized protocols, significant growth stimulation of a mefenoxam and propamocarb resistant isolate of P. aphanidermatum was observed when it was exposed to multiple doses of ethanol and the fungicides propamocarb and cyazofamid (Figure 3). Pradhan et al. (18) reported significant hormetic responses in mefenoxam and propamocarb resistant isolates of Globisporangium ultimum and G. irregulare when exposed 124 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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to subinhibitory doses of mefenoxam. These results further support hormesis as a common phenomenon in oomycetes.

Figure 2. Area under the disease progress curves (AUDPC) of damping-off of geranium seedlings caused by a Pythium aphanidermatum. Exposure of a mefenoxam and propamocarb resistant isolate to subtoxic doses of mefenoxam resulted in increased disease levels (a). Significant differences are represented by non-overlapping notches. (5). Reproduced with permission from reference Garzon, C. D., Molineros, J. E., Yánez, J. M., Flores, F. J., Jiménez-Gasco, M. D. M., and Moorman, G. W. 2011. Sublethal doses of mefenoxam enhance Pythium damping-off of geranium. Plant Disease, 95: 1233–1238. (Reproduced with permission from APS Press, 2011) One of the main challenges faced during assessment of hormetic responses in microbiological systems is the reproducibility of stimulatory responses at specific doses. Reproducibility can be a challenge due to technical issues during fungicide solution preparation, as well as obtaining accurate endpoint measurements. A relatively simple solution to minimize experimental error is the careful and consistent preparation of fresh fungicide stock solutions to be used on the same day or soon after. This practice is fundamental to ensure result reproducibility in chemical hormesis studies, but in particular those focused on fungicide formulations at high concentrations, since the small volumes needed to prepare stock solutions increase the risk of random errors in experimental measurements (8, 24). Endpoint measurement accuracy can be affected by intrinsic properties of the biological systems under study, physiological changes due to environmental variability, as well as by technical difficulties during data collection. Systematic and meticulous screening and selection of target organisms, optimal mycelial age, chemical stressors and appropriate endpoints are required elements in experimental design for hormesis studies. It is not uncommon in fungicide hormesis studies to observe radial growth stimulation of 10% to 25% at doses under the NOAEL, with maximum stimulation dose varying from repetition to repetition, which can result in statistically non-significant stimulation across repetitions (5, 24). Pradhan et al. (18) obtained improved result reproducibility using alternative endpoints, like total mycelial dry mass weight 125 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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and total growth area. In particular, the calculation of total growth area using image measuring software minimized measurement biases due to experimental error and optimized reproducibility of results in the study, thus this endpoint and measurement protocol were recommended for future studies of fungicide hormesis for assessment of growth stimulation.

Figure 3. Observed values of radial growth (% control) in vitro of P. aphanidermatum in response to subtoxic doses of cyazofamid (ln of ppb), and modeled curve using the Brain-Cousens non-linear regression model (4).Reproduced with permission from reference Flores, F.J., and Garzon, C.D. 2013. Detection and assessment of chemical hormesis on the radial growth in vitro of oomycetes and fungal plant pathogens. Dose-Response 11:361-373. (Reproduced with permission from SAGE Publishing, 2013).

Hormesis in Plant Pathogenic Fungi The first evidence of hormesis on a fungal pathogen was reported by Southam and Ehrlich (21), who demonstrated growth stimulation in Fomes officinalis cultured in vitro at low doses of red-cedar heartwood extract. Nearly a decade later, Hessayon (9) examined the production on different soils of an antifungal compound produced by Trichothecium roseum, and found that sublethal doses of the studied trichothecene induced mycelial growth in Fusarium oxysporum. Baraldi et al. (15) studied 41 thiabendazole (TBZ) resistant isolates of Penicillium expansum and observed improved conidia germination in seven isolates when compared to the control, and proposed that such stimulatory response might have resulted from metabolization of the fungicide as a nutrient compound. A 2014 study of plant extracts as potential alternatives for filamentous fungi control found that although extracts of Acalipha subviscida and Echeveria acutifolia had excellent potential for control of Fusarium oxisporum and Alternaria alternata, they had biphasic dose effects on A. alternata, with growth inhibition at high 126 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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doses and growth stimulation at low doses (25). Noguerol-Pato et al. (26) reported significant increases in ethanol production in the yeast Saccharomyces cerevisiae exposed to the maximum residual level of dimethomorph (3.0 mg/kg) established by the European legislation in wine grapes. Studies designed to assess hormetic responses in fungi to sublethal doses of antifungal agents are few and recent. Nonetheless, these studies have confirmed the potential for hormetic responses to chemical stressors in basidiomycetes and ascomycetes (4, 7, 8, 24, 27, 28). Flores and Garzon (4) reported radial growth stimulation in Rhizoctonia spp. at subtoxic doses of ethanol. Audenaert et al. (27) observed stimulatory effects of subtoxic doses of the fungicide prothioconazole on Fusarium graminearum production of the mycotoxin deoxynivalenol (DON) in vitro and in planta. Low doses of prothioconazole+fluoxastrobin increased the production of the hydrogen peroxide, which enhanced DON production (Figure 4).

