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Saccharin Provides Protection and Activates Defense Mechanisms in Wheat Against the Hemibiotrophic Pathogen Zymoseptoria tritici

    Authors and Affiliations
    • Samara Mejri1
    • Maryline Magnin-Robert2
    • Beatrice Randoux2
    • Alina Ghinet3 4
    • Patrice Halama1
    • Ali Siah1
    • Philippe Reignault2
    1. 1UMR-Transfrontalière 1158 BioEcoAgro, Junia, Université de Lille, Université d’Artois, ULCO, UPJV, Université de Liège, INRAE, 59000 Lille, France
    2. 2Unité de Chimie Environnementale et Interactions sur le Vivant, Université de Littoral Côte d’Opale, 62228 Calais, France
    3. 3Laboratoire de Chimie Durable et Santé, Yncréa Hauts-de-France, Heath & Environment Department, Team Sustainable Chemistry, 59046 Lille, France
    4. 4Faculty of Chemistry, University of Iasi, 700506 Iasi, Romania

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    Plant resistance inducers are among the most promising alternatives to develop sustainable crop protection. Here, we examined the ability of saccharin, a metabolite derived from probenazole, to protect wheat against Zymoseptoria tritici, the most frequently occurring and damaging foliar pathogen on this crop. The experiments were performed in the greenhouse by treating seedlings of the wheat cultivar ‘Alixan’ with 15 mM of saccharin 2 days before challenge inoculation with the Z. tritici pathogenic strain T02596. Foliar application of saccharin resulted in 77% lower disease severity than in nontreated control plants. In vitro and in planta assays showed that saccharin did not exhibit any direct antifungal effect on spore germination or hyphal growth. Molecular investigations from 2 to 7 days posttreatment (dpt) revealed that saccharin treatment upregulates the expression of genes encoding for lipoxygenase (LOX) at all sampled time points and pathogenesis-related protein 1 (PR1) at 7 dpt, in both noninfectious and infectious contexts, as well as peroxidase (POX2) in noninfectious conditions. However, saccharin did not induce significant change in the expression of PAL gene encoding for phenylalanine ammonia-lyase. Our findings report for the first time the potential of saccharin to confer protection in wheat against Z. tritici through an elicitation and priming of LOX and PR gene-related defense pathways. Additional investigations would provide a better deciphering of defense mechanisms activated by this molecule in wheat against Z. tritici.

    Plants have the ability to defend themselves from pathogen attacks with a range of responses, based on constitutive defenses and other inductive ones. The speed and the intensity of such defense responses are important factors that may determine the success or failure of the plant colonization by the pathogen (Dixon et al. 1994). The first description of plant defense mechanisms was reported in the 1970s, with the discovery of microbial molecules able to induce in the plant the production of phytoalexins, secondary metabolites with antimicrobial proprieties (Keen et al. 1972). Since then, such molecules have been called elicitors (Keen et al. 1972), and their subsequent use in practice by the 1980s led to the emergence of the concept of resistance inducers (Kuc 1982). Resistance inducers can activate the plant immunity system by two means, elicitation or priming. Elicitation leads to a significant induction of plant defense responses upon treatment with a resistance inducer, whereas priming is characterized by an enhanced (faster or stronger) expression of defense reactions in response to infection upon pretreatment with a resistance inducer (Mauch-Mani et al. 2017).

