The Nematode Signaling Molecule ascr#18 Induces Prepenetration Defenses in Wheat Against a Leaf Rust Fungus

The Nematode Signaling Molecule ascr#18 Induces Prepenetration Defenses in Wheat Against a Leaf Rust Fungus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Nematode Signaling Molecule ascr#18 Induces Prepenetration Defenses in Wheat Against a Leaf Rust Fungus Akshita Kamboj, Jennifer Thielmann, Saba Delfan, Tim Kloppe, Philipp Schulz, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4224139/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2024 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted 6 You are reading this latest preprint version Abstract Leaf rust, caused by the pathogenic biotrophic rust fungus Puccinia triticina ( Pt ), is one of the most destructive wheat diseases worldwide; its negative impact on crop yields is exacerbated by increasing temperatures due to climate change. Ascarosides are nematode pheromones that induce resistance to microbial pathogens and pests in a wide range of crops, making them valuable components in biocontrol scenarios. We investigated the effect on infection of various wheat ( Triticum aestivum ) genotypes with the virulent Pt race 77WxR by ascr#18, the major ascaroside secreted into the rhizosphere by plant-parasitic nematodes. Spraying the leaves with ascr#18 24 hours before inoculation with fungal uredospores slowed disease development and resulted in a reduction of the number of rust pustules on treated compared to untreated leaves. Dose-response analysis over the nano- and micromolar range revealed a broad optimum concentration down to 0.01 nM ascr#18. Microscopic analysis showed very early arrest of the fungus at the appressorial stage, with associated enhanced local accumulation of H 2 O 2 and abortive stoma penetration. The results of this study are consistent with and extend previous research that has shown that ascr#18 activates plant immunity and thus protects plants from pathogens even at very low doses. Ascaroside biocontrol crop protection induced resistance Puccinia triticina Triticum wheat Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In the face of climate change, political crises, and a growing population, there is an urgent need to secure and even increase wheat production to ensure sustainable food security (Poore and Nemecek 2018; OECD 2020). Leaf rust caused by Puccinia triticina ( Pt ) is one of the most common diseases affecting wheat worldwide (Bolton et al. 2008; Huerta-Espino et al. 2011; Kolmer 2013) and is exacerbated by temperature increases (Helfer 2014; Junk et al. 2016; Caubel 2017). Although widely successful in the past, control of leaf rust using conventional measures involving resistance genes ( R genes) and/or synthetic pesticides becomes ineffective over time because of strong selection pressures on fungal populations in intensive agricultural production systems to overcome R gene function or develop resistance or tolerance to pesticides (Andersen et al. 2018; Hawkins et al. 2019; van Esse et al. 2020). Modern integrative crop protection strategies include methods that rely on the plant's natural immune system (Galli et al. 2024). Plants, like animals, have evolved various innate and acquired immune strategies to combat microbial diseases (Sharrock and Sun 2020; Mermigka et al. 2020). Plants depend primarily on two interconnected layers of the innate immune system to recognize and respond to pathogens (Jones and Dangl 2006; Spoel and Dong, 2012; Han 2019). Firstly, pattern-recognition receptors (PRRs) recognize microbe-associated molecular patterns (MAMPs) from a wide spectrum of microbes, resulting in pattern-triggered immunity (PTI). A second layer involves disease resistance (R) proteins that recognize pathogen effector molecules or their activities on host targets, resulting in effector-triggered immunity (ETI). In addition, plants can acquire disease resistance by previous encounters with microbes or natural compounds such as plant hormones resulting in systemic acquired resistance (SAR) or induced systemic resistance (ISR), depending on whether the salicylic acid or jasmonate defense pathway is activated (Ryals et al. 1996; Pieterse et al. 2014; Klessig et al. 2018). This knowledge has led to the commercial development of synthetic resistance inducers such as Benzothiadiazol (BTH) or Probenazol that mimic the activity of a natural inducer and help to protect plants against various diseases (Nakashita et al. 2002; Kogel et al. 1994; Görlach et al. 1996; Vlot et al. 2021). More recently it was discovered that some synthetic resistance inducers and natural compounds such as salicylic acid, β-aminobutyric acid (BABA) or acyl-homoserine lactones (AHLs) at low concentrations trigger induced resistance via defense priming in plants leading to a physiological state that enables plants to respond more rapidly and/or more robustly to a challenge inoculation after exposure to biotic or abiotic stress (detailed reviews see Conrath et al. 2015; Balmer et al. 2015; Baccelli and Mauch-Mani 2016; Cooper and Ton 2022). An early response of primed plants is their production of reactive oxygen species (ROS) at the site of attack when they recognise a pathogen or pest, which is associated with the termination of the invasion or infestation (Balmer et al., 2015). Since a primed plant has only a very limited part of its defense system activated, priming could be a approach to protect plants from diseases and pests while minimizing energy expenditure and thus yield losses (Westman et al. 2019; Schenck et al. 2014; Jung and Cecchini 2023). Several recent studies have shown that a family of nematode-derived pheromones called ascarosides induce resistance in many plants against a broad spectrum of pathogens and pests by upregulating specific defense signaling pathways (Manosalva et al. 2015; Ali et al. 2018; Klessig et al. 2019; Ning et al. 2020). The term ascaroside originally referred to a distinct type of lipid first detected in parasitic roundworms of the family Ascaridia more than 100 years ago (Flury 1912). Ascarocides serve a wide range of biological functions which is facilitated by a great diversity of ascaroside chemical structures (Ludewig and Schroeder 2013). These are based on the sugar ascarylose, which is linked to fatty acid-like side chains of varying lengths and often decorated further with building blocks derived from amino acids, folate, and other primary metabolites. Plants can metabolize ascarosides and thereby change their chemical message, generating ascaroside mixtures that repel diseases and pests and reduce infection (Manohar et al. 2020; Yu et al. 2021). Here we report on the biocontrol effect of ascr#18, the most abundant ascaroside secreted by plant parasitic nematodes into the plant rhizosphere (Manosalva et al. 2015), on Puccinia triticina infections of various wheat ( Triticum aestivum ) genotypes. Importantly, ascr#18 was effective in the nano- and micromolar range, indicating a broad optimal concentration for controlling Pt . Our finding identifies a novel mode of ascr#18-induced resistance by triggering the accumulation of H 2 O 2 at attacked stomata, a characteristic also observed with other resistance inducers (Schenck et al. 2015). Materials and methods Plant material, fungal inoculation, and ascr#18 treatment The Puccinia triticina -susceptible wheat varieties Triticum aestivum cv. Chinese Spring (spring wheat) and winter wheat Arina LR (both provided by the Julius Kuehn Institute (JKI) Kleinmachnow, Germany), Zentos, Chinofuz (provided by the JKI Quedlinburg, Germany) and Boolani (Seed and Plant Improvement Institute, Karaj, Iran) were used. The leaf rust Pt race 77WxR (Serfling et al. 2013) was a gift of the JKI Quedlinburg. The virulence/avirulence profile of race77WxR used in field trials and seedling test are found in Rollar et al. (2021). The wheat cv. Boolani is susceptible to Pt race PKTTS (Delfan et al. 2022). The profile of virulence/avirulence of race PKTTS is shown in Table S1. The ascaroside ascr#18 was a gift from Ascribe Biosciences, 95 Brown Rd, Ithaca, NY, USA. For all experiments, wheat plants were grown in a pot containing fine-structured soil (Fruhstorfer Erde type T; HAWITA Gruppe GmbH, Vechta, Germany) in a growth chamber under controlled conditions with the temperature set to 18/20°C (night/day), light period of 16 h and 65% relative humidity. Ten-day-old seedlings were sprayed with ascr#18 in an aqueous solution containing 0.1% ethanol until run-off using a hand sprayer (Carl Roth, Germany); control plants were sprayed with 0.1% ethanol. After 24 h, leaves were inoculated by brushing with two-week-old Pt uredospores isolated from Triticum aestivum cv. Kanzler, using a mix of rust uredospores and talcum powder (Alliance Chemical, Germany) in a concentration of 1:4 (McIntosh et al. 1995). The inoculated seedlings were grown at 18/20°C (night/day) with 16 h of photoperiod, 95% relative humidity for three days, followed by 65% relative humidity for seven days. The number of uredinia were evaluated on one leaf per plant in an area of 0.5 cm² after 10 days post inoculation (dpi) by use of a binocular (Leica Microsystems GmbH, Wetzlar, Germany). Test on direct toxicity of ascr#18 To test whether there is a direct effect of ascr#18 on the germination of Puccinia triticina , 9-cm petri dish plates of water agar (3% w/v Agar) were pretreated with 2 mL of 1 µM ascr#18 dispensed in 0.1 % v/v ethanol using a sprayer (Preval, Art.