Mechanism of the antifungal activity of the diterpenoid aldehyde traversianal against the cucumber anthracnose fungus Colletotrichum orbiculare

Mechanism of the antifungal activity of the diterpenoid aldehyde traversianal against the cucumber anthracnose fungus Colletotrichum orbiculare | 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 Mechanism of the antifungal activity of the diterpenoid aldehyde traversianal against the cucumber anthracnose fungus Colletotrichum orbiculare Masatoshi Ino, Junichi Kihara, Atsushi Ishihara, Makoto Ueno This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6608584/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2025 Read the published version in Journal of General Plant Pathology → Version 1 posted 4 You are reading this latest preprint version Abstract The development of fungicides with novel modes of action is essential for addressing the emergence of drug resistance in plant pathogens caused by the long-term use of chemical pesticides. In this study, we investigated the mechanisms underlying the antifungal activity of traversianal, a diterpenoid aldehyde isolated from a culture filtrate of Cercospora sp. ME202, against Colletotrichum orbiculare , the causal agent of cucumber anthracnose. In antibacterial assays using two Pseudomonas species and one Bacillus species, traversianal showed growth-inhibitory activity against the Bacillus species. The fungicidal activity of traversianal against C. orbiculare was attenuated by the addition of lecithin, which consists primarily of phospholipids. Compared with that in the control, we observed a significant increase in the number of colonies of C. orbiculare on potato sucrose agar following treatment with traversianal in the presence of lecithin, thus indicating the competitive inhibition of fungicidal activity by lecithin. However, this antagonistic effect was not observed with the addition of l-α-phosphatidylcholine dioleoyl, whereas it was detected in the presence of l-α-phosphatidylethanolamine dioleoyl. These findings provide evidence that the cell membrane component phosphatidylethanolamine may be specifically targeted in the fungicidal activity of traversianal, which is consistent with our transmission electron microscopy observations revealing the fragmentation of plasma membranes and disappearance of cell organelles in the conidia of C. orbiculare treated with traversianal. These findings thus indicate that traversianal may have potential application as a novel fungicide that targets phosphatidylethanolamine. Mechanism of action Antifungal activity Colltotrichum orbiculare Traversianal phospholipids Antagonistic effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The inappropriate use of synthetic chemical fungicides has led to the development of drug resistance in numerous plant pathogens, thereby presenting substantial challenges for the control of plant diseases. For example, among species of Colletotrichum fungi, strains resistant to benzimidazole-based and strobilurin fungicides have been reported (Forcelini et al. 2016 ; Torres-Calzada et al. 2015 ). Moreover, the occurrence of cross-resistance and multi-drug resistant strains among numerous economically important plant pathogens, including Botrytis cinerea , Pyricularia oryzae , and Fusarium species, has become a source of particular concern (Alzohairy et al. 2023 ; Becher et al. 2010 ; Vicentini et al. 2022 ). Consequently, there is an urgent need to develop novel fungicides with modes of action that differ from those of existing agents. In this regard, given their diverse chemical structures, fungal secondary metabolites have attracted considerable attention as valuable biological resources with potential utility in the development of novel fungicides and pharmaceutical agents. These secondary metabolites, including polyketides, terpenoids, and alkaloids, produced via different biosynthetic pathways (Cox 2007 ; Panaccione 2023 ; Quin et al. 2014 ), are also characterized by diversity in terms of biological properties, including antibacterial, antifungal, and cytotoxic activities (Conrado et al. 2022 ; Zhong and Xiao 2009 ). Given their structural and functional diversity, these fungal metabolites are accordingly considered promising sources for the discovery of bioactive compounds with novel mechanisms of action. Terpenoids, which represent the largest category of natural products and are considered a rich source of compounds with antibacterial and antifungal activities (Gershenzon and Dudareva 2007 ), are biosynthesized from isoprene units and have a wide range of structures, including monoterpenes, sesquiterpenes, and diterpenes. We have previously reported that traversianal, a diterpenoid aldehyde produced by Cercospora sp. ME202, is characterized antifungal activity, including fungicidal effects, with inhibition of the conidial germination and mycelial growth of the cucumber anthracnose fungus Colletotrichum orbiculare (Ino et al. 2024 ). Traversianal has also been reported to have toxic effects against brine shrimps and snails, and mild hemolytic activity against human erythrocytes, although shows no toxicity toward chicks and plants (Stoessl et al. 1989 ). At present, however, our knowledge regarding the biological activities of traversianal remains relatively limited, and its antibacterial activity and mechanisms underlying its toxic and antifungal activities have yet to be sufficiently elucidated. Traversianal is a member of a group of compounds, the basic structure of which is a 5-8-5 ring carbon skeleton, that have structural similarities to ophiobolins and fusicoccins (Cimmino et al. 2014 ). Among these compounds, ophiobolin A has been established to have anticancer activity by specifically combining and forming a complex with phosphatidylethanolamine (PE) in the membranes of cancer cells via the 1,4-dicarbonyl groups present in its chemical structure (Chidley et al. 2016 ). This is based on observations that mutations in the Kennedy pathway for PE biosynthesis are associated with reduced sensitivity to ophiobolin A and that phospholipid supplementation antagonistically reduces ophiobolin A cytotoxicity (Chidley et al. 2016 ). Given that traversianal also contains a 1,4-dicarbonyl group, it is speculated that its antifungal activity may be mediated via specific interactions with PE, similar to the mechanism observed for ophiobolin A. Accordingly, in this study, we sought to elucidate the mechanisms underlying the antifungal activity of traversianal against C. orbiculare by evaluating its antibacterial properties, determining the antagonistic effects of lecithin and different phospholipids, and examining changes in the conidial ultrastructure of traversianal-treated C. orbiculare using transmission electron microscopy. Materials and methods Chemicals Traversianal was isolated from a culture filtrate of Cercospora sp. ME202 via silica gel column chromatography and high-performance liquid chromatography, as previously described (Ino et al. 2024 ). The compound was stored at -20°C and used for experiments by dissolving in dimethyl sulfoxide (DMSO) at appropriate concentrations. Egg yolk lecithin, l-α-phosphatidylcholine dioleoyl (DOPC), and l-α-phosphatidylethanolamine dioleoyl (DOPE) were purchased from Fujifilm Wako Pure Chemical Corporation for antagonistic assay. These chemicals were prepared as 10 mg/ml stock solutions in chloroform and stored at 4°C for subsequent use. Fungal and bacterial strains For the purposes of this study, we used Colletotrichum orbiculare (strain CO-01), preserved on potato sucrose agar (PSA) slants at the Plant Pathology Laboratory at Shimane University, Japan. The fungus was cultured on rice bran agar (rice bran 50 g/1, sucrose 20 g/1, agar 20 g/1, and distilled water) for conidiation at 25 ± 2°C for 7 days. The conidia were suspended in sterile distilled water (DW), the concentration of which was adjusted to 1.0 × 10 5 conidia/ml with the aid of a hemocytometer (Thoma, Hirschmann, Germany). The