    The plant immunity system relies on the specific recognition, by transmembrane plant protein recognition receptors, of relatively conserved molecular motifs of the pathogen, called pathogen-associated molecular patterns (Jones and Dangl 2006). Likewise, plant immunity could be activated through the recognition, by protein recognition receptors, of microbe-associated molecular patterns released by beneficial microorganisms, or of damage-associated molecular patterns associated to cellular damage induced in the plant (Jones and Dangl 2006; Siah et al. 2018). The perception of these motifs leads to the establishment of several defense reactions in the plant. This plant immunity begins with a cascade of earlier responses, including an influx of calcium (Galon et al. 2010), rapid extracellular alkalinization (Felle 2001), mitogen-activated protein kinase cascade involved in sequence phosphorylation events (Pedley and Martin 2005), and oxidative burst (Bolwell et al. 1995). Thereafter, subsequent transcriptional changes in attack-responsive genes lead to the synthesis of pathogenesis-related (PR) proteins, and the production of low–molecular mass secondary metabolites such as phytoalexins or antimicrobial peptides (van Loon et al. 2006). The activation of localized defense provides a local acquired resistance against the attacking pathogen but also initiates the transmission of a systemic signal, which may induce defense responses in distal parts of the plant, to provide protection against subsequent infections. This natural phenomenon whereby local prior infection by a pathogen confers a protection in spatially distant organs against subsequent attacks from a broad range of pathogens is known as systemic acquired resistance (Ross 1961). The phytohormone salicylic acid (SA-2-hydroxybenzoic acid) is considered a key signal for the activation of acquired resistance (local and systemic acquired resistance) in many plant species (Ádám et al. 2018). Infection signal spreading, to the healthy tissues of the infected plant and to other neighboring plants, occurs (in part) via a biologically inactive, nonpolar, and volatile form of SA, methyl salicylate SA, because the latter is more hydrophobic than SA and can therefore cross membranes more readily than SA (Forouhar et al. 2005). After reaching its destination via the phloem flow, this molecule is converted back to SA (Park et al. 2007). Several studies have demonstrated that exogenous application of SA activates the expression of PR protein-encoding genes and the accumulation of reactive oxygen species in plants (Faize and Faize 2018). Moreover, SA has been shown to confer plant immunity toward a wide range of phytopathogenic organisms, such as fungi, bacteria, and viruses (Glazebrook 2005; Vlot et al. 2009). This suggests that biological signaling molecules known to mediate plant resistance induction may be exploited as a tool to control plant pests and diseases through their defense-stimulating effect. Although SA is an effective inducer of plant defense reactions, rapid glycosylation often reduces its long-lasting efficacy (Faize and Faize 2018).

    Various functional analogs of SA were considered for their potential to induce plant defense responses (Tripathi et al. 2019). Reignault and Walters (2007) suggested that >30 different synthetic derivatives related to the bioactivity of SA have been shown to provide plant protection. For instance, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), also known as acibenzolar-S-methyl, and 2,6-dichloroisonicotinic acid have already been shown to induce plant resistance against a wide range of biotic stresses (Faize and Faize 2018; Lyon 2007; Tripathi et al. 2019). Application of BTH has been reported to be effective on 32 agricultural crops (Vallad and Goodman 2004). Another resistance inducer, probenazole (3-(allyloxy)benzo[d]isothiazole 1,1-dioxide), has been reported to confer protection against diseases in several plants, such as tobacco, tea, and rice (Iwata 2001; Nakashita et al. 2002; Yoshida et al. 2010). Probenazole is the active ingredient of the commercial product Oryzemate (developed by Meiji Holdings, Co., Ltd. Japan), used to control rice blast caused by the hemibiotrophic Magnaporthe grisea. In addition, probenazole appears to be metabolized readily in plants by hydrolytic dealkylation of the allyl group to form saccharin (1,1-dioxo-1,2-benzothiazol-3-one), which is then N-glycosylated (Roberts 1998). Saccharin may be the active metabolite of probenazole. However, saccharin has a chemical structure (five-membered heterocyclic ring attached to a phenyl) different from the other salicylic-related molecules (phenyl unit decorated with a phenol and a carboxylic acid [SA] or ester [methyl salicylate SA]), BTH, or 2,6-dichloroisonicotinic acid, previously studied for plant resistance induction (Fig. 1). Saccharin has also been reported as an inducer of systemic resistance in various host–pathogen systems (Boyle et al. 2009; Boyle and Walters 2005, 2006; Siegrist et al. 1998; Srivastava et al. 2011). In barley, saccharin did not induce defense directly but rather primes defense responses against the biotrophic fungus Blumeria graminis f. sp. hordei (Boyle and Walters 2006).

    Fig. 1.

    Fig. 1. Structure of the main synthetic molecules previously investigated in crop protection (salicylic acid, acibenzolar-S-methyl, 2,6-dichloroisonicotinic acid, and probenazole), and of saccharin targeted in the current study on the wheat–Zymoseptoria tritici pathosystem.

