Toxic Effects of Thiram on Fusarium phytopathogens, L929 Fibroblast Cell Lines, and Wheat Seeds

Articles in Press

Document Type : Original Research

Authors
Mehmet Arslan 1
Tuğba Teker 2
Büşra Nur Çetin 1
Emre Yörük 3
Gülruh Albayrak 4
1 Programme of Molecular Biotechnology and Genetics, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul, Türkiye.
2 Department of Molecular Biology and Genetics, Faculty of Engineering and Natural Sciences, Istanbul Atlas University, Istanbul, Türkiye.
3 Department of Molecular Biology and Genetics, Faculty of Arts and Science, Istanbul Yeni Yuzyil University, Istanbul, Türkiye
4 Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul, Türkiye.
Abstract
Antifungal and cytotoxic effects of thiram (THR) were investigated on Fusarium graminearum PH-1 and F. culmorum FcUK99, and mouse fibroblast cell line L929, respectively. THR’s minimum inhibitory concentration (MIC) against PH-1 and FcUK99 was 165 μg/mL. The MIC25, MIC50, and MIC75 significantly decreased the linear growth rates (LGRs) of both strains (P<0.0001) and reduced cell viability by 18.30%, 32.06%, and 45.45% in PH-1 and 33.53%, 51.68%, and 76.1% in FcUK99, respectively. The dose-dependent increase in intense green fluorescence observed in both Fusarium strains after 2',7'-dichlorodihydrofluorescein diacetate staining, indicated oxidative stress. The apoptosis-like process was visualized through the increase in orange fluorescence emissions with acridine orange/ethidium bromide dual staining. THR reduced deoxynivalenol (DON) and 15-ADON production in PH-1 while DON and 3-ADON production in FcUK99. THR doses (0.32-165 μg/mL corresponding to 1.34-686.24 μM) decreased cell viability of the L929 cells by 4.56% (P<0.0001)-97.95% (P>0.05) at 24th hour and from 4.88% (P>0.05)-88.68% (P<0.0001) at 48th hour. The MIC did not significantly affect (P> 0.05) the germination rates of wheat seeds, root and plantlet lengths, and water loss. Although THR decreased the growth and mycotoxin production of phytopathogens and didn’t adversely affect plant development, because of its inhibitory effect on the cell line, it must be evaluated as a potential risk to public health, and its utilization ought to be regulated through legislation.

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1.Akpinar, R., Arslan, M., Teker, T., Shikhaliyeva, I., Albayrak, G., Ozkorucuklu, S.P. 2025. Evaluation of B-type Trichothecene Production by Fusarium graminearum and F. culmorum strains. J Food Compos Anal., 148.
2.Albayrak, G., Yörük, E., Teker, T. and Sefer, Ö. 2023. Investigation of antifungal activities of myrcene on Fusarium reference strains. Arch. Microbiol., 205 (3):82.
3.Alisaac, E. and Mahlein, A.K. 2023. Fusarium Head Blight on Wheat: Biology, Modern Detection and Diagnosis and Integrated Disease Management. Toxins, 15 (3):192.
4.Becher, R., Hettwer, U., Karlovsky, P., Deising, H.B. and Wirsel, S.G. 2010. Adaptation of Fusarium graminearum to tebuconazole yielded descendants diverging for levels of fitness, fungicide resistance, virulence, and mycotoxin production. Phytopathol., 100 (5):444-53.
5.Berridge, M.V., Herst, P.M. and Tan, A.S. 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev., 11:127-152.  
6.Bouaicha, O., Laraba, I. and Boureghda, H. 2022. Identification, in vitro growth and pathogenicity of Microdochium spp. associated with wheat crown rot in Algeria. Plant Pathol. J., 104 (4):1431-1442.
7.Cargill: Cargill World Mycotoxin Report Feeding Intelligence. 2022. https://www.cargill.com/2022-cargill-world-mycotoxin-report. Accessed 18 May 2025
8.Cereser, C., Boget, S., Parvaz, P. and Revol, A. 2001. An evaluation of thiram toxicity on cultured human skin fibroblasts. Toxicol., 162 (2):89-101.  