conidial suspension was then used for antagonistic assays and transmission electron microscopy (TEM). Pseudomonas syringae pv. lachrymans (MAFF301315), and Pseudomonas alliivorans (MAFF301157) were obtained from the MAFF Genebank, and Bacillus sp. strain MA3 was obtained from laboratory stocks. All bacterial strains were initially stored at -80°C as glycerol stocks (15% glycerol) and cultured on LBA plates (yeast extract 5 g/l, tryptone 10 g/l, NaCl 10 g/l, agar 20 g/l and distilled water) prior to use in antibacterial assays. Evaluation of antibacterial activity The three bacterial strains were incubated in Luria–Bertani (LB) liquid medium (2 ml) at 25 ± 2°C for 24 h with constant shaking on a rotary shaker (160 rpm). To prepare bacterial suspensions, the bacterial cultures were adjusted to OD 600 = 0.1, and subsequently diluted 100-fold with LB medium, and thereafter mixed with traversianal (2, 4, 10, 20, 40, and 60 ppm) at a 1:1 ratio, static incubation at 25 ± 2°C for 24 h. Bacterial growth was assessed visually based on turbidity. As control treatments, we used a bacterial suspension mixed with 0.6% DMSO as a positive control and LB medium mixed with 0.6% DMSO as a negative control. Furthermore, to evaluate bactericidal activity, 50 µl of the post-incubation bacterial cultures, characterized by traversianal-induced growth inhibition, was spread on LBA plates and incubated at 25 ± 2°C for 48 h, followed by examination of colony formation. Two LBA plates were used for each treatment, which was repeated two times. Determination of the antagonistic effects of phospholipids Egg yolk lecithin (10 mg/ml) was diluted five-fold with methanol and then with DW, and DOPC and DOPE (10 mg/ml) were similarly diluted 10-fold. The organic solvent was removed using an evaporator and the concentration in water (1 ml) was adjusted to 400 ppm for lecithin and 100, 200, and 300 ppm for DOPC and DOPE. The lecithin solution was mixed in a 1:1 ratio with traversianal (2, 4, 10, and 20 ppm) and 0.2% DMSO (traversianal 0 ppm), and the DOPC and DOPE solutions were mixed in a 1:1 ratio with traversianal (10 ppm) for the fungicidal activity assays. As controls, we used mixtures of traversianal with DW or DMSO. Fungicidal activity assays were performed according to previously published methods (Ino et al. 2022 ). C. orbiculare conidia (1.0 × 10 5 conidia/ml) were suspended with mixed traversianal solutions and incubated at 25 ± 2°C for 24 h. Following centrifugation, the liquid phase was replaced with DW, and the suspensions (10 µl) were spread on PSA plates containing chloramphenicol (20 ppm). All plates were incubated at 25 ± 2°C for 3 days, after which, the number of colonies were counted. For each treatment, we assessed five plates, and treatments were performed in duplicate. Transmission electron microscopy C. orbiculare conidia (1.0 × 10 5 conidia/ml) were suspended in traversianal (10 ppm) and maintained at 25 ± 2°C for 24 h, with DMSO (0.1%) used as a control. The treated conidia were collected by centrifugation and pre-fixed with 2.5% glutaraldehyde in 0.05 M phosphate-buffered saline (PBS, pH 7.2) at 4°C for at least 24 h. Having subsequently rinsed three times with 0.1 M cacodylate buffer (pH 7.4), samples were embedded in agarose and post-fixed with 2% osmium tetraoxide in 0.1 M PBS for 2 h. After rinsing two times with 0.1 M cacodylate buffer (pH 7.4), the samples were sequentially dehydrated using a gradient series of ethanol solutions (50%, 70%, 90%, 100%, 100%, and 100%), and thereafter embedded in Spurr’s low-viscosity embedding medium (Polysciences Inc.). Ultrathin sections (approximately 70–80 nm) were cut using an ultramicrotome (Leica EM UC7; Leica Microsystems, Wetzlar, Germany) and double-stained with a 3% uranyl acetate and lead staining solutions (Sigma-Aldrich). The samples were observed using a JEM-1400 Plus transmission electron microscope (JEOL, Tokyo, Japan) operated at 80.0 kV. Statistical analysis The data shown in Figs. 2 and 3 are presented as the means and standard deviations, and were analyzed for statistically significant differences using SPSS Statistics ver. 22.0 for Windows (IBM, Armonk, NY, USA). Differences in the means of different treatment groups were analyzed for significance using a t -test ( P < 0.05) (Fig. 2 ) and Tukey’s HSD test ( P < 0.05), as shown in Fig. 3 . Results Antibacterial activity of traversianal against three target bacterial strains At all assessed concentrations of traversianal, bacterial growth (turbidity) was observed in mixtures of the two Pseudomonas species ( P. syringae pv. lachrymans and P. allivorans ) (Table 1). In contrast, we observed a notable reduction in the turbidity of Bacillus sp. cultured in the presence of 5 ppm traversianal. Moreover, at traversianal concentrations exceeding 10 ppm, the levels of turbidity were comparable to those observed for the negative control (Table 1). Furthermore, whereas Bacillus sp. treated with 1, 2, 5, and 10 ppm traversianal and 0.3% DMSO (positive control) grew abundantly on LBA plates, there was a significant reduction in the number of colonies following treatment at 20 ppm, and colony formation was completely inhibited at 30 ppm (Fig. 1 ). Antagonistic activity of phospholipids against the fungicidal activity of traversianal In the absence of lecithin, we observed reductions in the number of colonies of C. orbiculare with increases in the concentration of traversianal (Fig. 2 a), with high fungicidal activity observed at 5 and 10 ppm, resulting in 0.2 ± 0.4 and 0.1 ± 0.3 colonies/plate, respectively (Fig. 2 b). In contrast, compared with the control, in the presence of lecithin, there were significant increases in colony formation (Fig. 2 a), with 461.8 ± 24.1 and 381.4 ± 59.6 colonies/plate being counted at 2 and 5 ppm traversianal, respectively (Fig. 2 b). The antagonistic effect of lecithin on fungicidal activity was particularly pronounced at 5 ppm. Compared with traversianal alone (as a control), we observed no significant differences in the number of colonies in the combined treatment with DOPC (no colony formation), and no antagonistic activity of DOPC was detected (Fig. 3 a). In treatments with mixtures of DOPC (50, 100, and 150 ppm) and traversianal (5 ppm), the number of colonies were 0.1 ± 0.3, 0.2 ± 0.4 and 3.6 ± 3.6 colonies/plate, respectively (Fig. 3 b). In contrast, treatments with mixtures of DOPE (50 and 100 ppm) and traversianal (5 ppm) resulted in 2.2 ± 0.9 and 20.8 ± 13.8 colonies/plate, respectively (Fig. 3 b). The number of colonies in the mixture containing DOPE (150 ppm) and traversianal was 212.8 ± 90.1 colonies/plate (Fig. 3 b), which was significantly higher compared with the number detected following treatment with traversianal alone. When applied individually, neither DOPC nor DOPE exhibited any discernible effect on the colony formation of C. orbiculare at any of the concentrations tested, as compared to DMSO without the addition of traversianal (Fig. 3 b). The effects of traversianal on the ultrastructural transformation of C. orbiculare Changes in the cytology of C. orbiculare treated with traversianal (examined using TEM) revealed that the internal structure of the conidia of C. orbiculare treated with 0.1% DMSO (as a control) was characterized by clearly observable normal organelles, as evidenced by nuclei surrounded by a double membrane and structurally sound mitochondria (Fig. 4 a). In addition, we observed no abnormalities in either cell walls or plasma membranes (Fig. 4 b). In contrast, conidia treated with traversianal were found to be characterized by numerous ultrastructural changes, including fragmentation of the plasma membrane, loss of cell organelles, and vacuolation due to cytoplasmic leakage (Fig. 4 c, d), although with no evident changes in the appearance of the cell wall compared with that in the control (Fig. 4 d). Discussion The Fungicide Resistance Action Committee (FRAC) classifies fungicides based on their biochemical modes of action with respect to