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    Wheat is one of the most cultivated crops worldwide. Septoria tritici blotch (STB), caused by the hemibiotrophic fungus Zymoseptoria tritici, is the most frequently occurring and one of the most economically damaging diseases on wheat crops worldwide, especially in Europe, where agroenvironmental conditions are suitable for disease development (Jørgensen et al. 2014). Severe disease epidemics in wheat may lead to >50% of yield losses (Ponomarenko et al. 2011). Currently, there is no completely resistant wheat cultivar, and the control of STB relies mainly on the use of conventional fungicides. Meanwhile, the durability of this control strategy is at risk of being compromised in the field because Z. tritici displays a strong capacity of adaptation and often succeeds in developing new resistances to fungicides and in the overcoming of host resistances (e.g., Cheval et al. 2017; Cowger et al. 2000). Therefore, looking for more sustainable agricultural practices, such as the use of biocontrol compounds including resistance inducers, is currently encouraged. Even though saccharin has already been tested on a broad spectrum of phytopathosystems, it has never been investigated on wheat against Z. tritici. The aim of the present study was to examine the protection efficacy of saccharin, an artificial sweetener, as a preventive treatment on wheat against Z. tritici. Furthermore, the mode of action of saccharin was investigated by assessing its direct antifungal activity toward Z. tritici by using both in vitro and in planta bioassays and by monitoring, during the early stages of treatment and infection, the relative expression of four genes involved in different defense pathways. These genes include a lipoxygenase-encoding gene (LOX) involved in the octadecanoid pathway, a phenylalanine ammonia-lyase-encoding gene (PAL) involved in the phenylpropanoid pathway, and two PR protein-encoding genes, including PR9 encoding for peroxidase (POX2), playing a role in the reactive oxygen species metabolism, and PR1 encoding for the pathogenesis-related protein 1.

    Materials and Methods

    Plant growth, treatment, and inoculation.

    Grains of the STB-susceptible wheat cultivar ‘Alixan’ (‘Limagrain’) were pregerminated in Petri dishes (12 × 12 cm) on moist filter paper, and sprouted grains were placed in 3-liter pots filled with universal loam. Replicates of three pots with 12 grains per pot were used for each condition, and two repetitions were done separately in time for each experiment. Experiments were performed in the greenhouse under semicontrolled conditions at 18°C (±2°C) with a day–night cycle of 16/8 h and supplementary lighting. When the third leaf of the plants was fully expanded, exogenous treatments were applied with a manual hand sprayer. In all experiments, saccharin was first dissolved in 1% ethanol (vol/vol) of final distilled water volume. Each pot containing 12 plants received 30 ml of saccharin solution at 15 mM, amended with 0.15% Cantor (Grandes Cultures, Jouffray Drillaud, France) as a wetting agent. Control plants were sprayed with distilled water alone or amended with 0.15% Cantor (vol/vol) and 1% ethanol (vol/vol). Two days after treatments, plants were inoculated by spraying a spore suspension of Z. tritici at 106 spores/ml in distilled water amended with 0.05% polyoxyethylene-sorbitan monolaurate. Spore suspensions were produced on potato dextrose agar (PDA) medium according to Siah et al. (2010a), with the Z. tritici strain T02596 isolated in 2014 from northern France and selected for its pathogenicity on the cultivar ‘Alixan’ in preliminary experiments. Immediately after inoculation, each pot was covered with a clear polyethylene bag for 3 days to ensure a water-saturated atmosphere necessary for a good infection development. We scored disease severity 21 days postinoculation (dpi) by assessing the percentage of the third leaf area covered by lesions (chlorosis or necrosis) bearing pycnidia.

    In vitro antifungal assay.