9.Cinar, A. and Onbaşı, E. 2020. Mycotoxins: The Hidden Danger in Foods. IntechOpen, 1-21.  
10.  Djekoun, M., Berrebbah, H., Saib, A. and Djebar, M.R. 2016. Determination of median effective inhibitory concentration of three fungicides widely used for treatment of wheat on the target pest Fusarium sp. Res. J. Environ. Toxicol., 10:109-114.  
11.  DSM: World Mycotoxin Survey 2022 Report. 2022. https://www.dsm.com/anh/news/downloads/whitepapers-and-reports/dsm-world-mycotoxin-survey-2022-report.html. Accessed 18 May 2025
12.  Duan, Y., Lu, F., Zhou, Z., Zhao, H., Zhang, J., Mao, Y., Li, M., Wang, J. and Zhou, M. 2020. Quinone outside inhibitors affect DON biosynthesis, mitochondrial structure and toxisome formation in Fusarium graminearum. J. Hazard Mater., 398:122908.
13.  Ergin, N., Kulan, E. G., & Kaya, M. D. (2021). The effects of fungicidal seed treatments on seed germination, mean germination time and seedling growth in safflower (Carthamus tinctorius L.). Selcuk Journal of Agriculture and Food Sciences, 35(2), 139-143.
14.  Eskola, M., Kos, G., Elliott, C. T., Hajšlová, J., Mayar, S., and Krska, R. 2020. Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited ‘FAO estimate’of 25%. Crit. Rev. Food Sci. 60(16), 2773-2789.
15.  FAO. 1977. Perspective on mycotoxins Food and Agriculture Organization of the United Nations, Rome. Accessed 05 February 2026
16.  Fravel, D.R., Deahl, K.L. and Stommel, J.R. 2005. Compatibility of the biocontrol fungus Fusarium oxysporum strain CS-20 with selected fungicides. Biol. Control, 34 (2):165-169.  
17.  Freiberg, J.A., Ludwig, M.P. and Girotto, E. 2021. Does seed treatment affect wheat yield components?. Cad. Ciên. Agrá., 13:1-5.
18.  Ghafourian, M., Tamri, P. and Hemmati, A. 2015. Enhancement of human skin fibroblasts proliferation as a result of treating with quince seed mucilage. Jundishapur J. Nat. Pharm. Prod., 10 (1):e18820.
19.  Ghasemian, E., Naghoni, A., Tabaraie, B. and Tabaraie, T. 2012. In vitro susceptibility of filamentous fungi to copper nanoparticles assessed by rapid XTT colorimetry and agar dilution method. J. Mycol. Med., 22 (4):322-328.  
20.  Ghimire, B., Sapkota, S., Bahri, B.A., Martinez-Espinoza, A.D., Buck, J.W. and Mergoum, M. 2020. Fusarium Head Blight and Rust Diseases in Soft Red Winter Wheat in the Southeast United States: State of the Art, Challenges and Future Perspective for Breeding. Front. Plant Sci., 11:1080.
21.  Grosicka, E., Sadurska, B., Szumiło, M., Grzela, T., Łazarczyk, P., Niderla-Bielińska, J. and Rahden-Staroń, I. 2005. Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts. Int. Immunopharmacol., 5 (13-14):1945-1956.
22.  Horsfall, J.G. 1956. The Principles of fungicidal actions. USA: Massachusetts and Hafner Publishing Company, p. 279.  
23.  Jamdagni, P., Rana, J.S. and Khatri, P. 2018. Comparative study of antifungal effect of green and chemically synthesised silver nanoparticles in combination with carbendazim, mancozeb, and thiram. IET Nanobiotechnol., 12 (8):1102-1107.  
24.  Jamil, A. and Ashraf, S. 2020. Utilization of chemical fungicides in managing the wilt disease of chickpea caused by Fusarium oxysporum f. sp. cicero. Arch. Phytopathol. Plant Prot., 53 (17-18):876-898.  
25.  Lewis, R.E., Wiederhold, N.P. and Klepser, M.E. 2005. In vitro pharmacodynamics of amphotericin B, itraconazole, and voriconazole against Aspergillus, Fusarium, and Scedosporium spp. Antimicrob. Agents Ch., 49 (3):945-951.  