the biosynthetic pathways in plant pathogens and the associated risks of resistance (Fungicide Resistance Action Committee 2024 ). In plant disease control using fungicides, elucidating the mechanisms of action and target sites of active constituents is particularly important from the perspective of assessing the risk of emerging drug-resistant pathogens and the effects of these compounds on non-target organisms. The excessive application of fungicides with similar mechanisms of action can contribute to promoting genetic adaptation among pathogenic fungi, thereby reduce the sustainability of control efficacy (Bradshaw et al. 2021 ). Consequently, the discovery of novel modes of action that are not currently included in the FRAC classification system can potentially contribute to the establishment of more sustainable plant disease management strategies by mitigating the risk of resistance development. At present, our understanding regarding the bioactivity of traversianal remains comparatively limited, and addressing this knowledge gap will be of particular importance for the further development of this compound as a fungicide. In this study, we established that traversianal has growth-inhibitory effects against Bacillus sp. at concentrations in excess of 10 ppm (Table 1) and bactericidal activity at concentrations higher than 20 ppm (Fig. 1 ). To the best of our knowledge, this is the first report of the antibacterial activity of traversianal. Contrastingly, however, traversianal was found to have no evident antibacterial activity against the two Pseudomonas species when assessed at concentrations within the range of 1 to 30 ppm (Table 1). We speculate that this differential sensitivity of bacteria to traversianal may be attributed primarily to difference in the structures of the cell walls of gram-positive and -negative bacteria, and to genus-specific defense mechanisms. Gram-negative Pseudomonas species are characterized by a cell wall structure consisting of an outer membrane and a peptidoglycan layer on the exterior of the inner membrane. The outer membrane, which consists of a lipid bilayer and lipopolysaccharides, restricts the permeation of numerous hydrophobic substances (Delcour 2009 ; O’Shea and Moser 2008 ; Pagès et al. 2008 ). In addition, Pseudomonas species harbor multiple drug efflux pump systems, among which are resistance-nodulation-division (RND) family efflux pumps that can expel a broad spectrum of antimicrobial compounds from cells (Zahedi Bialvaei et al. 2021 ). These mechanisms may not only contribute to preventing the migration of relatively hydrophobic compounds, such as traversianal, across the plasma membrane and into the cell interior but also potentially facilitate the rapid expulsion of compounds that are able to penetrate the cell. In contrast, the gram-positive bacilli have a relatively simple cell wall structure comprising a thick peptidoglycan layer and an inner membrane. Unlike gram-negative bacteria, these bacteria lack an outer membrane, and it is conceivable that traversianal can pass readily through the peptidoglycan layer and interact directly with the cell membrane. However, the presence of multidrug resistance pumps in Bacillus species has also been reported (Neyfakh et al. 1991 ), which may indicate that differences in substrate specificity and the expression levels of these pumps also contribute to observed differences in the sensitivity to traversianal. Consequently, to comprehensively assess the antibacterial spectrum of traversianal, future studies should accordingly examine a broader range of bacterial species. A notable finding in this study was our observation of a significant reduction in the fungicidal activity of traversianal against C. orbiculare in the presence of lecithin (200 ppm) and DOPE (150 ppm), whereas no comparable reduction was detected in co-treatments with DOPC (Fig. 3 ). Furthermore, a similar pattern was detected in the inhibitory effect of traversianal on conidial germination (Fig. S1 and S2). The structures of both DOPC and DOPE are characterized by glycerol backbones with two oleic acid chains, although they differ with respect to a choline (DOPC) or ethanolamine (DOPE) group attached to the phosphate group bound to the glycerol backbone. This structural distinction could indicate that the ethanolamine group plays a prominent role in the antagonistic activity of DOPE against the fungicidal effects of traversianal. Indeed, a similar DOPE-specific antagonistic activity has been reported with respect to the antagonistic activity of different phospholipids against the cytotoxicity of ophiobolin A (Chidley et al. 2016 ), which has been established to have potential anticancer activity by forming complexes via specific binding to PE in the cell membrane, mediated by its 1,4-dicarbonyl structure (Chidley et al. 2016 ). Consequently, the results obtained in this study would tend to indicate that the fungicidal activity of traversianal is associated with its binding to PE in the cell membrane of C. orbiculare via the 1,4-dicarbonyl structure. This proposed fungicidal mechanism of traversianal is consistent with our TEM observations of the internal structure of C. orbiculare conidia treated with traversianal, which were found to be characterized by distinct cytological changes, including vacuolization and fragmentation of the plasma membrane (Fig. 4 c, d). Furthermore, an absence of any evident changes in the integrity of cell wall structures and the disappearance of cell organelles containing lipid bilayers, such as mitochondria and nuclei, provide evidence to indicate that traversianal targets cell membranes. We have previously evaluated the fungicidal activity of traversianal against C. orbiculare via fluorescent double-staining using fluorescein diacetate (FDA) and propidium iodide (PI), which revealed a significant increase in the proportion of conidia characterized by PI-derived red fluorescence following treatment with traversianal (Ino et al. 2024 ). When taken up into cells, FDA is degraded by esterase and accumulates intracellularly as the fluorescent compound fluorescein (Franklin et al. 2001 ). Consequently, cells with esterase activity can be detected by green fluorescence when excited at 488 nm. However, PI is typically unable to penetrate the membranes of viable cells, and thus only enters cells with compromised membrane integrity (dead cells), wherein it intercalates with the DNA double helix structure to produce the characteristic intense red fluorescence (Brana et al. 2002 ). Accordingly, simultaneous staining with FDA and PI facilitates an assessment of cell viability based on membrane integrity (Liu et al. 2020 ; Palma-Guerrero et al. 2009 ). Our findings thus provide evidence to indicate that by targeting cell membranes, the antimicrobial activity of traversianal against C. orbiculare is mediated by inducing alterations in membrane permeability and damage. Ophiobolin A forms a complex with PE containing a pyrrole ring that exhibits a characteristic absorption maximum at 580 nm (Chidley et al. 2016 ). Further studies using a microplate reader to measure the absorbance of the reaction product of trabacyanal with PE are necessary to confirm similar complex formation. In conclusion, our findings in this study revealed that traversianal, a diterpenoid aldehyde produced by Cercospora sp. ME202, has antibacterial activity in addition to its previously established antifungal properties. The fungicidal activity of this compound against C. orbiculare is suggested to be associated with a specific targeting of phosphatidylethanolamine within the cell membrane, thereby identifying this membrane component as a potential target site for the development of novel fungicides. Declarations Acknowledgments The authors thank the Faculty of Life and Environmental Science at Shimane University and SDGs Research Project of Shimane University for their financial support in publishing this report. Author contributions Conceptualization: MI, JK, and MU; formal analysis and research: MI, MU, and AI; original draft preparation: MI, MU, and AI; review and editing: MI, MU, and AI; funding acquisition: MU; supervision: KJ, MU, and AI. Funding This study was funded by Shimane University, Japan. Compliance with Ethical Standard Conflicts of interest The authors declare that they have no conflicts of interest relevant to the contents of this article. 