-Nr. YC44.1). Subsequently, a suspension of Pt isolate 77WxR (5 mg of uredospores in 25 mL 0.1 % w/v agar) was sprayed onto the agar plates either 15 min or 24 h or after ascr#18 application. Inoculated plates were incubated in dark at 25°C for 10 h at 100% relative humidity. Three plates were prepared for each treatment and 100 uredospores were examined for germination on each plate. Uredospores were rated as germinated when germ tubes were visible and at least 5 times the size of the uredospore. To test whether there was a direct effect of ethanol on the germination, 0.1% ethanol was used in absence of ascr#18 as additional control. Water agar with 0.16 g/L prothioconazol (Proline, Bayer CropScience) was used as positive control for inhibition of Pt germination. Statistics For statistical analysis, data were checked for normality. The t test for normalized data and Mann-Whitney test for unnormalized data were performed in experiments with two groups to compare. For dose effect experiments, the analysis was done by one-way ANOVA and multiple comparison was carried out using Tukey’s post‐hoc test (p < 0.05). All statistical analyses and graphs were done with GraphPad Prism 8 software. For the germination test, data were fitted to a linear model using the function aov (Chambers et al.1992) in R. Tukey honest significant difference test was conducted on the fitted model using the TukeyHSD function (p < 0.05; Miller 1981). Microscopy Pt -infected leaves (10 dpi) of ascr#18-treated and mock-treated (0.1% ethanol) control plants were fixed in 4% paraformaldehyde (in PBS buffer) or 0.15% trichloroacetic acid (in chloroform:ethanol 20:80, v/v). Fungal structures were visualized using chitin-specific staining with WGA-AF488 (wheat germ agglutinin; Molecular Probes, Karlsruhe, Germany). Leaves were investigated under an epifluorescence microscope (Axio Imager.A2, Carl Zeiss, Oberkochen, Germany) and a confocal laser scanning microscope (CLSM; TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany) by use of ZEISS ZEN 3.8 and Leica LAS X software, respectively. WGA-AF488 was visualized at λ exc 494 nm, λ em 515 and fluorescence control settings were set to λ exc 631 nm, λ em 642. For H 2 O 2 detection, Pt -infected leaves of ascr#18- and mock-treated plants were collected 12 hpi, 24 hpi, 48 hpi and 96 hpi, and samples were stained with 3,3′-Diaminobenzidine (DAB)-tetrahydrochloride (Hückelhoven et al. 1999) and subsequently kept in 0.15% trichloroacetic acid (in chloroform:ethanol 20:80, v/v). To evaluate the DAB stained area, the average size of precipitates on the attacked stomata was quantified using ImageJ free software (https://imagej.net/ij/). Results Ascr#18 induces resistance against Puccinia triticina in all tested wheat cultivars The effect of ascr#18 on wheat against leaf rust was first tested with the four cultivars (cvs.) Zentos, Chinese Spring, Arina LR, and Chinofuz. Leaves of 10-day-old seedlings were sprayed with 1 µM ascr#18 and 24 h later inoculated with uredospores Pt race 77WxR. Ascr#18 significantly reduced the number of Pt uredinia on all four wheat genotypes as compared to mock treatment ( t -test for normalized data, Mann-Whitney test for unnormalized data; p < 0.05): Zentos (70%), Chinese Spring (71%), Arina LR (77%), and Chinofuz (81%) (Fig. 1; Supplementary Fig. 1). To exclude the possibility that the effect on Pt development was due to direct toxic effects of ascr#18, we exposed the fungus to 1 µM ascr#18 on water agar plates for 10 h. Consistent with previous reports on other fungal pathogens (Manosalva et al. 2015; Klessig et al. 2019), Pt 's germination rate was unaffected by ascr#18 and was comparable to the control treatments (Table 1). We concluded that ascr#18 induces resistance to leaf rust fungus in the wheat cultivars tested. Ascr#18 induces resistance against Puccinia triticina in the nM range Next, we conducted a dose-response experiment in the concentration range between 0.000001 to 10µM ascr#18. Leaves of 10-day-old seedlings of cvs. Chinese Spring and Zentos were sprayed with the respective concentrations of ascr#18 and 24 h later inoculated with Pt race 77WxR. Ascr#18 significantly reduced the number of uredinia on both wheat genotypes down to a concentration of 0.01 nM (One-way ANOVA, Tukey’s post‐hoc test; p < 0.05) (Fig. 2). To broaden the agronomic relevance, we extended our investigation to the wheat cv. Boolani, which is susceptible to Pt race PKTTS. Ten-day-old seedlings were sprayed with ascr#18 and the dose effect on the number of uredinia at 10 dpi was analysed. As expected, the number of uredinia was greatly reduced over a wide range of concentrations, suggesting that the effect of ascr#18 on Pt is not race-specific (Supplementary Fig. 2). Ascr#18 induces impaired appressorial stoma penetration Next we examined microscopically how fungal growth was inhibited in response to ascr#18-treatement. To this end, cv. Chinese Spring was inoculated with Pt race 77WxR and 10 days later stained with chitin-specific WGA-AF488 to detect fungal hyphae and infection structures. Fluorescence microscopy at low magnification showed that fungal mycelium formation was greatly reduced and the density of uredinia on the examined leaf section was consistently very low after treatment with ascr#18 (Fig. 3a,b). Moreover, hyphae on the leaf surface were much shorter and barely branched (Fig. 3c,d). Further analysis using confocal laser microscopy (CLSM) showed that penetration of the fungus from an appressorium into the substomatal cavity often failed. Examples of the penetration failure on ascr#18-treated leaves are shown in Fig. 3e,f,g,h. Either the fungus did not penetrate the leaf at all (Fig. 3e,f) or, in rare cases, was arrested at the stage of substomatal vesicle formation (Fig. 3g,h). In agreement with this, 3D analysis of leaves using CLSM at 10 dpi showed a strong fungal invasion in the mesophyll of the control plants (Fig. 3i), whereas hardly any fungal structures were found in the mesophyll after ascr#18 treatment (Fig. 3j). Ascr#18-mediated resistance is associated with enhanced early H 2 O 2 accumulation at stomata A previous study on the priming activities of AHLs showed that N -3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for accumulation of phenolic compounds, lignification of cell walls and promoted closure of stomata in response to Pseudomonas syringae infection (Schenck et al. 2014). For wheat leaf rust, R gene-mediated prehaustorial resistance in Triticum monococcum was also reported to be associated with H 2 O 2 accumulation at sites of attempted infection (Serfling et al. 2016). Since we did not detect a hypersensitive reaction (HR) of epidermal or mesophyll cells, nor papillae formation in ascr#18-treated leaves at sites of attempted stomata penetration, we tested the possibility that arrest of the fungus at this early stage of infection is associated with enhanced H 2 O 2 . Indeed, DAB-stained leaf samples collected at different times after inoculation with race 77WxR uredopsores showed much more H 2 O 2 accumulation as revealed by brown precipitate at attacked stomata of plants treated with 1 µM ascr#18 than at stomata of control plants at 12, 24 and 48 hpi (Table. 