26.  Liu, K., Li, Y., Iqbal, M., Tang, Z. and Zhang, H. 2022. Thiram exposure in environment: A critical review on cytotoxicity. Chemosphere, 295:133928.
27.  Meletiadis, J., Mouton, J.W., Meis, J.F., Bouman, B.A., Donnelly, J.P., Verweij, P.E. and EUROFUNG Network 2001. Colorimetric assay for antifungal susceptibility testing of Aspergillus species. J. Clin. Microbiol., 39 (9):3402-3408.
28.  Montoya, A.M., Rodríguez-Grimaldo, J.E., López-Jácome, L.E., Bonifaz, A., Enríquez-Domínguez, E., Castañón-Olivares, L.R., Charles-Niño, C.L., Rodríguez-Rodríguez, A., Treviño-Rangela, R.J., Rojas, O.C. and González, G.M. 2024. Species distribution and antifungal susceptibility profiles of clinical and environmental Fusarium isolates from Mexico: A multicenter study. Mycol., 116(2):258–266.
29.  Osman, A.G., Sherif, A.M., Elhussein, A.A. and Mohamed, A.T. 2012. Sensitivity of some nitrogen fixers and the target pest Fusarium oxysporum to fungicide thiram. Interdiscip. Toxicol., 5 (1):25-29.
30.  Oufensou, S., Dessì, A., Dallocchio, R., Balmas, V., Azara, E., Carta, P., Migheli, Q. and Delogu, G. 2021. Molecular Docking and Comparative Inhibitory Efficacy of Naturally Occurring Compounds on Vegetative Growth and Deoxynivalenol Biosynthesis in Fusarium culmorum. Toxins, 13 (11):759.
31.  PPDB. 2025. https://sitem.herts.ac.uk/aeru/ppdb/. Accessed 18 May 2025
32.  PubChem. 2025. https://pubchem.ncbi.nlm.nih.gov/. Accessed 18 May 2025
33.  Radko, L., Cybulski, W. and Rzeski, W.  2017. The protective effects of silybin on the cytotoxicity of thiram in human, rat and chicken cell cultures. Pestic. Biochem. Physiol., 143:154-160.
34.  Renvoize, C., Biola, A., Pallardy, M. and Breard, J. 1998. Apoptosis: identification of dying cells. Cell Biol. Toxicol., 14:111-120.
35.  Ribble, D., Goldstein, N.B., Norris, D.A. and Shellman, Y.G. 2005. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol., 5:1-7.  
36.  Rose, S., Parker, M. and Punja, Z.K. 2003. Efficacy of Biological and Chemical Treatments for Control of Fusarium Root and Stem Rot on Greenhouse Cucumber. Plant Dis., 87 (12):1462-1470. 
37.  Sankowska, M., Gajek, A., Celiński, M. and Sałasińska, K. 2017. Determination of gaseous products of thermal degradation of thiram. J. Therm. Anal. Calorim., 128:1639-1647.
38.  Sharma, V.K., Aulakh, J.S. and Malik, A.K. 2003. Thiram: degradation, applications and analytical methods. JEM, 5 (5):717-723.
39.  Shikhaliyeva, I., Teker, T. and Albayrak, G. 2020. Masked Mycotoxins of Deoxynivalenol and Zearalenone- Unpredicted Toxicity. Biomed. J. Sci. Tech. Res., 29: 22288-22293.
40.  Shivankar, S.K., Shivankar, R.S. and Nagone, A.H. 2000. Effect of fungicidal seed treatment on the germination, shoot and root length of wheat seed infected by black point disease. ASD, 20 (3):205-206.
41.  Singh, M., McKenzie, K. and Ma, X. 2017. Effect of dimethyl sulfoxide on in vitro proliferation of skin fibroblast cells. J. Biotech. Res., 8:78.
42.  Smith, J.E., Solomons, G., Lewis, C. and Anderson, J.G. 1995. Role of mycotoxins in human and animal nutrition and health. Nat. Toxins, 3 (4):187-192.  