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Microb Pathog 153:104789. https://doi.org/10.1016/j.micpath.2021.104789 Zhong JJ, Xiao JH (2009) Secondary metabolites from higher fungi: Discovery, bioactivity, and bioproduction. Adv Biochem Eng Biotechnol 113:79–150. https://doi.org/10.1007/10_2008_26 Table Table 1 is available in the Supplementary Files section Supplementary Files InoetalTable.docx InoetalSupplementalData.docx Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2025 Read the published version in Journal of General Plant Pathology → Version 1 posted Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 12 May, 2025 Editor assigned by journal 07 May, 2025 First submitted to journal 06 May, 2025 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. 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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-6608584","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455623260,"identity":"448d968e-f6b9-4dcf-acf5-f389d31d77bc","order_by":0,"name":"Masatoshi Ino","email":"","orcid":"","institution":"Tottori University: Tottori Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Masatoshi","middleName":"","lastName":"Ino","suffix":""},{"id":455623261,"identity":"4564a88c-4a9d-4fa0-8460-2e03c82bc07e","order_by":1,"name":"Junichi Kihara","email":"","orcid":"","institution":"Shimane Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Junichi","middleName":"","lastName":"Kihara","suffix":""},{"id":455623262,"identity":"86ce94aa-05ce-44e7-b1b7-91550b468a10","order_by":2,"name":"Atsushi Ishihara","email":"","orcid":"","institution":"Tottori University: Tottori Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Ishihara","suffix":""},{"id":455623263,"identity":"b46f65c1-585d-4ad6-9027-7610e0c63b8c","order_by":3,"name":"Makoto Ueno","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9358-4487","institution":"Shimane Daigaku","correspondingAuthor":true,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Ueno","suffix":""}],"badges":[],"createdAt":"2025-05-07 06:18:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6608584/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6608584/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10327-025-01249-w","type":"published","date":"2025-09-11T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82771947,"identity":"f9fbf2cd-b722-46de-8f14-0e53c9e3c303","added_by":"auto","created_at":"2025-05-15 06:29:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBactericidal activity of traversianal against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA culture suspension of \u003cem\u003eBacillus\u003c/em\u003e sp. in LB medium was mixed with traversianal (2, 4, 10, 20, 40, or 60 ppm) at 1:1 ratios followed by static incubation at 25 ± 2°C for 24 h. Negative control (NC: LB liquid without bacteria) and positive control (PC: bacterial suspension without traversianal) treatments were also assessed. Following incubation, the bacterial mixtures were spread on LBA plates and incubated at 25 ± 2°C for 48 h, followed by examination for colony formation\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/5ff408b6111a9bceeca17e16.png"},{"id":82771951,"identity":"fe90b124-a6d5-4d54-95ce-d8c1aaf52f17","added_by":"auto","created_at":"2025-05-15 06:29:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":287162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntagonistic effects of lecithin on the fungicidal activity of traversianal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConidia of \u003cem\u003eColletotrichum orbiculare\u003c/em\u003e (1.0 × 10\u003csup\u003e5\u003c/sup\u003e conidia/ml) were suspended in mixtures of equal parts lecithin (400 ppm), traversianal (2, 4, 10, or 20 ppm), and 0.2% DMSO (traversianal 0 ppm) for 24 h and then spread on PSA plates. (a) After 3 days, colony development was examined, and (b) the number of colonies counted. Mixtures of sterile distilled water and traversianal (2, 4, 10, or 20 ppm) or equal amounts of 0.2% DMSO were used as controls. The bars above columns represent the standard deviations of the means. Asterisks indicate significant differences among treatments, whereas ns indicates no significant difference (\u003cem\u003et\u003c/em\u003e-test: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/caf18e69027d55a57f58a5d0.png"},{"id":82771968,"identity":"7d54f0b8-69a0-4a44-97ae-12ea9d25fdac","added_by":"auto","created_at":"2025-05-15 06:29:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":270955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntagonistic effects of phospholipids on the fungicidal activity of traversianal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConidia of \u003cem\u003eColletotrichum orbiculare\u003c/em\u003e (1.0 × 10\u003csup\u003e5\u003c/sup\u003e conidia/ml) were suspended in mixtures of equal parts DOPC or DOPE (100, 200, or 300 ppm) and traversianal (10 ppm) for 24 h and then spread on PSA plates. (a) After 3 days, colony development was examined, and (b) the number of colonies counted. Mixtures of sterile distilled water and 0.1% DMSO or traversianal (10 ppm) in equal volumes were used as controls. Error bars represent standard deviations of the mean. Different letters after the means indicate significant differences between treatments (Tukey–Kramer test: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/bbfd4e9d2e6658524ddd8611.png"},{"id":82771970,"identity":"9053a35f-fa76-4366-854b-8d173c365a47","added_by":"auto","created_at":"2025-05-15 06:29:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":493396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransmission electron micrographs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eColletotrichum orbiculare\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etreated with or without traversianal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) Conidia of\u003cem\u003e C. orbiculare\u003c/em\u003e treated with 0.1% DMSO for 24 h as a control. (c, d) Conidia of\u003cem\u003eC. orbiculare\u003c/em\u003e treated with traversianal (10 ppm) for 24 h. CW: cell wall, PM: plasma membrane, N: nucleus, M: mitochondrion, V: vacuole. Bars represent 2.0 μm (a, c) and 200.0 nm (b, d)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/efe8f545098209be458c3776.png"},{"id":91358976,"identity":"514de47f-f0db-4cc2-8b7c-4aaef02069ba","added_by":"auto","created_at":"2025-09-15 16:02:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1985472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/92dac89b-8888-407a-991b-baa6081d45d7.pdf"},{"id":82771949,"identity":"fe767ca8-72c1-43cb-b25b-5b4e5969f1ff","added_by":"auto","created_at":"2025-05-15 06:29:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39741,"visible":true,"origin":"","legend":"","description":"","filename":"InoetalTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/25d64108a9fe4d0dc6d630f7.docx"},{"id":82771950,"identity":"5a47d1d4-e4d9-4bc2-9e19-11d23148711f","added_by":"auto","created_at":"2025-05-15 06:29:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":745702,"visible":true,"origin":"","legend":"","description":"","filename":"InoetalSupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6608584/v1/d55361e8aa349be20e824e1c.docx"}],"financialInterests":"","formattedTitle":"Mechanism of the antifungal activity of the diterpenoid aldehyde traversianal against the cucumber anthracnose fungus Colletotrichum orbiculare","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe inappropriate use of synthetic chemical fungicides has led to the development of drug resistance in numerous plant pathogens, thereby presenting substantial challenges for the control of plant diseases. For example, among species of \u003cem\u003eColletotrichum\u003c/em\u003e fungi, strains resistant to benzimidazole-based and strobilurin fungicides have been reported (Forcelini et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Torres-Calzada et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the occurrence of cross-resistance and multi-drug resistant strains among numerous economically important plant pathogens, including \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003ePyricularia oryzae\u003c/em\u003e, and \u003cem\u003eFusarium\u003c/em\u003e species, has become a source of particular concern (Alzohairy et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Becher et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Vicentini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, there is an urgent need to develop novel fungicides with modes of action that differ from those of existing agents.