2). Notably, at later time points (96 hpi) little H 2 O 2 was detected byDAB staining detect only, suggesting that the accumulation of hydrogen peroxide is only transiently and limited to the site of attempted penetration (Fig. 4). Discussion Here we demonstrate broad and highly efficient resistance-inducing activity of the currently best-studied and most active ascaroside, ascr#18, using a representative set of wheat cultivars and two Puccinia triticina races with very different virulence spectra. Recording Pt infection on infected leaves showed that spray-pretreatment with ascr#18 significantly reduced the number of uredinia as compared to mock-pretreated Pt -inoculated plants. A dose-response analysis over the nano- and micromolar concentration range revealed a unusually broad optimum concentration down to 0.01 nM for the control of wheat leaf rust indicating that ascr#18 is a very potent resistance inducer. Moreover, microscopic analysis showed very early abortion of the fungus in the prepenetration stage. This was associated with local accumulation of H 2 O 2 as visualized by DAB staining at attacked stomata. It is noteworthy that no papilla formation or HR of epidermal or mesophyll cells could be detected at the site of the attempted penetration. Instead, the fungus did not overcome the appressoric stage in many penetration attempts with the formation of substomatal vesicles in only rare cases. Overall, our results are consistent with the current view that H 2 O 2 accumulation and the resulting strengthening of the cell wall and regulation of stomata play a key role in the very early defence responses of plants triggered by resistance inducers (Schenck et al 2014; for review Balmer et al, 2015). Previous reports showed the strong resistance-inducing effect of ascr#18 in plant protection against a virus (Turnip Crinkle Virus), a bacterium ( Pseudomonas syringae pv. tomato ), a fungus (e.g. Blumeria graminis f sp. hordei ), an oomycete ( Phytophthora infestans ) and two nematodes ( Heterodera schachtii and Meloidogyne incognita ) in four plant species (barley, potato, tomato, and Arabidopsis ) (Manosalva et al. 2015). In another report, ascr#18 was shown to induce resistance to four crops (wheat, soybean, rice, and tomato) against eight pathogens/pests, including one virus, bacteria, fungi, an oomycete, and a nematode (Klessig et al. 2019), overall suggesting that ascarosides are effective tools that can be used in crop production. There are only a few reports on the mode of action of resistance-inducing agents that are effective in controlling rust fungi on cereal crops. The bacterium Ensifer (syn. Sinorhizobium ) meliloti induces resistance via priming against Puccinia hordei (Matros et al. 2023). Interestingly the authors compared the priming activity of strain E. meliloti expR+chthat which produced large amounts of the AHL 3- oxo-C14-HSL with a transformed strain E. meliloti attM that does not accumulate AHL, suggesting an AHL-induced P. hordei resistance. Interestingly, oxo-C14-HSL in Arabidopsis can induce the oxylipin/SA signalling pathway and thus a stomata defence response and cell wall strengthening, preventing pathogen invasion (Schenck and Schikora 2015). This mode of action is similar to the effect of ascr#18 in our analysis, although a more detailed molecular investigation of the similarities and differences between AHL and ascr#18 is required. 7-oxosterols and the 7-hydroxysterols also can induce resistance toward Puccinia striiformis and Puccinia hordei in barley and wheat when sprayed onto primary leaves using 10 -4 M in 1% ethanol (Schadbach et al. 2014) two days prior to challenge inoculation with the pathogen. It was suggested that the sterol derivatives selectively activate plant defence mechanisms that impair the development or differentiation of infection structures. Thus, changes in the morphology or chemistry of the cuticle that prevent the formation of appressoria at the stomata could suppress the fungus. Induced resistance against Puccinia triticina has also achieved by treating wheat (cv. Arina) with the beneficia bacterium Pseudomonas protegens CHA0 (by seed coating) and the compound β-aminobutyric acid (BABA) (soil drenching) (Bellameche et al. 2021). BABA was tested at high concentrations (10-20 mM), and a dose-dependent reduction of pustule formation was observed with greatest protection at 20 mM. In light of these results, previous and our current work shows that ascr#18 acts at many orders of magnitude lower concentrations (Fig. 2; Manosalva et al. 2015; Klessig et al. 2019). Similar to our study, accumulation of H 2 O 2 in both CHA0- and BABA-treated plants was mostly detected in host guard cells at penetration sites; and both treatments reduced fungus penetration and haustorium formation. The authors suggested that during recognition or formation of appressoria, generation of H 2 O 2 in guard cells is induced, possibly following secretion of rust effectors, and mechanical forces during adhesion of appressoria over stomata may also elicit H 2 O 2 generation in guard cells (Bellameche et al. 2021). In Arabidopsis, H 2 O 2 accumulation in guard cells was involved in signal transduction during ABA-mediated stomatal closing (Sun et al. 2017). Similarly, appressorium formation of P. triticina also caused stoma closure in wheat leaves (Bolton et al., 2008). In conclusion, ascr#18 enables induction and modulation of different signaling pathways to activate immune responses in plants art very low concentrations. Thus, ascr#18 has a intersting potential as biological control agent to reduce disease damage and increase sustainable food security. Declarations Authors' contributions K.H.K. designed the study. A.K. conducted the experimental analysis of ascr#18 effects on uredinia formation in wheat. J.T. and A.K. conducted the microscopic anlysis of ascr#18 acitivity on Pt infections. S.D. conducted experiments with cv Boolani. T.K. and P.S. conducted the toxicity analysis. A.K., J.T., K.H.K., MM. DK and FS analyzed the data and wrote the manuscript. All authors reviewed the final manuscript. Compliance with Ethical Standards: Conflict of Interest: The research described in the manuscript was not funded by private partners or industry. Author Akshita Kamboj declares that she has no conflict of interest. Author Jennifer Thielmann declares that she has no conflict of interest. Author Saba Delfan declares that she has no conflict of interest. Authors Murli Manohar, Frank Schroeder, and Daniel Klessig are co-founders of Ascribe Bioscience, a company that develops plant treatments based on small molecules from microbiota. Author Karl-Heinz Kogel declares that he has no conflict of interest. Consent for publication All authors declare consent of publication. Availability of data and material All data generated or analyzed during this study are included in this published article and its supplementary information files. Code availability (software application or custom code) Not applicable. Acknowledgements We thank Christina Birkenstock for technical assistance. This work was funded in the project PrimedWeizen FKZ 2818409B19 of the Bundesministerium für Ernährung und Landwirtschaft (BMEL), Germany, to KHK and the project WheatInterfere of the Bundesministerium für Bildung und Forschung (BMBF), Germany to KHK. References Ali MA, Anjam MS, Nawaz MA, Lam HM, Chung G (2018) Signal transduction in plant–nematode interactions. International Journal of Molecular Sciences 19(6):1648.‏ Andersen EJ, Ali S, Byamukama E, Yen Y, Nepal MP (2018) Disease resistance mechanisms in plants. Genes 9(7):339. 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(2020) Modular and scalable synthesis of nematode pheromone ascarosides: implications in eliciting plant defense response. Organic Biomolecular Chemistry 18(26):4956-4961.‏ OECD (2020) COVID-19 and International Trade: Issues and Actions, https://read.oecd-ilibrary.org/view/?ref=128_128542-3ijg8kfswh&title=COVID-19-and-international-trade-issues-and-actions. Pieterse CM, Zamioudis C, Berendsen RL, Weller DM, Van Wees SC, Bakker PA (2014) Induced systemic resistance by beneficial microbes. Annu Rev Phytopathol 52(1):347–375. doi:10.1146/annurev-phyto-082712-102340. Poore J, Nemecek T (2018) Reducing food’s environmental impacts through producers and consumers. Science 360(6392):987-992. Rollar S, Serfling A, Geyer M, Hartl L, Mohler V, Ordon F (2021) QTL mapping of adult plant and seedling resistance to leaf rust ( Puccinia triticina Eriks.) in a multiparent advanced generation intercross (MAGIC) wheat population. Theor Appl Genet 134(1):37-51. 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Nature Protocols 3(6):1101-1108.‏ Serfling A, Krämer I, PerovicD, OrdonF (2013) Broadening the genetic base of leaf rust ( Puccinia triticina f. sp. tritici ) resistance in wheat ( Triticum aestivum ). Journal für Kulturpflanzen 65(7):262–272.