Figure 4. Production of deoxynivalenol (DON) by Fusarium graminearum during exposure to serial dilutions of prothioconazole+fluoxastrobin. DON production increased 48 h after fungicide treatment. (Audenaert et al. (27)). Conidia (106 conidia/ml) were challenged with tenfold dilutions of prothioconazole+fluoxastrobin starting from the field rate of 0.5 g/l + 0.5 g/l, in absence of 1000 U/ml catalase. The experiment included two repetitions and was replicated twice. Statistical analysis with a Kruskall-Wallis and Mann-Whitney test with a sequential Bonferroni correction for multiple comparisons found significant differences between treatments as reflected by different letters over the bars. (Modified from Audenaert et al. (27)). Reproduced with permission from reference Audenaert, K., Callewaert, E., Höfte, M., De Saeger, S., Haesaert, G. 2010. Hydrogen peroxide induced by the fungicide prothioconazole triggers deoxynivalenol (DON) production by Fusarium graminearum. BMC Microbiology 10:112. DOI: 10.1186/1471-2180-10-112. (Reproduced with permission from Springer Nature, 2010). 127 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Figure 5. Infection progress time course of Sclerotinia sclerotiorum isolates (AH-17 and LJ-86) on detached rapeseed leaves after treatment with subtoxic doses of carbendazim (0.2 and 1 ug/ml respectively) (Di et al. (8)) . Reproduced with permission from reference Di, Y.L., Lu, X.M., Zhu, Z.Q., and Zhu F.X. 2016. Time course of carbendazim stimulation on pathogenicity of Sclerotinia sclerotiorum indicates a direct stimulation mechanism. Plant Dis. 100:1454-1459. (Reproduced with permission from APS Press, 2016).

Zhou et al. (6) observed hormetic effects of dimethachlon fungicide on different isolates of Sclerotinia sclerotiorum. Eighteen out of 58 isolates had increased growth rates compared to the non-treated control when grown on media amended with 0.5 - 4 µg/ml dimethachlon. They also found increased virulence on detached leaves of oilseed rape after spraying plants with dimethachlon at a concentration of 2 µg/ml. Di et al. (7) reported statistically significant stimulation of the virulence of two carbendazim resistant S. sclerotiorum isolates on rapeseed detached leaves (up to 31% virulence increase) and on potted plants (23% increase) at doses of carbendazim between 0.2 and 5 µg/ml. Although this 128 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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study attempted to elucidate the potential pathogenicity mechanisms involved in stimulation, hormetic doses failed to increase oxalic acid production or isolate tolerance to hydrogen peroxide, two known pathogenicity mechanisms of S. sclerotiorum. In a subsequent study, Di et al. (8) assessed the time course of virulence stimulation by subtoxic doses of carbendazim. They observed larger lesion diameters caused by two S. sclerotiorum isolates on detached leaves treated with 0.2 and 1 µg/ml of carbedazim, compared to the non-treated control, after 12, 18 and 48 hours post inoculation (Figure 5). They also reported that virulence stimulation was significant from one to seven days after application (DAA) of 400 µg/ml of carbendazim on potted plants, with a reduction in stimulation magnitude at 10 DAA, and no significant stimulation observed 14 days DAA. Mycelium grown on PDA medium amended with 400 µg/ml of carbendazim lost its enhanced virulence on detached rapeseed leaves two days after being transferred to non-amended medium. However, a new attempt to elucidate the pathogenicity mechanism enhanced at hormetic doses failed when no significant effects were observed on cell-wall- degrading enzymes.