    Direct antifungal effect of saccharin was assessed in clear and sterile flat-bottomed polystyrene microplates, according to Siah et al. (2010b). Plate wells were filled with 150 μl of liquid glucose peptone medium (14.3 g/liter dextrose, 7.1 g/liter Bacto-Peptone [Difco Laboratories], and 1.4 g/liter yeast extract) amended with saccharin at 15 mM (final concentrations in 200 μl of medium). Aliquots of 50 μl containing 2.105 spores/ml of the Z. tritici strain T02596 were added to each well. Eight wells were used as replicates for each condition. Inoculated (i) and noninoculated (ni) media without saccharin were used as experimental controls. The plate was incubated for 6 days at 20°C in the dark while being shaken at 140 rpm. Fungal growth was measured with a plate reader at 405 nm. For the wetting agent Cantor, direct antifungal activity was assessed on PDA medium according to Siah et al. (2010b) because this product alone strongly influenced the optical density when compared with control medium during microplate reading. Cantor was added to PDA medium at 50°C after autoclaving at the same concentration used in the greenhouse (0.15%), corresponding to the approved field concentration. Aliquots of 5 μl of 5.105 spores/ml were spotted on the PDA plates. Five Petri dishes with one spot on each were used as replicates. After incubation for 10 days at 20°C in the dark, the colony perpendicular diameters were measured for each spot and compared with spots developed on control medium.

    In planta cytological assay.

    The assessment of spore germination and epiphytic hyphal growth was performed with Fluorescence Brightener 28 (Calcofluor, Sigma Aldrich). Three third-leaf segments (4 cm), from different plants and different pots, were used as replicates for each condition. Segments were collected at 1 and 5 dpi from control plants, plants treated with saccharin, and plants treated with Cantor. Segments were stained and prepared as described by Mejri et al. (2018a) and were observed microscopically under ultraviolet illumination. Observations were performed on 100 different and randomly observed fungal spores on each leaf segment. We measured the effect of saccharin on spore germination at 1 dpi by scoring the percentage of germinated spores. We assessed the effect of saccharin on hyphal growth at 5 dpi by noting four classes of fungal germination events: class 1, nongerminated spore; class 2, germinated spore with a small germ tube; class 3, germinated spore with a developed germ tube; and class 4, germinated spore with extensively developed epiphytic hyphae.

    Plant RNA extraction.

    To examine the plant gene expression, leaf samples were collected at 2, 3, 4, and 7 days posttreatment (dpt) for both (i) and (ni) conditions, immediately frozen in liquid nitrogen, and stored at −80°C. Samples were ground to a fine powder in liquid nitrogen, and total RNA was extracted from 100 mg of leaf tissues with an RNeasy Plant Mini Kit (Qiagen, The Netherlands). Genomic DNA contaminating the samples was removed with an RNase-Free DNase Set (Qiagen, The Netherlands). The obtained RNA was suspended in 60 µl of RNase-free water and quantified by measuring the absorbance at 260 nm (Eppendorf AG, BioPhotometer). Three third leaves from different pots were used as independent biological replicates for each condition.

    Real-time reverse transcription PCR analysis of gene expression.

    The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used to ensure reverse transcription of obtained RNA, according to the manufacturer’s protocol. The PCRs were conducted on subsequent obtained complementary DNA to amplify four wheat genes (Table 1). Actin (ACT) and β-tubulin (TUB)–encoding genes were used as housekeeping genes after preliminary assays performed on different samples. Primer efficiencies were calculated via real-time PCR on several dilutions of complementary DNA samples corresponding to different conditions (Table 1). Reactions were carried out in a real-time PCR detector under the following thermal conditions: a denaturation cycle at 95°C for 3:10 min, followed by an amplification and quantification cycle repeated 39 times (95°C for 3 s for annealing, 60°C for 30 s for extension). Melting curve assays were performed from 65 to 95°C with 0.5°C/s, and melting peaks were visualized to check the specificity of each amplification. Each quantitative PCR was performed in duplicate (two technical replicates) for each sample.

    Table 1. Primers used in quantitative reverse transcription PCR assays

    Data analyses.