\u003c/p\u003e \u003cp\u003eIn this regard, given their diverse chemical structures, fungal secondary metabolites have attracted considerable attention as valuable biological resources with potential utility in the development of novel fungicides and pharmaceutical agents. These secondary metabolites, including polyketides, terpenoids, and alkaloids, produced via different biosynthetic pathways (Cox \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Panaccione \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Quin et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), are also characterized by diversity in terms of biological properties, including antibacterial, antifungal, and cytotoxic activities (Conrado et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhong and Xiao \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Given their structural and functional diversity, these fungal metabolites are accordingly considered promising sources for the discovery of bioactive compounds with novel mechanisms of action.\u003c/p\u003e \u003cp\u003eTerpenoids, which represent the largest category of natural products and are considered a rich source of compounds with antibacterial and antifungal activities (Gershenzon and Dudareva \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), are biosynthesized from isoprene units and have a wide range of structures, including monoterpenes, sesquiterpenes, and diterpenes. We have previously reported that traversianal, a diterpenoid aldehyde produced by \u003cem\u003eCercospora\u003c/em\u003e sp. ME202, is characterized antifungal activity, including fungicidal effects, with inhibition of the conidial germination and mycelial growth of the cucumber anthracnose fungus \u003cem\u003eColletotrichum orbiculare\u003c/em\u003e (Ino et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Traversianal has also been reported to have toxic effects against brine shrimps and snails, and mild hemolytic activity against human erythrocytes, although shows no toxicity toward chicks and plants (Stoessl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). At present, however, our knowledge regarding the biological activities of traversianal remains relatively limited, and its antibacterial activity and mechanisms underlying its toxic and antifungal activities have yet to be sufficiently elucidated.\u003c/p\u003e \u003cp\u003eTraversianal is a member of a group of compounds, the basic structure of which is a 5-8-5 ring carbon skeleton, that have structural similarities to ophiobolins and fusicoccins (Cimmino et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Among these compounds, ophiobolin A has been established to have anticancer activity by specifically combining and forming a complex with phosphatidylethanolamine (PE) in the membranes of cancer cells via the 1,4-dicarbonyl groups present in its chemical structure (Chidley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This is based on observations that mutations in the Kennedy pathway for PE biosynthesis are associated with reduced sensitivity to ophiobolin A and that phospholipid supplementation antagonistically reduces ophiobolin A cytotoxicity (Chidley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Given that traversianal also contains a 1,4-dicarbonyl group, it is speculated that its antifungal activity may be mediated via specific interactions with PE, similar to the mechanism observed for ophiobolin A. Accordingly, in this study, we sought to elucidate the mechanisms underlying the antifungal activity of traversianal against \u003cem\u003eC. orbiculare\u003c/em\u003e by evaluating its antibacterial properties, determining the antagonistic effects of lecithin and different phospholipids, and examining changes in the conidial ultrastructure of traversianal-treated \u003cem\u003eC. orbiculare\u003c/em\u003e using transmission electron microscopy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eTraversianal was isolated from a culture filtrate of \u003cem\u003eCercospora\u003c/em\u003e sp. ME202 via silica gel column chromatography and high-performance liquid chromatography, as previously described (Ino et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The compound was stored at -20\u0026deg;C and used for experiments by dissolving in dimethyl sulfoxide (DMSO) at appropriate concentrations.\u003c/p\u003e \u003cp\u003eEgg yolk lecithin, l-α-phosphatidylcholine dioleoyl (DOPC), and l-α-phosphatidylethanolamine dioleoyl (DOPE) were purchased from Fujifilm Wako Pure Chemical Corporation for antagonistic assay. These chemicals were prepared as 10 mg/ml stock solutions in chloroform and stored at 4\u0026deg;C for subsequent use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFungal and bacterial strains\u003c/h3\u003e\n\u003cp\u003eFor the purposes of this study, we used \u003cem\u003eColletotrichum orbiculare\u003c/em\u003e (strain CO-01), preserved on potato sucrose agar (PSA) slants at the Plant Pathology Laboratory at Shimane University, Japan. The fungus was cultured on rice bran agar (rice bran 50 g/1, sucrose 20 g/1, agar 20 g/1, and distilled water) for conidiation at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 7 days. The conidia were suspended in sterile distilled water (DW), the concentration of which was adjusted to 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/ml with the aid of a hemocytometer (Thoma, Hirschmann, Germany). The conidial suspension was then used for antagonistic assays and transmission electron microscopy (TEM).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003elachrymans\u003c/em\u003e (MAFF301315), and \u003cem\u003ePseudomonas alliivorans\u003c/em\u003e (MAFF301157) were obtained from the MAFF Genebank, and \u003cem\u003eBacillus\u003c/em\u003e sp. strain MA3 was obtained from laboratory stocks. All bacterial strains were initially stored at -80\u0026deg;C as glycerol stocks (15% glycerol) and cultured on LBA plates (yeast extract 5 g/l, tryptone 10 g/l, NaCl 10 g/l, agar 20 g/l and distilled water) prior to use in antibacterial assays.\u003c/p\u003e\n\u003ch3\u003eEvaluation of antibacterial activity\u003c/h3\u003e\n\u003cp\u003eThe three bacterial strains were incubated in Luria\u0026ndash;Bertani (LB) liquid medium (2 ml) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 24 h with constant shaking on a rotary shaker (160 rpm). To prepare bacterial suspensions, the bacterial cultures were adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1, and subsequently diluted 100-fold with LB medium, and thereafter mixed with traversianal (2, 4, 10, 20, 40, and 60 ppm) at a 1:1 ratio, static incubation at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 24 h. Bacterial growth was assessed visually based on turbidity. As control treatments, we used a bacterial suspension mixed with 0.6% DMSO as a positive control and LB medium mixed with 0.6% DMSO as a negative control. Furthermore, to evaluate bactericidal activity, 50 \u0026micro;l of the post-incubation bacterial cultures, characterized by traversianal-induced growth inhibition, was spread on LBA plates and incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 48 h, followed by examination of colony formation. Two LBA plates were used for each treatment, which was repeated two times.