‏ Serfling A, Templer SE, Winter P, Ordon F (2016) Microscopic and molecular characterization of the prehaustorial resistance against wheat leaf rust ( Puccinia triticina ) in Einkorn ( Triticum monococcum ). Front Plant Sci. 7:1668. doi: 10.3389/fpls.2016.01668. Sharrock J, Sun JC (2020) Innate immunological memory: from plants to animals. Curr Opin Immunol 62:69-78. doi: 10.1016/j.coi.2019.12.001. Spoel SH, Dong X (2012) How do plants achieve immunity? Defence without specialized immune cells. Nat Rev Immun 12(2):89-100.‏ Sun L, Li Y, Miao W, Piao T, Hao Y, Hao FS (2017) NADK2 positively modulates abscisic acid-induced stomatal closure by affecting accumulation of H 2 O 2 , Ca 2+ and nitric oxide in Arabidopsis guard cells. Plant Science 262:81-90. van Esse HP, Reuber TL, van der Does D (2020) Genetic modification to improve disease resistance in crops. New Phytol 225:70-86. Vlot AC, Sales J H, Lenk M, Bauer K, Brambilla A, Sommer A et al. (2021) Systemic propagation of immunity in plants. New Phytol 229(3):1234-1250.‏ Westman SM, Kloth KJ, Hanson J et al. (2019) Defence priming in Arabidopsis – a Meta-Analysis. Sci Rep 9:13309. Yandell BS (1997) Practical Data Analysis for Designed Experiments. Eds. Chapman & Hall. Routledge. Yu Y, Zhang YK, Manohar M, Artyukhin AB, Kumari A, Tenjo-Castano FJ, Nguyen H, Routray P, Choe A, Klessig DF, Schroeder FC (2021) Nematode signaling molecules are extensively metabolized by animals, plants, and microorganisms. ACS Chem Biol 16(6):1050-1058. Tables Tables 1 to 2 are available in the Supplementary Files section Supplementary Files SupplementoryFig1.png Supplement Figure 1 The ascaroside ascr#18 induces resistance to Puccinia triticina on wheat ( Triticum aestivum ). Leaves of ten-day-old wheat cvs. Zentos, Chinese spring, Arina LR, and Chinofuz were sprayed with 1 µM ascr#18 in 0.1% ethanol and 24 h later inoculated with uredospores of Pt race 77WxR. Controls (Con) were treated with 0.1% ethanol. Images of infected leaves were taken at 10 dpi. Kambojetalfinalfigsuppl.pptx Supplement Figure 2 Dose-response analysis of the effect of the ascaroside ascr#18 on uredinia numbers in wheat cv. Boolani. Leaves of ten-day-old seedings were sprayed with the indicated concentration of ascr#18 in 0.1% ethanol and 24 h later inoculated with uredospores of Puccinia triticina race PKTTS. Controls were treated with 0.1% ethanol. Number of uredinia were counted at 10 dpi. Treatments were done on 10 seedlings, each represented by a single dot in the boxplot. Minimum/maximum values are represented by whiskers and centre represents the median in the boxplot. Statistics was performed with one-way ANOVA and boxplots with different letters are significantly different according to Tukey’s post‐hoc test (p < 0.05). Tablesfinal.docx subtable1.pptx Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2024 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted Editorial decision: Minor revisions 17 May, 2024 Reviewers agreed at journal 15 Apr, 2024 Reviewers invited by journal 15 Apr, 2024 Editor invited by journal 09 Apr, 2024 Editor assigned by journal 08 Apr, 2024 First submitted to journal 05 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4224139","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291265655,"identity":"adb6adfc-239f-40a3-b848-bc4defbe7b82","order_by":0,"name":"Akshita Kamboj","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Akshita","middleName":"","lastName":"Kamboj","suffix":""},{"id":291265656,"identity":"e325c43f-3533-4611-98b9-9e97b05727f0","order_by":1,"name":"Jennifer Thielmann","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Thielmann","suffix":""},{"id":291265657,"identity":"609200b0-34c7-42ed-bfcc-fa76f107788b","order_by":2,"name":"Saba Delfan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Saba","middleName":"","lastName":"Delfan","suffix":""},{"id":291265658,"identity":"3b681a1b-1e44-40f1-b88c-6490dcd985a0","order_by":3,"name":"Tim Kloppe","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tim","middleName":"","lastName":"Kloppe","suffix":""},{"id":291265659,"identity":"862900c9-bbde-4b98-b13c-5ab0e5706bff","order_by":4,"name":"Philipp Schulz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Schulz","suffix":""},{"id":291265660,"identity":"7ab76159-1a74-463f-9e77-3073ef1d7337","order_by":5,"name":"Murli Manohar","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Murli","middleName":"","lastName":"Manohar","suffix":""},{"id":291265661,"identity":"0a7362fc-f737-4faa-8839-97147192374d","order_by":6,"name":"Frank C. Schroeder","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"C.","lastName":"Schroeder","suffix":""},{"id":291265662,"identity":"b5853ccc-283c-47d2-a30e-e9801c43b804","order_by":7,"name":"Daniel F. Klessig","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"F.","lastName":"Klessig","suffix":""},{"id":291265663,"identity":"6372bbd8-1c6c-4c38-ac1f-f0583e42408e","order_by":8,"name":"Karl-Heinz Kogel","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1226-003X","institution":"Institut für Phytopathologie","correspondingAuthor":true,"prefix":"","firstName":"Karl-Heinz","middleName":"","lastName":"Kogel","suffix":""}],"badges":[],"createdAt":"2024-04-05 16:43:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4224139/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4224139/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s41348-024-00950-w","type":"published","date":"2024-07-02T15:43:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54924637,"identity":"5b85827f-6407-47e3-8a73-588500ab1129","added_by":"auto","created_at":"2024-04-18 16:14:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211595,"visible":true,"origin":"","legend":"\u003cp\u003eThe ascaroside ascr#18 reduces the number of uredinia in wheat leaves inoculated with the leaf rust fungus \u003cem\u003ePuccinia triticina\u003c/em\u003e. Leaves of ten-day-old wheat cvs. Zentos, Chinese Spring, Arina LR, and Chinofuz were sprayed with 1 µM ascr#18 in 0.1% ethanol and 24 h later inoculated with uredospores of \u003cem\u003ePt\u003c/em\u003erace 77WxR. Controls (Con) were treated with 0.1% ethanol. Number of uredinia were counted at 10 dpi. Per experiment 15 seedlings were treated, each represented by a single dot in the boxplot. The experiment was repeated twice. Minimum/maximum values are represented by whiskers and centre represents the median in the boxplot. Statistics was performed with t-test for normalized data and Mann-Whitney test for unnormalized data (p \u0026lt; 0.05). Asterisks indicate significant differences to the control group (*** p ≤ 0.0001).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/e1e898900f7081cce791043d.png"},{"id":54924636,"identity":"3e50c03e-d26f-4a52-a177-5bfee5abad27","added_by":"auto","created_at":"2024-04-18 16:14:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312101,"visible":true,"origin":"","legend":"\u003cp\u003eDose-response analysis of the effect of ascr#18 on the formation of uredinia in wheat. Leaves of ten-day-old seedlings of cvs. Chinese Spring and Zentos were sprayed with the indicated concentration of ascr#18 dissolved in 0.1% ethanol and 24 h later inoculated with uredospores of \u003cem\u003ePuccinia triticina\u003c/em\u003e race 77WxR. Control seedlings were treated with 0.1% ethanol. The number of uredinia was counted at 10 dpi. Treatments were done on 15 seedlings, each represented by a single data point in the boxplot. The experiment was repeated twice. Minimum/maximum values are represented by whiskers and centre represents the median in the boxplot. Statistics was performed with one-way ANOVA and box-plots with different letters are significantly different according to Tukey’s post‐hoc test (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/6ea3294f69b509d64ade2317.png"},{"id":54925335,"identity":"b197d939-0ae3-498a-8a69-7fca657f8c54","added_by":"auto","created_at":"2024-04-18 16:30:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2908487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePt\u003c/em\u003e structures on leaves of wheat cv. Chinese Spring as visualized by WGA-AF488 staining. Plants were treated with 0.1% ethanol (a,c: control) or 1 µM ascr#18 in 0.1 % ethanol(b,d,e,g,f,h) and 10 days later harvested for microscopic investigation. On ascr#18-treated leaves only a few or no uredinia were formed, hyphal development was impaired and germ-tubes were short and barely branched (b,d). Many appressoria in ascr#18-treated leaves were not able to penetrate the stomata as revealed by CLSM inspection of the fungal infection structures in different layers under the appressorium (e,g,f,h). In the rare cases penetration of stomata in ascr#18-treated leaves was successful, substomatal vesicles were visible in layers below the appressorium, but formation of primary infection hyphae was not seen (g,h). 