    Comparisons between percentage of disease severity, in vitro fungal growth, in planta spore germination, and in planta hyphal growth were carried out with the Tukey test at a significance level of P ≤ 0.05, in XLSTAT software (Addinsoft, France). For gene expression, the relative levels of each gene expression (normalized to the housekeeping genes) were determined in CFX96 Touch software. Gene expression results were represented, at each time point, as log10 relative expression of treated (ni) plants compared with nontreated (ni) control plants (expression value in the control fixed at 1) in both noninfectious and infectious contexts, and as log10 relative expression of treated (i) plants compared with nontreated (i) control plants. To assess the effect of the fungus alone on gene expression, nontreated and (i) control plants were compared with nontreated and (ni) control plants, at each time point. Significant differences between the gene expression levels (means from three biological independent replicates, with each replicate being performed as a duplicate PCR) at each time point were determined via Student t test at P ≤ 0.05, in GraphPad Prism version 8.0 (GraphPad Software, Inc., CA).


    Treatment with saccharin protects wheat against Z. tritici.

    A foliar treatment of the susceptible wheat cultivar ‘Alixan’ with 15 mM saccharin, with Cantor used as a wetting agent, was carried out in the greenhouse 2 days before inoculation with the pathogenic Z. tritici strain T02596 in order to evaluate the protection efficacy of this molecule. A high level of disease severity (55.6% of diseased leaf area) was scored at 21 dpi on nontreated inoculated control plants. Plants treated with Cantor alone were not any different than control plants treated with water alone (Fig. 2). However, a significant reduction in disease severity was recorded on plants treated with saccharin, with a protection level that reached 77% disease severity reduction when compared with control plants (Fig. 2). Saccharin-treated plants did not display any visual impact on their vigor when compared with control plants.

    Fig. 2.

    Fig. 2. Disease severity level on plants of the wheat cultivar ‘Alixan’ treated or not with saccharin (15 mM) 2 days before challenge inoculation with the pathogenic Zymoseptoria tritici strain T02596. Cantor, used as a wetting agent at the approved field dosage (0.15%), was tested as an independent treatment. We recorded disease symptoms 21 days after inoculation by scoring the percentage of the third leaf area covered with lesions bearing pycnidia. Means tagged with the same letter are not significantly different under the Tukey test at P ≤ 0.05.

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    Saccharin did not affect directly Z. tritici in vitro or in planta.

    The concentration of saccharin (15 mM) tested in protection assays and the approved field concentration of the wetting agent Cantor (0.15%) were examined for their direct antifungal effect against Z. tritici. In vitro notations revealed no direct effect of saccharin on Z. tritici development at the tested concentration (0.94 ± 0.09 vs. 0.93 ± 0.08 optical density values recorded at 405 nm in the corresponding saccharin and control microplate wells, respectively). Likewise, the wetting agent, Cantor, did not exhibit a direct biocide effect toward Z. tritici. On the contrary, Cantor slightly increased the size of fungal in vitro colonies because of its wetting effect, observed during the inoculation (i.e., deposition of fungal spore suspension) of PDA-amended plates with fungal inoculum (10.7 ± 1.5 mm vs. 6.9 ± 1.3 mm colony perpendicular diameters scored in the corresponding Cantor and control Petri dishes, respectively).

    In planta microscopic notations performed on the surface of wheat leaves showed that at 1 dpi, 73% of spores were germinated on control plants. Cantor (0.15%) and saccharin (15 mM) did not significantly reduce the rate of germinated spores, with an observed percentage of germinated spores of 80 and 82%, respectively. At 5 dpi, fungal germ tubes were randomly oriented on the leaf surface without any specific positioning toward stomata, and no chemotropism or thigmotropism was observed (data not shown). No significant differences in the patterns of epiphytic hyphal growth were highlighted on the leaves treated with saccharin and Cantor when compared with the inoculated control plants (Fig. 3).

    Fig. 3.

    Fig. 3. In planta effect of saccharin and Cantor treatments on the epiphytic hyphal growth of the Zymoseptoria tritici strain T02596 on the third leaf surface of the wheat cultivar ‘Alixan’ at 5 days after inoculation. Four different classes of Calcofluor-stained germinated spores were assessed from 100 different spores on each leaf segment for each condition: class 1, nongerminated spores; class 2, germinated spores with a small germ tube; class 3, germinated spores with a developed germ tube; and class 4, germinated spores with a strongly developed germ tube. Within each class, bars with common letters are not significantly different under the Tukey test at P ≤ 0.05.

