Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC2 and FfSdhD

doi:10.1016/j.jia.2024.01.004

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Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC₂ and FfSdhD

Yang Sun^{1, 3, 4, 5*}, Yu Liu^{1, 3, 4, 5*}, Li Zhou^{1, 3, 4, 5}, Xinyan Liu^{1, 3, 4, 5}, Kun Wang^{1, 3, 4, 5}, Xing Chen^{1, 3, 4, 5}, Chuanqing Zhang^2#, Yu Chen^{1, 3, 4, 5#}

¹ School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

² Department of Plant Pathology, Zhejiang Agriculture and Forest University, Hangzhou 311300, China

³ Key Laboratory of Integrated Crop Pest Management of Anhui Province, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

⁴Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

⁵ Anhui Province Engineering Laboratory for Green Pesticide Development and Application, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

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摘要水稻恶苗病是一种严重危害水稻生产的种传病害，其优势病原菌为藤仓镰孢菌（Fusarium fujikuroi）。为明确新型琥珀酸脱氢酶抑制剂类杀菌剂三氟吡啶胺用于防治水稻恶苗病的应用潜力，评估抗性风险并解析病原抗药性分子机制，本研究采用菌丝生长速率法和孢子萌发抑制法测定了72株藤仓镰孢菌对三氟吡啶胺的敏感性，并建立敏感性基线，继而探究药剂对病原菌形态学和相关生理生化指标的影响，并测定在活体盆栽条件下药剂防效，评估其用于恶苗病防治潜力。进一步采用药剂驯化获得抗性突变体并评估抗性风险及与常用药剂交互抗性，比对分析抗性菌株与亲本菌株Sdh基因序列，鉴定抗性突变位点，最终采用同源替换与分子对接技术揭示抗药性分子机制。研究发现三氟吡啶胺对藤仓镰孢菌具有显著生物活性，生长速率法和孢子萌发法测定EC₅₀分别为0.0410 ± 0.0470 μg mL^-1和0.0038 ± 0.0015 μg mL^-1，且敏感性频率呈正态分布。三氟吡啶胺可破坏病原菌菌丝和孢子形态，提升细胞膜透性，降低病菌的毒素合成，并抑制琥珀酸脱氢酶活性。此外，活体盆栽条件下三氟吡啶胺对水稻恶苗病的保护作用显著优于对照药剂氰烯菌酯和嘧菌酯。以上结果表明三氟吡啶胺可作为防治水稻恶苗病的理想候选药剂。通过药剂驯化共获得6株抗性突变体菌株，突变频率为3.75 × 10^-4，抗性菌株生物适合度均显著低于其亲本菌株，且对SDHI类杀菌剂氟唑菌酰羟胺和氟唑菌苯胺存在交互抗性，而与非同类药剂氰烯菌酯、咯菌腈、咪鲜胺和嘧菌酯等无交互抗性，表明藤仓镰孢菌对三氟吡啶胺存在中等抗性风险。抗性机制研究发现SdhB亚基H248D突变可赋予病原菌中等水平抗药性（20< RF <100）；SdhC₂亚基A83V突变可赋予高水平抗药性（RF >100）；SdhD亚基S106F和E166K突变赋予菌株低水平抗性（RF <10）。研究结果可为三氟吡啶胺在水稻恶苗病防治上的科学使用及抗药性治理提供理论依据。