3D analysis of infected leaves by CLSM revealed heavily infected mesophyll tissue in control plants (i), while in ascr#18-treated leaves barely any fungal structures were found in the mesophyll tissue (j). White arrows: uredinia on control leaf.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/16df468022f83c55a6fce7f0.png"},{"id":54924639,"identity":"4b3f225d-e61b-4a62-9dbc-b710ab8a428a","added_by":"auto","created_at":"2024-04-18 16:14:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4665068,"visible":true,"origin":"","legend":"\u003cp\u003eDAB-mediated visualization of hydrogen peroxide accumulation at the sites of attempted penetration, indicated by a brown precipitate. \u003cem\u003ePt\u003c/em\u003e was stained with WGA-AF488. For each sample, brightfield images (top row) show the DAB-based brown precipitate and fluorescence images (bottom row) show WGA-AF488 fluorescence of the fungal chitin structures. Plants were treated with 01 % ethanol (Con) or 1 µM ascr#18 in 0.1 % ethanol, inoculated 24 h later with \u003cem\u003ePt\u003c/em\u003e race 77WxR and sampled at the time indicated in the image. After 12, 24 and 48 h, a higher accumulation of hydrogen peroxide was observed in the leaves treated with ascr#18 compared to the control samples. After 96 hpi, only little accumulation of hydrogen peroxide was detected in ascr#18 treated plants. White arrows indicate attacked stomata.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/2d05bb202c8e0798a03ffb14.png"},{"id":59508296,"identity":"6370ef4a-7a01-4928-965b-e8d885d7e416","added_by":"auto","created_at":"2024-07-02 15:44:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10138645,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/02e0952b-e3e3-413e-b810-9a8715a3de79.pdf"},{"id":54924635,"identity":"d55d9e3f-8503-4d55-900c-7cf3fda15c52","added_by":"auto","created_at":"2024-04-18 16:14:26","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3528887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplement Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ascaroside ascr#18 induces resistance to \u003cem\u003ePuccinia triticina \u003c/em\u003eon wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e). Leaves of ten-day-old wheat cvs. Zentos, Chinese spring, Arina LR, and Chinofuz were sprayed with 1 µM ascr#18 in 0.1% ethanol and 24 h later inoculated with uredospores of \u003cem\u003ePt\u003c/em\u003e race 77WxR. Controls (Con) were treated with 0.1% ethanol. Images of infected leaves were taken at 10 dpi.\u003c/p\u003e","description":"","filename":"SupplementoryFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/f2189eb5c455235867a0bf93.png"},{"id":54924641,"identity":"d2e39e83-85d7-466a-b1a8-fa1498aac6b7","added_by":"auto","created_at":"2024-04-18 16:14:26","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":86925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplement Figure 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDose-response analysis of the effect of the ascaroside ascr#18 on uredinia numbers in wheat cv. Boolani. Leaves of ten-day-old seedings were sprayed with the indicated concentration of ascr#18 in 0.1% ethanol and 24 h later inoculated with uredospores of \u003cem\u003ePuccinia triticina\u003c/em\u003e race PKTTS. Controls were treated with 0.1% ethanol. Number of uredinia were counted at 10 dpi. Treatments were done on 10 seedlings, each represented by a single dot in the boxplot. Minimum/maximum values are represented by whiskers and centre represents the median in the boxplot. Statistics was performed with one-way ANOVA and boxplots with different letters are significantly different according to Tukey’s post‐hoc test (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Kambojetalfinalfigsuppl.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/184bd031cb8aece6701eeb0c.pptx"},{"id":54925048,"identity":"7c46b5a3-7c4c-4393-8525-98e8f2b4d614","added_by":"auto","created_at":"2024-04-18 16:22:26","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19028,"visible":true,"origin":"","legend":"","description":"","filename":"Tablesfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/b8de9b6a06c5cc4afa9d7a93.docx"},{"id":54924642,"identity":"b107d0a9-941d-4d7b-9e1b-8c31ec182d7f","added_by":"auto","created_at":"2024-04-18 16:14:26","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":42216,"visible":true,"origin":"","legend":"","description":"","filename":"subtable1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4224139/v1/24f25a1364630c1abd071209.pptx"}],"financialInterests":"","formattedTitle":"The Nematode Signaling Molecule ascr#18 Induces Prepenetration Defenses in Wheat Against a Leaf Rust Fungus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the face of climate change, political crises, and a growing population, there is an urgent need to secure and even increase wheat production to ensure sustainable food security (Poore and Nemecek 2018; OECD 2020). Leaf rust caused by \u003cem\u003ePuccinia triticina\u003c/em\u003e (\u003cem\u003ePt\u003c/em\u003e) is one of the most common diseases affecting wheat worldwide (Bolton et al. 2008; Huerta-Espino et al. 2011; Kolmer 2013) and is exacerbated by temperature increases (Helfer 2014; Junk et al. 2016; Caubel 2017). Although widely successful in the past, control of leaf rust using conventional measures involving resistance genes (\u003cem\u003eR\u0026nbsp;\u003c/em\u003egenes) and/or synthetic pesticides becomes ineffective over time because of strong selection pressures on fungal populations in intensive agricultural production systems to overcome \u003cem\u003eR\u003c/em\u003e gene function or develop resistance or tolerance to pesticides (Andersen et al. 2018; Hawkins et al. 2019; van Esse et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eModern integrative crop protection strategies include methods that rely on the plant's natural immune system (Galli et al. 2024). Plants, like animals, have evolved various innate and acquired immune strategies to combat microbial diseases (Sharrock and Sun 2020; Mermigka et al. 2020). Plants depend primarily on two interconnected layers of the innate immune system to recognize and respond to pathogens (Jones and Dangl 2006;\u0026nbsp;Spoel and Dong, 2012; Han 2019). Firstly, pattern-recognition receptors (PRRs) recognize microbe-associated molecular patterns (MAMPs) from a wide spectrum of microbes, resulting in pattern-triggered immunity (PTI). A second layer involves disease resistance (R) proteins that recognize pathogen effector molecules or their activities on host targets, resulting in effector-triggered immunity (ETI). In addition, plants can acquire disease resistance by previous encounters with microbes or natural compounds such as plant hormones resulting in systemic acquired resistance (SAR) or induced systemic resistance (ISR), depending on whether the salicylic acid or jasmonate defense pathway is activated (Ryals et al. 1996; Pieterse et al. 2014; Klessig et al. 2018). This knowledge has led to the commercial development of synthetic resistance inducers such as Benzothiadiazol (BTH) or Probenazol that mimic the activity of a natural inducer and help to protect plants against various diseases (Nakashita et al. 2002; Kogel et al. 1994; Görlach et al. 1996; Vlot et al. 2021). More recently it was discovered that some synthetic resistance inducers and natural compounds such as salicylic acid, β-aminobutyric acid (BABA) or acyl-homoserine lactones (AHLs) at low concentrations trigger induced resistance via defense priming in plants leading to a physiological state that enables plants to respond more rapidly and/or more robustly to a challenge inoculation after exposure to biotic or abiotic stress (detailed reviews see Conrath et al. 2015; Balmer et al. 2015; Baccelli and Mauch-Mani 2016; Cooper and Ton 2022).\u0026nbsp;An early response of primed plants is their production of reactive oxygen species (ROS) at the site of attack when they recognise a pathogen or pest, which is associated with the termination of the invasion or infestation (Balmer et al., 2015).\u0026nbsp;Since a primed plant has only a very limited part of its defense system activated, priming could be a approach to protect plants from diseases and pests while minimizing energy expenditure and thus yield losses (Westman et al. 2019; Schenck et al. 2014; Jung and Cecchini 2023).