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    Saccharin elicits and primes defenses in wheat leaves against Z. tritici.

    The relative expression of LOX, PAL, POX2, and PR1 genes was examined via quantitative reverse transcription PCR in treated and nontreated plants in both (ni) and (i) conditions and was normalized to the ACT and TUB gene expression. In (ni) conditions, marked changes in the expression profiles of LOX, POX2, and PR1 were observed in saccharin (ni) treated plants when compared with the (ni) control plants, and no significant overexpression was recorded for PAL at all tested time points (Fig. 4). For the LOX gene, the expression level in saccharin-treated plants was upregulated with 15-, 48-, 11-, and 27-fold changes at 2, 3, 4, and 7 dpt, respectively. POX2 was first downregulated at 2 dpt (with a 0.30-fold change), then the gene was significantly upregulated at the following time points, with five-, three-, and fourfold changes at 3, 4, and 7 dpt, respectively. Regarding PR1, it was downregulated at 2 dpt with 0.07-fold change before being highly upregulated at 7 dpt, with a 296-fold change. For Cantor (ni) treated plants, slight changes in gene expression patterns were observed at all sampled dates, characterized mainly by downregulations (Fig. 4). Only a significant upregulation, observed for POX2 at 7 dpt, was scored on treatment with this wetting agent (Fig. 4).

    Fig. 4.

    Fig. 4. Relative expression of four selected defense-related genes in wheat leaves pretreated or not with saccharin or Cantor and inoculated or not with the Zymoseptoria tritici strain T02596. The relative expression of each modality was calculated as log10 fold change compared with the nontreated noninoculated (nt, ni) control at each time point. The expression value of the (nt, ni) control at each time point was fixed at 1. Bars represent the mean of three biological replicates. Error bars show ± standard deviation of the mean. The presence of an asterisk indicates a significant difference compared with the (nt, ni) control at the corresponding time point, according to Student’s t test at P ≤ 0.05. (i), inoculated; (ni), noninoculated.

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    The specific effect of the inoculation in (i) control plants was also assessed to highlight the way in which the fungus alone influences the targeted gene expression (Fig. 4). The nontreated (i) control plants showed a significant downregulation of LOX expression at all sampled dates, with 0.22-, 0.23-, and 0.05-fold changes at 3, 4, and 7 dpt, corresponding to 1, 2, and 5 dpi, respectively (Fig. 4). By contrast, POX2 was highly upregulated, with 154-, 34-, and 21-fold changes at 3, 4, and 7 dpt, respectively. PR1 was significantly upregulated (with an 11-fold change) only at 7 dpt, corresponding to 5 dpi. No significant change in PAL gene expression was detected at all tested time points in (i) control plants when compared with (ni) control plants (Fig. 4).

    The effect of treatments on plant gene expression in the infectious context was also examined at 3, 4, and 7 dpt, corresponding to 1, 2, and 5 dpi, respectively (Fig. 4). In saccharin-treated plants, a significant upregulation of LOX, POX2, and PR1 genes was observed at all examined time points, except for LOX at 4 dpt, and no significant change in PAL gene expression was highlighted at all tested time points (Fig. 4). The changes detected for LOX, POX2, and PR1 genes consisted of 19-, 2-, and 33-fold changes for LOX; 123-, 31-, and 9-fold changes for POX2; and 4-, 3-, and 156-fold changes for PR1, at 3, 4, and 7 dpt, respectively. Regarding Cantor, it exhibited overall expression patterns in infectious conditions similar to those found for the nontreated (i) control for all targeted genes, except for LOX gene at 7 dpt in (i) conditions where Cantor did induce any significant induction of this gene at this time point (Fig. 4).

    Finally, to investigate a possible priming effect of saccharin treatment, we performed a comparison between treated (i) plants and nontreated (i) control plants (Fig. 5). Results showed significant changes only in the expression of LOX and PR1 genes, whereas no significant change was scored for PAL and POX2 genes at all sampled dates. LOX was upregulated at all time points, with 88-, 10-, and 624-fold changes at 3, 4, and 7 dpt, corresponding to 1, 2, and 5 dpi, respectively. PR1 was upregulated with 3- and 13-fold changes at 3 and 7 dpt, corresponding to 1 and 5 dpi, respectively. Concerning Cantor in the infectious context, only changes in LOX gene expression (0.14-fold and ninefold changes at 4 and 7 dpt, respectively) were highlighted in plants treated with this wetting agent when compared with nontreated (i) control plants (Fig. 5).