Abstract Rice bakanae disease (RBD) is a devastating plant disease caused by Fusarium fujikuroi. This study aimed to evaluate the potential of cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI), to control RBD, and determine the risk and mechanism of resistance to cyclobutrifluram in F. fujikuroi. In vitro experiments showed that cyclobutrifluram significantly inhibited mycelial growth and spore germination, and altered the morphology of mycelia and conidia. Treatment with cyclobutrifluram significantly decreased mycotoxin production and increased cell membrane permeability in F. fujikuroi. The baseline sensitivity of 72 F. fujikuroi isolates to cyclobutrifluram was determined using mycelial growth and spore germination inhibition assays, which revealed EC₅₀ values of 0.0114 – 0.1304 μg mL^-1 and 0.0012 – 0.016 μg mL^-1, with mean EC₅₀ values of 0.0410 ± 0.0470 μg mL^-1 and 0.0038 ± 0.0015 μg mL^-1, respectively. Pot experiments demonstrated that the protective effect of cyclobutrifluram against F. fujikuroi was more significant than that of phenamacril and azoxystrobin, indicating that cyclobutrifluram is a promising antifungal agent for the control of RBD. Six cyclobutrifluram-resistant mutants of F. fujikuroi were obtained via fungicide adaptation. Moreover, these mutants exhibited weaker fitness than their parental isolate and positive cross-resistance with other SDHI fungicides, including pydiflumetofen and penflufen; however, no cross-resistance was detected with other classes of fungicides, including phenamacril, fludioxonil, prochloraz, or azoxystrobin. These results indicated that the resistance risk of F. fujikuroi to cyclobutrifluram might be moderate. Sequencing analysis revealed that mutations, including H248D in FfSdhB, A83V in FfSdhC₂, and S106F and E166K in FfSdhD, contributed to resistance, which was confirmed by molecular docking and homologous replacement experiments. The results suggest a high potential for cyclobutrifluram to control RBD and a moderate resistance risk of F. fujikuroi to cyclobutrifluram, which are meaningful findings for the scientific application of cyclobutrifluram.

Keywords: Fusarium fujikuroi rice bakanae disease cyclobutrifluram succinate dehydrogenase inhibitors resistance risk and mechanisms

Online: 25 January 2024

About author: #Correspondence Yu Chen, E-mail: chenyu66891@sina.com * Yang Sun and Yu Liu share joint first authorship for this work.

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Yang Sun, Yu Liu, Li Zhou, Xinyan Liu, Kun Wang, Xing Chen, Chuanqing Zhang, Yu Chen. 2024. Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC₂ and FfSdhD. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.01.004

Avenot H, Sellam A, Karaoglanidis G, Michailides T. 2008. Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternata from California pistachio. Phytopathology, 98, 736-742.

Avenot H F, Michailides T J. 2010. Progress in understanding molecular mechanisms and evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in phytopathogenic fungi. Crop Protection, 29, 643-651.

Avenot H F, Thomas A, Gitaitis R D, Langston J D B, Stevenson K L. 2012. Molecular characterization of boscalid and penthiopyrad‐resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram. Pest Management Science, 68, 645-651.

Bai Y, Gu C Y, Pan R, Abid M, Zang H Y, Yang X, Tan G J, Chen Y. 2021. Activity of a novel succinate dehydrogenase inhibitor fungicide pydiflumetofen against Fusarium fujikuroi causing rice bakanae disease. Plant Disease, 105, 3208-3217.

Chen L, Zhu S, Lu X, Pang Z, Cai M, Liu X. 2012. Assessing the risk that Phytophthora melonis can develop a point mutation (V1109L) in CesA3 conferring resistance to carboxylic acid amide fungicides. The Public Library of Science, e42069.

Chen W C, Wei L L, Zhao W C, Wang B R, Zheng H H, Zhang P C, Lou T C, Duan Y B, Hou Y P, Zhou M G, Chen C J. 2021. Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum. Pest Management Science, 77, 538-547.

Chen Z, Gao T, Liang S, Liu K, Zhou M, Chen C. 2014. Molecular mechanism of resistance of Fusarium fujikuroi to benzimidazole fungicides. Fems Microbiology Letters, 357, 77-84.

Cook N M, Chng S, Woodman T L, Warren R, Oliver R P, Saunders D G. 2021. High frequency of fungicide resistance‐associated mutations in the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici. Pest Management Science, 77, 3358-3371.

Cpn A, Mh C, As C, Lbg A, Al C, Gs C, Emdp B, Am C, Lhp A. 2020. Fusarium fujikuroi species complex in Brazilian rice: Unveiling increased phylogenetic diversity and toxigenic potential. International Journal of Food Microbiology, 330, 108667.

Di Pietro A, García‐Maceira F I, Méglecz E, et al. 2001. A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis. Molecular Microbiology, 39(5), 1140-1152.

Dong T, Qiao S, Xu J, Shi J, Qiu J, Ma G. 2023. Effect of abiotic conditions on growth, mycotoxin production, and gene expression by Fusarium fujikuroi species complex strains from maize. Toxins, 15, 260.

Gao X, Peng Q, Yuan K, Li Y, Shi M, Miao J, Liu X. 2022. Monitoring and characterization of prochloraz resistance in Fusarium fujikuroi in China. Pesticide Biochemistry and Physiology, 187, 105189.