\u003c/p\u003e\n\u003ch3\u003eDetermination of the antagonistic effects of phospholipids\u003c/h3\u003e\n\u003cp\u003eEgg yolk lecithin (10 mg/ml) was diluted five-fold with methanol and then with DW, and DOPC and DOPE (10 mg/ml) were similarly diluted 10-fold. The organic solvent was removed using an evaporator and the concentration in water (1 ml) was adjusted to 400 ppm for lecithin and 100, 200, and 300 ppm for DOPC and DOPE. The lecithin solution was mixed in a 1:1 ratio with traversianal (2, 4, 10, and 20 ppm) and 0.2% DMSO (traversianal 0 ppm), and the DOPC and DOPE solutions were mixed in a 1:1 ratio with traversianal (10 ppm) for the fungicidal activity assays. As controls, we used mixtures of traversianal with DW or DMSO.\u003c/p\u003e \u003cp\u003eFungicidal activity assays were performed according to previously published methods (Ino et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eC. orbiculare\u003c/em\u003e conidia (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/ml) were suspended with mixed traversianal solutions and incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 24 h. Following centrifugation, the liquid phase was replaced with DW, and the suspensions (10 \u0026micro;l) were spread on PSA plates containing chloramphenicol (20 ppm). All plates were incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 3 days, after which, the number of colonies were counted. For each treatment, we assessed five plates, and treatments were performed in duplicate.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eC. orbiculare\u003c/em\u003e conidia (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/ml) were suspended in traversianal (10 ppm) and maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 24 h, with DMSO (0.1%) used as a control. The treated conidia were collected by centrifugation and pre-fixed with 2.5% glutaraldehyde in 0.05 M phosphate-buffered saline (PBS, pH 7.2) at 4\u0026deg;C for at least 24 h. Having subsequently rinsed three times with 0.1 M cacodylate buffer (pH 7.4), samples were embedded in agarose and post-fixed with 2% osmium tetraoxide in 0.1 M PBS for 2 h. After rinsing two times with 0.1 M cacodylate buffer (pH 7.4), the samples were sequentially dehydrated using a gradient series of ethanol solutions (50%, 70%, 90%, 100%, 100%, and 100%), and thereafter embedded in Spurr\u0026rsquo;s low-viscosity embedding medium (Polysciences Inc.). Ultrathin sections (approximately 70\u0026ndash;80 nm) were cut using an ultramicrotome (Leica EM UC7; Leica Microsystems, Wetzlar, Germany) and double-stained with a 3% uranyl acetate and lead staining solutions (Sigma-Aldrich). The samples were observed using a JEM-1400 Plus transmission electron microscope (JEOL, Tokyo, Japan) operated at 80.0 kV.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e are presented as the means and standard deviations, and were analyzed for statistically significant differences using SPSS Statistics ver. 22.0 for Windows (IBM, Armonk, NY, USA). Differences in the means of different treatment groups were analyzed for significance using a \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and Tukey\u0026rsquo;s HSD test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial activity of traversianal against three target bacterial strains\u003c/h2\u003e \u003cp\u003eAt all assessed concentrations of traversianal, bacterial growth (turbidity) was observed in mixtures of the two \u003cem\u003ePseudomonas\u003c/em\u003e species (\u003cem\u003eP. syringae\u003c/em\u003e pv. \u003cem\u003elachrymans\u003c/em\u003e and \u003cem\u003eP. allivorans\u003c/em\u003e) (Table\u0026nbsp;1). In contrast, we observed a notable reduction in the turbidity of \u003cem\u003eBacillus\u003c/em\u003e sp. cultured in the presence of 5 ppm traversianal. Moreover, at traversianal concentrations exceeding 10 ppm, the levels of turbidity were comparable to those observed for the negative control (Table\u0026nbsp;1). Furthermore, whereas \u003cem\u003eBacillus\u003c/em\u003e sp. treated with 1, 2, 5, and 10 ppm traversianal and 0.3% DMSO (positive control) grew abundantly on LBA plates, there was a significant reduction in the number of colonies following treatment at 20 ppm, and colony formation was completely inhibited at 30 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntagonistic activity of phospholipids against the fungicidal activity of traversianal\u003c/h2\u003e \u003cp\u003eIn the absence of lecithin, we observed reductions in the number of colonies of \u003cem\u003eC. orbiculare\u003c/em\u003e with increases in the concentration of traversianal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with high fungicidal activity observed at 5 and 10 ppm, resulting in 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 and 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 colonies/plate, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, compared with the control, in the presence of lecithin, there were significant increases in colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with 461.8\u0026thinsp;\u0026plusmn;\u0026thinsp;24.1 and 381.4\u0026thinsp;\u0026plusmn;\u0026thinsp;59.6 colonies/plate being counted at 2 and 5 ppm traversianal, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The antagonistic effect of lecithin on fungicidal activity was particularly pronounced at 5 ppm.\u003c/p\u003e \u003cp\u003eCompared with traversianal alone (as a control), we observed no significant differences in the number of colonies in the combined treatment with DOPC (no colony formation), and no antagonistic activity of DOPC was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In treatments with mixtures of DOPC (50, 100, and 150 ppm) and traversianal (5 ppm), the number of colonies were 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 and 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 colonies/plate, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In contrast, treatments with mixtures of DOPE (50 and 100 ppm) and traversianal (5 ppm) resulted in 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 and 20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8 colonies/plate, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The number of colonies in the mixture containing DOPE (150 ppm) and traversianal was 212.8\u0026thinsp;\u0026plusmn;\u0026thinsp;90.1 colonies/plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), which was significantly higher compared with the number detected following treatment with traversianal alone. When applied individually, neither DOPC nor DOPE exhibited any discernible effect on the colony formation of C. \u003cem\u003eorbiculare\u003c/em\u003e at any of the concentrations tested, as compared to DMSO without the addition of traversianal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effects of traversianal on the ultrastructural transformation of\u003c/b\u003e \u003cb\u003eC. orbiculare\u003c/b\u003e\u003c/p\u003e \u003cp\u003eChanges in the cytology of \u003cem\u003eC. orbiculare\u003c/em\u003e treated with traversianal (examined using TEM) revealed that the internal structure of the conidia of \u003cem\u003eC. orbiculare\u003c/em\u003e treated with 0.1% DMSO (as a control) was characterized by clearly observable normal organelles, as evidenced by nuclei surrounded by a double membrane and structurally sound mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, we observed no abnormalities in either cell walls or plasma membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In contrast, conidia treated with traversianal were found to be characterized by numerous ultrastructural changes, including