    Fig. 5.

    Fig. 5. Relative expression of four selected defense-related genes in wheat leaves pretreated with saccharin or Cantor and inoculated with the Zymoseptoria tritici strain T02596, compared with wheat leaves nontreated and inoculated with Z. tritici, at 3, 4, and 7 days after treatment, corresponding to 1, 2, and 5 days after inoculation. The relative expression was calculated as log10 fold change compared with the nontreated inoculated (nt, i) control at each time point. Bars represent the mean of three biological replicates. Error bars show ± standard deviation of the mean. The presence of an asterisk indicates a significant difference compared with the (nt, i) control at the corresponding time point, according to Student’s t test at P ≤ 0.05. (i), inoculated.

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    The current controversy on the intensive use of conventional pesticides and the frequent adaptation of pathogens to control strategies requires the identification of new eco-friendly and sustainable control tools. Plant resistance inducers are considered one of the most promising alternatives satisfying such requirements. Our findings showed that saccharin, applied as a preventive foliar treatment on wheat, significantly decreases the disease severity caused by Z. tritici (>70% disease reduction) under greenhouse conditions, thus agreeing with previous results (Boyle and Walters 2005, 2006; Delgado et al. 2013; Koganezawa et al. 1998; Phuong et al. 2020a, b; Srivastava et al. 2011; Walters and Paterson 2012). Indeed, saccharin has been shown to confer protection to both monocot and dicot plants toward various phytopathogens, including fungi, bacteria, and viruses, with mostly biotrophic or hemibiotrophic infection processes. For instance, saccharin pretreatment increased resistance in wheat against B. graminis f. sp. tritici (Phuong et al. 2020a), in barley against both B. graminis f. sp. hordei (Boyle and Walters 2006) and Rhynchosporium commune (Walters and Paterson 2012), in bean against Phaseolus vulgaris (Delgado et al. 2013), in broad bean against Uromyces viciae-fabae (Boyle and Walters 2005), in soybean against Phakopsora pachyrhizi (Srivastava et al. 2011), in Arabidopsis thaliana against Pseudomonas syringae pv. tomato (Phuong et al. 2020b), and in tobacco against Tobacco mosaic virus (Koganezawa et al. 1998). In the case of barley, the state of induced resistance to R. commune in parental plants pretreated with saccharin was shown to be transmitted to the progeny, thus suggesting the possibility of producing disease-resistant plants by exposing parent plants to chemical elicitors (Walters and Paterson 2012).

    The protective effect of saccharin highlighted here in wheat plantlets against Z. tritici results from an activation of host defense mechanisms rather than from a direct antifungal activity, as demonstrated in both in vitro and in planta assays. No direct activity of saccharin was observed on both spore germination and epiphytic hyphal growth of the fungus on the wheat leaf surface. Likewise, it has been shown that, in both barley–B. graminis f. sp. hordei and A. thaliana–Colletotrichum higgingsianum pathosystems, pretreatment with saccharin by foliar application before fungal inoculation has no effect on conidial germination and fungal appressorium formation on the leaf surface (Boyle and Walters 2006; Phuong et al. 2020a). Moreover, Phuong and colleagues (2020b) confirmed that saccharin had no direct bactericidal effect on Pseudomonas syringae pv. tomato. Interestingly, a previous study reported that SA exhibits in vitro direct antifungal activity against Z. tritici (Mejri et al. 2018b), thus suggesting that SA and saccharin may have distinct direct effect–based biological activities toward this fungus.