\u003c/p\u003e\n\u003cp\u003eSeveral recent studies have shown that a family of nematode-derived pheromones called ascarosides induce resistance in many plants against a broad spectrum of pathogens and pests by upregulating specific defense signaling pathways (Manosalva et al. 2015; Ali et al. 2018; Klessig et al. 2019; Ning et al. 2020). The term ascaroside originally referred to a distinct type of lipid first detected in parasitic roundworms of the family \u003cem\u003eAscaridia\u003c/em\u003e more than 100 years ago (Flury 1912). Ascarocides serve a wide range of biological functions which is facilitated by a great diversity of ascaroside chemical structures (Ludewig and Schroeder 2013). These are based on the sugar ascarylose, which is linked to fatty acid-like side chains of varying lengths and often decorated further with building blocks derived from amino acids, folate, and other primary metabolites.\u0026nbsp;Plants can metabolize ascarosides and thereby change their chemical message, generating ascaroside mixtures that repel diseases and pests and reduce infection (Manohar et al. 2020; Yu et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we report on the biocontrol effect of ascr#18, the most abundant ascaroside secreted by plant parasitic nematodes into the plant rhizosphere (Manosalva et al. 2015), on \u003cem\u003ePuccinia triticina\u003c/em\u003e infections of various wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) genotypes. Importantly, ascr#18 was effective in the nano- and micromolar range,\u0026nbsp;indicating a broad optimal concentration for controlling \u003cem\u003ePt\u003c/em\u003e. Our finding identifies a novel mode of ascr#18-induced resistance by triggering the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at attacked stomata, a characteristic also observed with other resistance inducers\u0026nbsp;(Schenck et al. 2015).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material, fungal inoculation, and ascr#18 treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ePuccinia triticina\u003c/em\u003e-susceptible wheat varieties \u003cem\u003eTriticum aestivum\u003c/em\u003e cv. Chinese Spring (spring wheat) and winter wheat Arina LR (both provided by the Julius Kuehn Institute (JKI) Kleinmachnow, Germany), Zentos, Chinofuz (provided by the JKI Quedlinburg, Germany) and Boolani (Seed and Plant Improvement Institute, Karaj, Iran)\u0026nbsp;were used. The leaf rust \u003cem\u003ePt\u003c/em\u003e race 77WxR (Serfling et al. 2013) was a gift of the JKI Quedlinburg. The virulence/avirulence profile of\u0026nbsp;race77WxR used in field trials and seedling test are found in Rollar et al. (2021).\u0026nbsp;The wheat cv. Boolani is susceptible to \u003cem\u003ePt\u003c/em\u003e race PKTTS (Delfan et al. 2022). The profile of virulence/avirulence of race PKTTS is shown in Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ascaroside ascr#18 was a gift from Ascribe Biosciences, 95 Brown Rd, Ithaca, NY, USA. For all experiments, wheat plants were grown in a pot containing\u0026nbsp;fine-structured soil (Fruhstorfer Erde type T; HAWITA Gruppe GmbH, Vechta, Germany)\u0026nbsp;in a growth chamber under controlled conditions with the temperature set to\u0026nbsp;18/20\u0026deg;C (night/day), light period of 16 h and 65% relative humidity. Ten-day-old seedlings were sprayed with ascr#18 in an aqueous solution containing 0.1% ethanol until run-off using a hand sprayer (Carl Roth, Germany); control plants were sprayed with 0.1% ethanol. After 24 h, leaves were inoculated by brushing with two-week-old \u003cem\u003ePt\u0026nbsp;\u003c/em\u003euredospores isolated from \u003cem\u003eTriticum aestivum\u003c/em\u003e cv. Kanzler, using a mix of rust uredospores and talcum powder (Alliance Chemical, Germany) in a concentration of 1:4 (McIntosh et al. 1995). The inoculated seedlings were grown at 18/20\u0026deg;C (night/day) with 16 h of photoperiod, 95% relative humidity for three days, followed by 65% relative humidity for seven days. The number of uredinia were evaluated on one leaf per plant in\u0026nbsp;an area of 0.5 cm\u0026sup2; after 10 days post inoculation (dpi) by use of a binocular (Leica Microsystems GmbH, Wetzlar, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest on direct toxicity of ascr#18\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether there is a direct effect of ascr#18 on the germination of \u003cem\u003ePuccinia triticina\u003c/em\u003e, 9-cm petri dish plates of water agar (3% w/v Agar) were pretreated with 2 mL of 1 \u0026micro;M ascr#18 dispensed in 0.1 % v/v ethanol using a sprayer (Preval, Art.-Nr. YC44.1). Subsequently, a suspension of \u003cem\u003ePt\u003c/em\u003e isolate 77WxR (5 mg of uredospores in 25 mL 0.1 % w/v agar) was sprayed onto the agar plates either 15 min or 24 h or after ascr#18 application. Inoculated plates were incubated in dark at 25\u0026deg;C for 10 h at 100% relative humidity. Three plates were prepared for each treatment and 100 uredospores were examined for germination on each plate. Uredospores were rated as germinated when germ tubes were visible and at least 5 times the size of the uredospore. To test whether there was a direct effect of ethanol on the germination, 0.1% ethanol was used in absence of ascr#18 as additional control. Water agar with 0.16 g/L prothioconazol (Proline, Bayer CropScience) was used as positive control for inhibition of \u003cem\u003ePt\u003c/em\u003e germination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor statistical analysis, data were checked for normality. The \u003cem\u003et\u003c/em\u003etest for normalized data and Mann-Whitney test for unnormalized data were performed in experiments with two groups to compare. For dose effect experiments, the analysis was done by one-way ANOVA and multiple comparison was carried out using Tukey\u0026rsquo;s post‐hoc test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All statistical analyses and graphs were done with GraphPad Prism 8 software.\u0026nbsp;For the germination test, data were fitted to a linear model using the function aov (Chambers et al.1992) in R. Tukey honest significant difference test was conducted on the fitted model using the TukeyHSD function (p \u0026lt; 0.05; Miller 1981).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePt\u003c/em\u003e-infected leaves (10 dpi) of ascr#18-treated and mock-treated (0.1% ethanol) control plants were fixed in\u0026nbsp;4% paraformaldehyde (in PBS buffer) or 0.15% trichloroacetic acid (in chloroform:ethanol 20:80, v/v). Fungal structures were visualized using chitin-specific staining with WGA-AF488 (wheat germ agglutinin; Molecular Probes, Karlsruhe, Germany). Leaves were investigated under an epifluorescence microscope (Axio Imager.A2, Carl Zeiss, Oberkochen, Germany) and a confocal laser scanning microscope (CLSM; TCS SP8,\u0026nbsp;Leica Microsystems GmbH, Wetzlar, Germany) by use of ZEISS ZEN 3.8 and Leica LAS X software, respectively. WGA-AF488 was visualized at\u0026nbsp;\u0026lambda;\u003csub\u003eexc\u003c/sub\u003e 494 nm,\u0026nbsp;\u0026lambda;\u003csub\u003eem\u003c/sub\u003e 515 and fluorescence control settings were set to\u0026nbsp;\u0026lambda;\u003csub\u003eexc\u003c/sub\u003e 631 nm, \u0026lambda;\u003csub\u003eem\u003c/sub\u003e 642.\u0026nbsp;For H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detection,\u0026nbsp;\u003cem\u003ePt\u003c/em\u003e-infected leaves of ascr#18- and mock-treated plants were collected 12 hpi, 24 hpi, 48 hpi and 96 hpi, and samples were stained with 3,3\u0026prime;-Diaminobenzidine (DAB)-tetrahydrochloride (H\u0026uuml;ckelhoven et al. 1999) and subsequently kept in 0.15% trichloroacetic acid (in chloroform:ethanol 20:80, v/v). To evaluate the DAB stained area, the average size of precipitates on the attacked stomata was quantified using ImageJ free software (https://imagej.net/ij/).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eAscr#18 induces resistance against\u0026nbsp;\u003c/em\u003ePuccinia triticina\u003cem\u003e\u0026nbsp;in all tested wheat cultivars\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of ascr#18 on wheat against leaf rust was first tested with the four cultivars (cvs.) Zentos, Chinese Spring, Arina LR, and Chinofuz. Leaves of 10-day-old seedlings were sprayed with 1 µM ascr#18 and 24 h later inoculated with uredospores \u003cem\u003ePt\u003c/em\u003e race 77WxR. Ascr#18 significantly reduced the number of \u003cem\u003ePt\u003c/em\u003e uredinia on all four wheat genotypes as compared to mock treatment (\u003cem\u003et\u003c/em\u003e-test for normalized data, Mann-Whitney test for unnormalized data; p \u0026lt; 0.05): Zentos (70%), Chinese Spring (71%), Arina LR (77%), and Chinofuz (81%) (Fig. 1; Supplementary Fig. 1). To exclude the possibility that the effect on \u003cem\u003ePt\u003c/em\u003e development was due to direct toxic effects of ascr#18, we exposed the fungus to 1 µM ascr#18 on water agar plates for 10 h. Consistent with previous reports on other fungal pathogens (Manosalva et al. 2015; Klessig et al. 2019), \u003cem\u003ePt\u003c/em\u003e's germination rate was unaffected by ascr#18 and was comparable to the control treatments (Table 1). We concluded that ascr#18 induces resistance to leaf rust fungus in the wheat cultivars tested.