Unknown Risks, Challenges, and Future Directions Our current dependence on chemical management of plant diseases to maintain the current levels of agricultural productivity can serve as an indirect measure of the relevance of fungicide hormesis to agriculture and of the unaccounted economic losses occurring due to biphasic dose effects on fungal plant pathogens, derived from the lack of awareness about hormetic dose responses among growers, extension educators, plant scientists and plant pathologists in general. The study of fungicide hormesis is an emerging area of science with tremendous challenges ahead but also with imminent beneficial impact on agriculture and agriculture based economies. Although hormesis has been generalized as a universal phenomenon, scientists studying chemical hormesis face multiple challenges during the design and execution of experimental assays. Field, greenhouse and landscape observations of enhanced disease incidence and severity after application of certain fungicides often help to identify biological systems with potential hormetic responses, however, if the endpoint selected for assessment of biphasic dose responses is not appropriate, potential fruitful and high impact research can come to a halt. Too frequently, casual reports of increased virulence of fungal pathogens in the field offer opportunities to examine potential hormetic responses, but no evident stimulation is observed in vitro, even if multiple endpoints are examined (24, 28). Identifying an appropriate endpoint to evaluate the effects of subtoxic doses of inhibitory chemicals is not an intuitive task, and most of the time it is a trial and error discovery process. The selection of endpoint depends on the variability of the parameters observed and the resources available. Growth stimulation in vitro is the most frequently used endpoint in the fungicide hormesis literature (6–8, 18) since it is relatively simple to measure and it is often the most evident hormetic response. Growth parameters vary from study to study, 129 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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ranging from linear growth, to radial growth, to growth area and total dry mass (18). Important factors when selecting endpoints for a study are reproducibility and magnitude of stimulation (29). Reproducibility can be optimized by using standardized protocols that minimize experimental conditions variability (4), and using quantification tools that allow unbiased measurement of the target endpoints (18). In some biological systems, certain fungicides at low doses can produce consistently mild growth stimulation in vitro in response to a range of hormetic doses, but the absence of a maximum stimulation dose can result in a lack of statistical significance using standard statistical analyses (5). In those cases, the application of statistical tools capable of discriminating low but significant stimulation from background noise is necessary. Several statistical methods are available for assessment of hormetic responses, including parametric, non-parametric, and model based methods (17, 29–32). Pathogenicity, often measured in terms of disease severity quantified as area under the disease progress curve (AUDPC), is another important endpoint for assessment of fungicide hormesis in plant pathogenic oomycetes and fungi. Hormetic responses often result in highly significant increments in disease incidence and disease severity, which can easily be assessed for statistical significance using standard statistical methods (5–8). However, pathogenicity is a complex trait that fundamentally describes the ability of an organism to cause disease in its host. Pathogenicity can be modified by extrinsic factors, such as host genotype, environmental factors affecting pathogen and host metabolism, fungicide formulations and toxicity on the host, etc. Hence, to demonstrate statistically that changes in pathogenicity and disease severity were caused by subtoxic doses of fungicides, requires solid experimental design, strictly standardized experimental protocols with appropriate negative controls, extremely careful execution of methods and procedures, and multiple replicates and repetitions (4). Precise attention to detail during experiment execution is critical for reproducibility, which cannot be accomplished without careful data records, and critical observation analysis. Without these strict experimental conditions, seemingly minor methodological changes and small experimental errors can result in variability that will render extensive data sets, collected over several weeks or months, nearly if not completely unpublishable (5). Fungicide hormesis research builds scientific character. Although enhanced growth and increments in disease severity are relatively easy to detect and quantify, elucidating the mechanisms behind hormetic stimulation in fungal pathogens is elusive (7, 8). Attempts to identify mechanisms underlying hormesis in fungi and oomycetes have been so far fruitless ((7, 8), Garzon et al. unpublished). Pathogenicity factors such as production of oxalic acid, pectinase, polygalacturonidase, and cellulase, as well as tolerance to oxidative stresses have been examined as potential hormetic mechanisms in Sclerotinia sclerotiorum, however, no evidence has been found of their stimulation during exposure to sublethal doses of fungicides (7, 8). Nonetheless, since enhanced virulence was detected less than 24h after inoculation, Di et al. (8) concluded that direct stimulation and not adaptive compensation mechanisms may be involved in this hormetic response of S. sclerotiorum to sublethal doses of carbendazim. 130 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Hormetic responses result in modest endpoint increases, some peaking at over 60% of the control. Such increases reflect direct stimulation or overcompensation responses to mild injury caused by a stressor. Systematic search and critical analysis of hormesis literature, provides evidence that increments above hundred percent of the control are rare. Suggesting hormetic responses may result from allometrically based integrative biological processes (1). The impact of fungicide hormetic dose-responses of fungal plant pathogens on their host may be predicted within allometric parameters. Accordingly, disease severity increments of up to 60% were reported for Pythium damping off of geranium seedlings due to exposure to sublethal doses of mefenoxam (5); while disease severity increments of 20 to 30% were described for Sclerotinia sclerotiorum necrotic lesion expansion on rapeseed leaves due to exposure to low doses of carbendazim (8). This property of hormetic dose responses needs to be considered in future studies assessing the risk of deliberate and accidental use of fungicides at subinhibitory doses. Awareness of the risk associated to fungicide hormesis among growers, scientists and educators, is necessary to bring attention to preventable losses that may result from accidental stimulation of plant pathogens. Furthermore, a recent study suggested that exposure of Sclerotinia sclerotiorum to sublethal doses of fungicides may increase mutation rates in this clonal fungus, which could result in new phenotypes, potentially leading to the emergence of more virulent and fungicide resistant strains (33). Currently, there is a complete lack of information regarding crop losses due to fungicide hormesis, but a rough estimate of 30 to 60% increment on disease severity, can provide an indirect assessment of the potential economic impact of this toxicological phenomenon. Fungicide hormesis research is currently widely underrepresented in research initiatives and is underfunded. Hopefully, the increasing evidence of fungicide hormesis accumulating in the phytopathological and toxicological literature will help to remediate the challenges we presently face.

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