    Our findings revealed that saccharin induces the expression of the wheat defense-related genes LOX, POX2, and PR1, whereas PAL gene expression was not influenced by saccharin application in all tested conditions and at all examined time points, suggesting that PAL-mediated pathway, involved in SA biosynthesis, did not play a key role in saccharin-induced resistance in wheat. Similarly, Shetty et al. (2009) established that PAL gene expression is not involved in wheat–Z. tritici interaction and that there were no expression-level differences in susceptible and resistant wheat cultivars infected with Z. tritici, whereas Adhikari et al. (2007) recorded an induction of PAL gene expression in resistant wheat cultivars 3 to 6 h after inoculation. In barley, Boyle and Walters (2006) found that saccharin treatment decreases plant PAL activity. In the case of LOX and PR1 genes, our results highlighted that saccharin pretreatment induces their expression in absence and presence of the infection (Fig. 4), with stronger expression levels of both genes, as well as an early induction of PR1 expression, in inoculated conditions when compared with nontreated inoculated controls (Fig. 5). Such patterns of induction clearly suggest that saccharin primes the expression of these defense-related genes in wheat in response to Z. tritici infection, although the molecule also elicited the expression of both genes in absence of the infection. Regarding LOX, the priming effect on this gene conferred by saccharin results mainly from its downregulation caused by Z. tritici infection, because no marked difference was noticed between the saccharin-treated plants in both (ni) and (i) conditions (Fig. 4). Indeed, pretreatment with saccharin seems to invert (enhance), in early manner, the expression of this gene in the presence of the infection, thus probably contributing to the significant wheat plantlet protection toward the pathogen provided by the molecule. The LOX (13-LOX) gene is involved in jasmonic acid biosynthesis, and many studies have highlighted a downregulation of this gene in wheat on infection with Z. tritici (Rudd et al. 2015; Somai-Jemmali et al. 2016). Various other reports showed that exogenous application of elicitors, such as heptanoyl salicylic acid, Bion, Spirulina, and Milsana, known to reduce the symptoms of powdery mildew or STB on wheat, leads to an upregulation of LOX gene expression or the stimulation of LOX activities (Le Mire et al. 2019; Randoux et al. 2006; Renard-Merlier et al. 2007). Concerning PR1, we found that saccharin treatment highly elicited the expression of this gene at 7 dpt in the absence of infection and primed its expression during infection, which also could contribute to the observed disease severity reduction conferred by saccharin. Likewise, it has been shown that saccharin application affects multiple PR protein-encoding genes in wheat seedlings with or without B. graminis f. sp. hordei infection, with an association of gene upregulation patterns with resistance to this pathogen (Phuong et al. 2020a). Interestingly, our results indicate that saccharin elicits but does not prime POX2 expression in wheat to a subsequent Z. tritici infection. Nevertheless, POX genes have been associated with innate resistance against Z. tritici. For instance, Shetty et al. (2003) reported that resistant wheat cultivars accumulate POX transcripts and had a higher POX activity compared with susceptible cultivars when inoculated with Z. tritici. We could hypothesize that the strong accumulation of POX2 transcripts we observed in the inoculated conditions (induced mainly by fungal infection) were produced by the plant in order to limit the wheat leaf tissue colonization by Z. tritici during the early stages of infection.

    In conclusion, the current study reports for the first time the potential of saccharin to confer strong protection to wheat against Z. tritici (≤77% disease severity reduction) under semicontrolled conditions, providing new insights into the development of new resistance inducers for the management of this pathogen. In vitro and in planta assays demonstrated that the protection by saccharin results from a host resistance induction rather than to a direct antifungal effect. Gene expression assay confirmed this mode of action and revealed that saccharin can both elicit and prime defense reactions in wheat, through an elicitation and priming of LOX and PR gene-related defense pathways. However, further investigations are needed to decipher the defense-related mechanisms (elicitation and priming) activated by saccharin in the wheat–Z. tritici pathosystem.

    The author(s) declare no conflict of interest.

    Literature Cited

    A. S. and P. R. contributed equally to this work.

    The author(s) declare no conflict of interest.

    Funding: This research was conducted in the framework of the projects Optistim, funded by Fondation de la Catho de Lille–Matériaux Verts Fonctionnels program and Yncréa Hauts-de-France, CPER Alibiotech, funded by the European Union, the French State, and the French Region Hauts-de-France, and Bioscreen (Smartbiocontrol portfolio), funded by the European program Interreg V.