fragmentation of the plasma membrane, loss of cell organelles, and vacuolation due to cytoplasmic leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d), although with no evident changes in the appearance of the cell wall compared with that in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Fungicide Resistance Action Committee (FRAC) classifies fungicides based on their biochemical modes of action with respect to the biosynthetic pathways in plant pathogens and the associated risks of resistance (Fungicide Resistance Action Committee \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In plant disease control using fungicides, elucidating the mechanisms of action and target sites of active constituents is particularly important from the perspective of assessing the risk of emerging drug-resistant pathogens and the effects of these compounds on non-target organisms. The excessive application of fungicides with similar mechanisms of action can contribute to promoting genetic adaptation among pathogenic fungi, thereby reduce the sustainability of control efficacy (Bradshaw et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, the discovery of novel modes of action that are not currently included in the FRAC classification system can potentially contribute to the establishment of more sustainable plant disease management strategies by mitigating the risk of resistance development.\u003c/p\u003e \u003cp\u003eAt present, our understanding regarding the bioactivity of traversianal remains comparatively limited, and addressing this knowledge gap will be of particular importance for the further development of this compound as a fungicide. In this study, we established that traversianal has growth-inhibitory effects against \u003cem\u003eBacillus\u003c/em\u003e sp. at concentrations in excess of 10 ppm (Table\u0026nbsp;1) and bactericidal activity at concentrations higher than 20 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To the best of our knowledge, this is the first report of the antibacterial activity of traversianal. Contrastingly, however, traversianal was found to have no evident antibacterial activity against the two \u003cem\u003ePseudomonas\u003c/em\u003e species when assessed at concentrations within the range of 1 to 30 ppm (Table\u0026nbsp;1). We speculate that this differential sensitivity of bacteria to traversianal may be attributed primarily to difference in the structures of the cell walls of gram-positive and -negative bacteria, and to genus-specific defense mechanisms. Gram-negative \u003cem\u003ePseudomonas\u003c/em\u003e species are characterized by a cell wall structure consisting of an outer membrane and a peptidoglycan layer on the exterior of the inner membrane. The outer membrane, which consists of a lipid bilayer and lipopolysaccharides, restricts the permeation of numerous hydrophobic substances (Delcour \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; O\u0026rsquo;Shea and Moser \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Pag\u0026egrave;s et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In addition, \u003cem\u003ePseudomonas\u003c/em\u003e species harbor multiple drug efflux pump systems, among which are resistance-nodulation-division (RND) family efflux pumps that can expel a broad spectrum of antimicrobial compounds from cells (Zahedi Bialvaei et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These mechanisms may not only contribute to preventing the migration of relatively hydrophobic compounds, such as traversianal, across the plasma membrane and into the cell interior but also potentially facilitate the rapid expulsion of compounds that are able to penetrate the cell. In contrast, the gram-positive bacilli have a relatively simple cell wall structure comprising a thick peptidoglycan layer and an inner membrane. Unlike gram-negative bacteria, these bacteria lack an outer membrane, and it is conceivable that traversianal can pass readily through the peptidoglycan layer and interact directly with the cell membrane. However, the presence of multidrug resistance pumps in \u003cem\u003eBacillus\u003c/em\u003e species has also been reported (Neyfakh et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), which may indicate that differences in substrate specificity and the expression levels of these pumps also contribute to observed differences in the sensitivity to traversianal. Consequently, to comprehensively assess the antibacterial spectrum of traversianal, future studies should accordingly examine a broader range of bacterial species.\u003c/p\u003e \u003cp\u003eA notable finding in this study was our observation of a significant reduction in the fungicidal activity of traversianal against \u003cem\u003eC. orbiculare\u003c/em\u003e in the presence of lecithin (200 ppm) and DOPE (150 ppm), whereas no comparable reduction was detected in co-treatments with DOPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, a similar pattern was detected in the inhibitory effect of traversianal on conidial germination (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). The structures of both DOPC and DOPE are characterized by glycerol backbones with two oleic acid chains, although they differ with respect to a choline (DOPC) or ethanolamine (DOPE) group attached to the phosphate group bound to the glycerol backbone. This structural distinction could indicate that the ethanolamine group plays a prominent role in the antagonistic activity of DOPE against the fungicidal effects of traversianal. Indeed, a similar DOPE-specific antagonistic activity has been reported with respect to the antagonistic activity of different phospholipids against the cytotoxicity of ophiobolin A (Chidley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which has been established to have potential anticancer activity by forming complexes via specific binding to PE in the cell membrane, mediated by its 1,4-dicarbonyl structure (Chidley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consequently, the results obtained in this study would tend to indicate that the fungicidal activity of traversianal is associated with its binding to PE in the cell membrane of \u003cem\u003eC. orbiculare\u003c/em\u003e via the 1,4-dicarbonyl structure.\u003c/p\u003e \u003cp\u003eThis proposed fungicidal mechanism of traversianal is consistent with our TEM observations of the internal structure of \u003cem\u003eC. orbiculare\u003c/em\u003e conidia treated with traversianal, which were found to be characterized by distinct cytological changes, including vacuolization and fragmentation of the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). Furthermore, an absence of any evident changes in the integrity of cell wall structures and the disappearance of cell organelles containing lipid bilayers, such as mitochondria and nuclei, provide evidence to indicate that traversianal targets cell membranes. We have previously evaluated the fungicidal activity of traversianal against \u003cem\u003eC. orbiculare\u003c/em\u003e via fluorescent double-staining using fluorescein diacetate (FDA) and propidium iodide (PI), which revealed a significant increase in the proportion of conidia characterized by PI-derived red fluorescence following treatment with traversianal (Ino et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). When taken up into cells, FDA is degraded by esterase and accumulates intracellularly as the fluorescent compound fluorescein (Franklin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Consequently, cells with esterase activity can be detected by green fluorescence when excited at 488 nm. However, PI is typically unable to penetrate the membranes of viable cells, and thus only enters cells with compromised membrane integrity (dead cells), wherein it intercalates with the DNA double helix structure to produce the characteristic intense red fluorescence (Brana et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Accordingly, simultaneous staining with FDA and PI facilitates an assessment of cell viability based on membrane integrity (Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Palma-Guerrero et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Our findings thus provide evidence to indicate that by targeting cell membranes, the antimicrobial activity of traversianal against \u003cem\u003eC. orbiculare\u003c/em\u003e is mediated by inducing alterations in membrane permeability and damage. Ophiobolin A forms a complex with PE containing a pyrrole ring that exhibits a characteristic absorption maximum at 580 nm (Chidley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Further studies using a microplate reader to measure the absorbance of the reaction product of trabacyanal with PE are necessary to confirm similar complex formation.