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAscr#18 induces resistance against\u0026nbsp;\u003c/em\u003ePuccinia triticina in\u003cem\u003e\u0026nbsp;the nM range\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNext, we conducted a dose-response experiment in the concentration range between 0.000001 to 10µM ascr#18. Leaves of 10-day-old seedlings of cvs. Chinese Spring and Zentos were sprayed with the respective concentrations of ascr#18 and 24 h later inoculated with \u003cem\u003ePt\u003c/em\u003e race 77WxR. Ascr#18 significantly reduced the number of uredinia on both wheat genotypes down to a concentration of 0.01 nM (One-way ANOVA, Tukey’s post‐hoc test; p \u0026lt; 0.05) (Fig. 2).\u003c/p\u003e\n\u003cp\u003eTo broaden the agronomic relevance, we extended our investigation to the wheat cv. Boolani, which is susceptible to \u003cem\u003ePt\u003c/em\u003e race PKTTS. Ten-day-old seedlings were sprayed with ascr#18 and the dose effect on the number of uredinia at 10 dpi was analysed. As expected, the number of uredinia was greatly reduced over a wide range of concentrations, suggesting that the effect of ascr#18 on \u003cem\u003ePt\u003c/em\u003e is not race-specific (Supplementary Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAscr#18 induces impaired appressorial stoma penetration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNext we examined microscopically how fungal growth was inhibited in response to ascr#18-treatement. To this end, cv. Chinese Spring was inoculated with \u003cem\u003ePt\u003c/em\u003e race 77WxR and 10 \u0026nbsp;days later stained with chitin-specific WGA-AF488 to detect fungal hyphae and infection structures.\u0026nbsp;Fluorescence microscopy at low magnification showed that fungal mycelium formation was greatly reduced and the density of uredinia on the examined leaf section was consistently very low after treatment with ascr#18 (Fig. 3a,b). Moreover, hyphae on the leaf surface were much shorter and barely branched (Fig. 3c,d). Further analysis using confocal laser microscopy (CLSM) showed that penetration of the fungus from an appressorium into the substomatal cavity often failed. Examples of the penetration failure on ascr#18-treated leaves are shown in Fig. 3e,f,g,h. Either the fungus did not penetrate the leaf at all (Fig. 3e,f) or, in rare cases, was arrested at the stage of substomatal vesicle formation (Fig. 3g,h). In agreement with this, 3D analysis of leaves using CLSM at 10 dpi showed a strong fungal invasion in the mesophyll of the control plants (Fig. 3i), whereas hardly any fungal structures were found in the mesophyll after ascr#18 treatment (Fig. 3j).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAscr#18-mediated resistance is associated with enhanced early H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation at stomata\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA previous study on the priming activities of AHLs showed that \u003cem\u003eN\u003c/em\u003e-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for accumulation of phenolic compounds, lignification of cell walls and promoted closure of stomata in response to \u003cem\u003ePseudomonas syringae\u003c/em\u003e infection (Schenck et al. 2014). For wheat leaf rust, \u003cem\u003eR\u003c/em\u003e gene-mediated prehaustorial resistance in \u003cem\u003eTriticum monococcum\u003c/em\u003e was also reported to be associated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eaccumulation at sites of attempted infection (Serfling et al. 2016). Since we did not detect a hypersensitive reaction (HR) of epidermal or mesophyll cells, nor papillae formation in ascr#18-treated leaves at sites of attempted stomata penetration, we tested the possibility that arrest of the fungus at this early stage of infection is associated with enhanced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Indeed, DAB-stained leaf samples collected at different times after inoculation with race 77WxR uredopsores showed much more H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation as revealed by brown precipitate at attacked stomata of plants treated with 1 µM ascr#18 than at stomata of control plants at 12, 24 and 48 hpi (Table. 2). Notably, at later time points (96 hpi) little H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas detected byDAB staining detect only, suggesting that the accumulation of hydrogen peroxide is only transiently and limited \u0026nbsp;to the site of attempted penetration (Fig. 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we demonstrate broad and highly efficient resistance-inducing activity of the currently best-studied and most active ascaroside, ascr#18, using a representative set of wheat cultivars and two \u003cem\u003ePuccinia triticina\u003c/em\u003e races with very different virulence spectra. Recording \u003cem\u003ePt\u003c/em\u003e infection on infected leaves showed that spray-pretreatment with ascr#18 significantly reduced the number of uredinia as compared to mock-pretreated \u003cem\u003ePt\u003c/em\u003e-inoculated plants. A dose-response analysis over the nano- and micromolar concentration range revealed a unusually broad optimum concentration down to 0.01 nM for the control of wheat leaf rust indicating that ascr#18 is a very potent resistance inducer. Moreover, microscopic analysis showed very early abortion of the fungus in the prepenetration stage. This was associated with local accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as visualized by DAB staining at attacked stomata. It is noteworthy that no papilla formation or HR of epidermal or mesophyll cells could be detected at the site of the attempted penetration. Instead, the fungus did not overcome the appressoric stage in many penetration attempts with the formation of substomatal vesicles in only rare cases. Overall, our results are consistent with the current view that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation and the resulting strengthening of the cell wall and regulation of stomata play a key role in the very early defence responses of plants triggered by resistance inducers (Schenck et al 2014; for review Balmer et al, 2015).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious reports showed the strong resistance-inducing effect of ascr#18 in plant protection against a virus (Turnip Crinkle Virus), a bacterium (\u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e), a fungus (e.g. \u003cem\u003eBlumeria graminis\u003c/em\u003e f sp. \u003cem\u003ehordei\u003c/em\u003e), an oomycete (\u003cem\u003ePhytophthora infestans\u003c/em\u003e) and two nematodes (\u003cem\u003eHeterodera schachtii\u003c/em\u003e and \u003cem\u003eMeloidogyne incognita\u003c/em\u003e) in four plant species (barley, potato, tomato, and \u003cem\u003eArabidopsis\u003c/em\u003e) (Manosalva et al. 2015). In another report, ascr#18 was shown to induce resistance to four crops (wheat, soybean, rice, and tomato) against eight pathogens/pests, including one virus, bacteria, fungi, an oomycete, and a nematode (Klessig et al. 2019), overall suggesting that ascarosides are effective tools that can be used in crop production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are only a few reports on the mode of action of resistance-inducing agents that are effective in controlling rust fungi on cereal crops. The bacterium\u0026nbsp;\u003cem\u003eEnsifer\u003c/em\u003e (syn. \u003cem\u003eSinorhizobium\u003c/em\u003e) \u003cem\u003emeliloti induces resistance via priming against\u0026nbsp;\u003c/em\u003e\u003cem\u003ePuccinia hordei\u003c/em\u003e (Matros et al. 2023).\u0026nbsp;Interestingly the authors compared the priming activity of strain \u003cem\u003eE. meliloti expR+chthat which produced large amounts of the AHL 3-\u003c/em\u003e\u003cem\u003eoxo-C14-HSL\u003cem\u003e\u0026nbsp;with a transformed strain E. meliloti attM that does not accumulate AHL, suggesting an\u0026nbsp;\u003c/em\u003e\u003c/em\u003eAHL-induced \u003cem\u003eP. hordei\u003c/em\u003e resistance.