43.  Sobrova, P., Adam, V., Vasatkova, A., Beklova, M., Zeman, L. and Kizek, R. 2010. Deoxynivalenol and its toxicity. Interdiscip. Toxicol., 3 (3):94-99. 
44.  Streit, E., Naehrer, K., Rodrigues, I. and Schatzmayr, G. 2013. Mycotoxin occurrence in feed and feed raw materials worldwide: long-term analysis with special focus on Europe and Asia. J. Sci. Food Agric., 93 (12):2892-2899.
45.  Su, H., Hu, C., Cao, B., Qu, X., Guan, P., Mu, Y., Han, L. and Huang, X. 2020. A semisynthetic borrelidin analogue BN-3b exerts potent antifungal activity against Candida albicans through ROS-mediated oxidative damage. Sci. Rep., 10 (1):5081.
46.  Teker, T., Khalid, S.A., Yörük, E. and Albayrak, G. 2024. Physiological, genetic and transcriptional characterization of Fusarium graminearum isolates. Sigma J. Eng. Nat. Sci., 42 (2):450-458.
47.  Teker, T., Sefer, Ö., Gazdağlı, A., Yörük, E., Varol, G.İ. and Albayrak, G. 2021. α-Thujone exhibits an antifungal activity against F. graminearum by inducing oxidative stress, apoptosis, epigenetics alterations and reduced toxin synthesis. Eur. J. Plant Pathol., 160 (3):611-622.  
48.  Tobih, F.O., Bosah, B.O. and Nweke, F.U. 2015. Evaluation of the efficacy of Thiram and benomyl on radial growth, spore germination and sporulation density of Curvularia lunata and Fusarium semitectum. Int. J. Agric. Innov. Res., 4: 47-50.
49.  Wang, L., Ren, X., Guo, W., Wang, D., Han, L. and Feng, J. 2019. Oxidative Stress and Apoptosis of Gaeumannomyces graminis (Get) Induced by Carabrone. J. Agric. Food Chem., 67 (37):10448-10457.   
50.  Wang, L., Yan, Z., Zhou, H., Fan, Y., Wang, C., Zhang, J., Liao, Y. and Wu, A. 2021. Validation of LC-MS/MS Coupled with a Chiral Column for the Determination of 3- or 15-Acetyl Deoxynivalenol Mycotoxins from Fusarium graminearum in Wheat. Toxins, 13 (9):659.
51.  Wiederhold, N.P. and Lewis, R.E. 2009. Antifungal activity against Scedosporium species and novel assays to assess antifungal pharmacodynamics against filamentous fungi. Med. Mycol., 47(4):422-432.
52.  Xu, X.M., Nicholson, P., Thomsett, M.A., Simpson, D., Cooke, B.M., Doohan, F.M., Brennan, J., Monaghan, S., Moretti, A., Mule, G., Hornok, L., Beki, E., Tatnell, J., Ritieni, A. and Edwards S.G. 2008. Relationship between the fungal complex causing Fusarium head blight of wheat and environmental conditions. Phytopathol., 98 (1):69-78. 
53.  Yli-Mattila, T., Yörük, E., Abbas, A. and Teker, T. 2022. Overview on Major Mycotoxins Accumulated on Food and Feed. In: Deshmukh SK, Sridhareds KR (eds) Fungal Biotechnology: Prospects and Avenues, CRC Press, p. 310–343.
54.  Zhang, Y.J., Fan, P.S., Zhang, X., Chen, C.J. and Zhou, M.G. 2009 Quantification of Fusarium graminearum in harvested grain by real-time polymerase chain reaction to assess efficacies of fungicides on fusarium head blight, deoxynivalenol contamination, and yield of winter wheat. Phytopathol., 99 (1):95-100.
55.  Zubrod, J.P., Bundschuh, M., Arts, G., Brühl, C.A., Imfeld, G., Knäbel, A., Payraudeau, S., Rasmussen, J.J., Rohr, J., Scharmüller, A., Smalling, K., Stehle, S., Schulz, R. and Schäfer, R.B. 2019. Fungicides: an overlooked pesticide class?. Environ. Sci. Technol., 53 (7):3347-3365.
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Available Online from 11 May 2026