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings in this study revealed that traversianal, a diterpenoid aldehyde produced by \u003cem\u003eCercospora\u003c/em\u003e sp. ME202, has antibacterial activity in addition to its previously established antifungal properties. The fungicidal activity of this compound against \u003cem\u003eC. orbiculare\u003c/em\u003e is suggested to be associated with a specific targeting of phosphatidylethanolamine within the cell membrane, thereby identifying this membrane component as a potential target site for the development of novel fungicides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Faculty of Life and Environmental Science at Shimane University and SDGs Research Project of Shimane University for their financial support in publishing this report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: MI, JK, and MU; formal analysis and research: MI, MU, and AI; original draft preparation: MI, MU, and AI; review and editing: MI, MU, and AI; funding acquisition: MU; supervision: KJ, MU, and AI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Shimane University, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standard\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest relevant to the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were no procedures performed in this study involving human participants or animals\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlzohairy SA, Heger L, Nikzainalalam N, Miles TD (2023) Cross-resistance of succinate dehydrogenase inhibitors (SDHI) in \u003cem\u003eBotrytis cinerea\u0026nbsp;\u003c/em\u003eand development of molecular diagnostic tools for SDHI resistance detection. 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Nat Prod Rep 31:1449\u0026ndash;1473. https://doi.org/10.1039/C4NP00075G\u003c/li\u003e\n \u003cli\u003eStoessl A, Cole RJ, Abramowski Z, Lester HH, Towers GHN (1989) Some biological properties of traversianal, a strongly molluscicidal diterpenoid aldehyde from \u003cem\u003eCercospora traversiana\u003c/em\u003e. Mycopathologia 106:41\u0026ndash;46. https://doi.org/10.1007/BF00436925\u003c/li\u003e\n \u003cli\u003eTorres-Calzada C, Tapia-Tussell R, Higuera-Ciapara I, Martin-Mex R, Nexticapan-Garcez A, Perez-Brito D (2015) Sensitivity of \u003cem\u003eColletotrichum truncatum\u003c/em\u003e to four fungicides and characterization of thiabendazole-resistant isolates. Plant Dis 99:1590\u0026ndash;1595. https://doi.org/10.1094/PDIS-11-14-1183-RE\u003c/li\u003e\n \u003cli\u003eVicentini SNC, Casado PS, de Carvalho G, Moreira SI, Dorigan AF, Silva TC, Silva AG, Cust\u0026oacute;dio AAP, Gomes ACS, Nunes Maciel JL, Hawkins N, McDonald BA, Fraaije BA, Ceresini PC (2022) Monitoring of Brazilian wheat blast field populations reveals resistance to QoI, DMI, and SDHI fungicides. Plant Pathol 71:304\u0026ndash;321. https://doi.org/10.1111/ppa.13470\u003c/li\u003e\n \u003cli\u003eZahedi Bialvaei AZ, Rahbar M, Hamidi-Farahani R, Asgari A, Esmailkhani A, Mardani Dashti Y, Soleiman-Meigooni S (2021) Expression of RND efflux pumps mediated antibiotic resistance in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e clinical strains. Microb Pathog 153:104789. https://doi.org/10.1016/j.micpath.2021.104789\u003c/li\u003e\n \u003cli\u003eZhong JJ, Xiao JH (2009) Secondary metabolites from higher fungi: Discovery, bioactivity, and bioproduction. Adv Biochem Eng Biotechnol 113:79\u0026ndash;150. https://doi.org/10.1007/10_2008_26\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is 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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mechanism of action, Antifungal activity, Colltotrichum orbiculare, Traversianal, phospholipids, Antagonistic effect","lastPublishedDoi":"10.21203/rs.3.rs-6608584/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6608584/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of fungicides with novel modes of action is essential for addressing the emergence of drug resistance in plant pathogens caused by the long-term use of chemical pesticides. In this study, we investigated the mechanisms underlying the antifungal activity of traversianal, a diterpenoid aldehyde isolated from a culture filtrate of \u003cem\u003eCercospora\u003c/em\u003e sp. ME202, against\u003cem\u003e Colletotrichum orbiculare\u003c/em\u003e, the causal agent of cucumber anthracnose. In antibacterial assays using two \u003cem\u003ePseudomonas\u003c/em\u003e species and one \u003cem\u003eBacillus\u003c/em\u003e species, traversianal showed growth-inhibitory activity against the \u003cem\u003eBacillus\u003c/em\u003especies. The fungicidal activity of traversianal against \u003cem\u003eC. orbiculare\u003c/em\u003e was attenuated by the addition of lecithin, which consists primarily of phospholipids. Compared with that in the control, we observed a significant increase in the number of colonies of \u003cem\u003eC. orbiculare\u003c/em\u003e on potato sucrose agar following treatment with traversianal in the presence of lecithin, thus indicating the competitive inhibition of fungicidal activity by lecithin. However, this antagonistic effect was not observed with the addition of l-α-phosphatidylcholine dioleoyl, whereas it was detected in the presence of l-α-phosphatidylethanolamine dioleoyl. These findings provide evidence that the cell membrane component phosphatidylethanolamine may be specifically targeted in the fungicidal activity of traversianal, which is consistent with our transmission electron microscopy observations revealing the fragmentation of plasma membranes and disappearance of cell organelles in the conidia of \u003cem\u003eC. orbiculare\u003c/em\u003e treated with traversianal. These findings thus indicate that traversianal may have potential application as a novel fungicide that targets phosphatidylethanolamine.\u003c/p\u003e","manuscriptTitle":"Mechanism of the antifungal activity of the diterpenoid aldehyde traversianal against the cucumber anthracnose fungus Colletotrichum orbiculare","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 06:29:46","doi":"10.21203/rs.3.rs-6608584/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-14T00:18:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T00:53:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-08T00:15:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2025-05-07T02:17:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f765b48b-8e8d-4e10-b9bf-d64ff022f3aa","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T15:58:40+00:00","versionOfRecord":{"articleIdentity":"rs-6608584","link":"https://doi.org/10.1007/s10327-025-01249-w","journal":{"identity":"journal-of-general-plant-pathology","isVorOnly":false,"title":"Journal of General Plant Pathology"},"publishedOn":"2025-09-11 15:57:04","publishedOnDateReadable":"September 11th, 2025"},"versionCreatedAt":"2025-05-15 06:29:46","video":"","vorDoi":"10.1007/s10327-025-01249-w","vorDoiUrl":"https://doi.org/10.1007/s10327-025-01249-w","workflowStages":[]},"version":"v1","identity":"rs-6608584","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6608584","identity":"rs-6608584","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}