\u0026nbsp;Interestingly, oxo-C14-HSL in Arabidopsis can induce the oxylipin/SA signalling pathway and thus a stomata defence response and cell wall strengthening, preventing pathogen invasion (Schenck and Schikora 2015). This mode of action is similar to the effect of ascr#18 in our analysis, although a more detailed molecular investigation of the similarities and differences between AHL and ascr#18 is required.\u003c/p\u003e\n\u003cp\u003e7-oxosterols and the 7-hydroxysterols also can induce resistance toward \u003cem\u003ePuccinia striiformis\u003c/em\u003e and \u003cem\u003ePuccinia hordei\u003c/em\u003e in barley and wheat when sprayed onto primary leaves using 10\u003csup\u003e-4\u003c/sup\u003e M in 1% ethanol (Schadbach et al. 2014) two days prior to challenge inoculation with the pathogen. It was suggested that the sterol derivatives selectively activate plant defence mechanisms that impair the development or differentiation of infection structures. Thus, changes in the morphology or chemistry of the cuticle that prevent the formation of appressoria at the stomata could suppress the fungus.\u003c/p\u003e\n\u003cp\u003eInduced resistance against\u0026nbsp;\u003cem\u003ePuccinia triticina\u0026nbsp;\u003c/em\u003ehas also achieved by treating wheat (cv. Arina) with the beneficia bacterium\u003cem\u003e\u0026nbsp;Pseudomonas protegens\u0026nbsp;\u003c/em\u003eCHA0 (by seed coating) and the\u0026nbsp;compound β-aminobutyric acid (BABA) (soil drenching) (Bellameche et al. 2021).\u0026nbsp;BABA was tested at high concentrations (10-20 mM), and a dose-dependent reduction of pustule formation was observed with greatest protection at 20 mM. In light of these results, previous and our current work shows that ascr#18 acts at many orders of magnitude lower concentrations (Fig. 2; Manosalva et al. 2015; Klessig et al. 2019).\u003c/p\u003e\n\u003cp\u003eSimilar to our study,\u0026nbsp;accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in both CHA0- and BABA-treated plants was mostly detected in host guard cells at penetration sites; and both treatments reduced fungus penetration and haustorium formation. The authors suggested that during recognition or formation of appressoria, generation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in guard cells is induced, possibly following secretion of rust effectors, and mechanical forces during adhesion of appressoria over stomata may also elicit H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003egeneration in guard cells\u0026nbsp;(Bellameche et al. 2021). In Arabidopsis, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation in guard cells was involved in signal transduction during ABA-mediated stomatal closing (Sun et al. 2017). Similarly, appressorium formation of \u003cem\u003eP. triticina\u003c/em\u003e also caused stoma closure in wheat leaves (Bolton et al., 2008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, ascr#18 enables induction and modulation of different signaling pathways to activate immune responses in plants art very low concentrations. Thus, ascr#18 has a intersting potential as biological control agent to reduce disease damage and increase sustainable food security.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.H.K. designed the study. A.K. conducted the experimental analysis of ascr#18 effects on uredinia formation in wheat. J.T. and A.K. conducted the microscopic anlysis of ascr#18 acitivity on \u003cem\u003ePt\u0026nbsp;\u003c/em\u003einfections. S.D. conducted experiments with cv Boolani. T.K. and P.S. conducted the toxicity analysis. A.K., J.T., K.H.K., MM. DK and FS analyzed the data and wrote the manuscript. All authors reviewed the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe research described in the manuscript was not funded by private partners or industry. Author Akshita Kamboj\u0026nbsp;declares that she has no conflict of interest. Author Jennifer Thielmann\u0026nbsp;declares that she has no conflict of interest. Author\u0026nbsp;Saba Delfan declares that she has no conflict of interest.\u0026nbsp;Authors Murli Manohar, Frank Schroeder, and Daniel Klessig are co-founders of Ascribe Bioscience, a company that develops plant treatments based on small molecules from microbiota.\u0026nbsp;Author\u0026nbsp;Karl-Heinz Kogel declares that he has no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare consent of publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(software application or custom code)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Christina Birkenstock for technical assistance. This work was funded in the project PrimedWeizen FKZ 2818409B19 of the Bundesministerium für Ernährung und Landwirtschaft (BMEL), Germany, to KHK and the project WheatInterfere of the Bundesministerium für Bildung und Forschung (BMBF), Germany to KHK.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAli MA, Anjam MS, Nawaz MA, Lam HM, Chung G (2018) Signal transduction in plant\u0026ndash;nematode interactions. International Journal of Molecular Sciences 19(6):1648.\u0026rlm;\u003c/li\u003e\n\u003cli\u003eAndersen EJ, Ali S, Byamukama E, Yen Y, Nepal MP (2018) Disease resistance mechanisms in plants. Genes 9(7):339.\u003c/li\u003e\n\u003cli\u003eBaccelli I, Mauch-Mani B (2016) Beta-aminobutyric acid priming of plant defense; the role of ABA and other hormones. 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ACS Chem Biol 16(6):1050-1058. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ascaroside, biocontrol, crop protection, induced resistance, Puccinia triticina, Triticum, wheat ","lastPublishedDoi":"10.21203/rs.3.rs-4224139/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4224139/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeaf rust, caused by the pathogenic biotrophic rust fungus \u003cem\u003ePuccinia triticina\u003c/em\u003e (\u003cem\u003ePt\u003c/em\u003e), is one of the most destructive wheat diseases worldwide; its negative impact on crop yields is exacerbated by increasing temperatures due to climate change. Ascarosides are nematode pheromones that induce resistance to microbial pathogens and pests in a wide range of crops, making them valuable components in biocontrol scenarios. We investigated the effect on infection of various wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) genotypes with the virulent \u003cem\u003ePt\u003c/em\u003e race 77WxR by ascr#18, the major ascaroside secreted into the rhizosphere by plant-parasitic nematodes. Spraying the leaves with ascr#18 24 hours before inoculation with fungal uredospores slowed disease development and resulted in a reduction of the number of rust pustules on treated compared to untreated leaves. Dose-response analysis over the nano- and micromolar range revealed a broad optimum concentration down to 0.01 nM ascr#18. Microscopic analysis showed very early arrest of the fungus at the appressorial stage, with associated enhanced local accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and abortive stoma penetration. The results of this study are consistent with and extend previous research that has shown that ascr#18 activates plant immunity and thus protects plants from pathogens even at very low doses.\u003c/p\u003e","manuscriptTitle":"The Nematode Signaling Molecule ascr#18 Induces Prepenetration Defenses in Wheat Against a Leaf Rust Fungus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-18 16:14:21","doi":"10.21203/rs.3.rs-4224139/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-05-17T08:01:32+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-15T12:50:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-15T08:39:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2024-04-09T06:46:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-08T07:10:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2024-04-05T12:43:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ed64ad53-a822-4bb2-80c3-b6415e857d27","owner":[],"postedDate":"April 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-02T15:43:59+00:00","versionOfRecord":{"articleIdentity":"rs-4224139","link":"https://doi.org/10.1007/s41348-024-00950-w","journal":{"identity":"journal-of-plant-diseases-and-protection","isVorOnly":false,"title":"Journal of Plant Diseases and Protection"},"publishedOn":"2024-07-02 15:43:59","publishedOnDateReadable":"July 2nd, 2024"},"versionCreatedAt":"2024-04-18 16:14:21","video":"","vorDoi":"10.1007/s41348-024-00950-w","vorDoiUrl":"https://doi.org/10.1007/s41348-024-00950-w","workflowStages":[]},"version":"v1","identity":"rs-4224139","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4224139","identity":"rs-4224139","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}