Gregorio G, Senadhira D, Mendoza R, Manigbas N, Roxas J, Guerta C. 2002. Progress in breeding for salinity tolerance and associated abiotic stresses in rice. Field Crops Research, 76, 91-101.

Herron D A, Wingfield M J, Wingfield B D, Rodas C, Marincowitz S, Steenkamp E T. 2015. Novel taxa in the Fusarium fujikuroi species complex from Pinus spp. Studies in Mycology, 80, 131-150.

Horsefield R, Yankovskaya V, Sexton G, Whittingham W, Shiomi K, ōmura S, Byrne B, Cecchini G, Iwata S. 2006. Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction. Journal of Biological Chemistry, 281, 7309.

Hou Y P, Qu X P, Mao X W, Kuang J, Duan Y B, Song X s, Wang J X, Chen C J, Zhou M G. 2018. Resistance mechanism of Fusarium fujikuroi to phenamacril in the field. Pest Management Science, 74, 607-616.

Li M, Li T, Duan Y, Yang Y, Wu J, Zhao D, Xiao X, Pan X, Chen W, Wang J. 2018. Evaluation of phenamacril and ipconazole for control of rice bakanae disease caused by Fusarium fujikuroi. Plant Disease, 102, 1234-1239.

Liu Y, Ma T, Dong Y, Mao C, Wu J, Zhang C. 2022. Bioactivity of mefentrifluconazole against different Fusarium spp. Pesticide Biochemistry and Physiology, 186, 105169.

Liu Y, Sun Y, Bai Y, Cheng X, Li H, Chen X, Chen Y. 2023. Study on mechanisms of resistance to SDHI fungicide pydiflumetofen in Fusarium fujikuroi. Journal of Agricultural and Food Chemistry, 71(39), 14330-14341.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻^ΔΔCT method. Methods, 25, 402-408.

Mao X, Wu Z, Bi C, Wang J, Zhao F, Gao J, Hou Y, Zhou M. 2020. Molecular and biochemical characterization of pydiflumetofen-resistant mutants of Didymella bryoniae. Journal of Agricultural and Food Chemistry, 68, 9120-9130.

Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. 2022. ColabFold: making protein folding accessible to all. Nature Methods, 19, 679-682.

Miyamoto T, Ishii H, Stammler G, Koch A, Ogawara T, Tomita Y, Fountaine J, Ushio S, Seko T, Kobori S. 2010. Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid. Plant Pathology, 59, 873-881.

Munkvold G P, Proctor R H, Moretti A. 2021. Mycotoxin production in Fusarium according to contemporary species concepts. Annual Review of Phytopathology, 59, 373-402.

Muthayya S, Sugimoto J D, Montgomery S, Maberly G F. 2014. An overview of global rice production, supply, trade, and consumption. Annals of the New York Academy of Sciences, 1324, 7-14.

Niehaus E M, von Bargen K W, Espino J J, Pfannmüller A, Humpf H U, Tudzynski B. 2014. Characterization of the fusaric acid gene cluster in Fusarium fujikuroi. Applied Microbiology and Biotechnology, 98, 1749-1762.

Owen W J, Yao C, Myung K, Kemmitt G, Leader A, Meyer K G, Bowling A J, Slanec T, Kramer V J. 2017. Biological characterization of fenpicoxamid, a new fungicide with utility in cereals and other crops. Pest Management Science, 73, 2005-2016.

Qiu J, Lu Y, He D, Li Y W, Ji F, Xu J, Shi J. 2020. Fusarium fujikuroi species complex associated with rice, maize, and soybean from Jiangsu province, China: phylogenetic, pathogenic, and toxigenic analysis. Plant Disease, 104, 2193-2201.

Shao W, Wang J, Wang H, Wen Z, Liu C, Zhang Y, Zhao Y, Ma Z. 2022. Fusarium graminearum FgSdhC₁ point mutation A78V confers resistance to the succinate dehydrogenase inhibitor pydiflumetofen. Pest Management Science, 78, 1780-1788.

Sierotzki H, Scalliet G. 2013. A review of current knowledge of resistance aspects for the next-generation succinate dehydrogenase inhibitor fungicides. Phytopathology, 103, 880-887.

Simões K, Hawlik A, Rehfus A, Gava F, Stammler G. 2018. First detection of a SDH variant with reduced SDHI sensitivity in Phakopsora pachyrhizi. Journal of Plant Diseases and Protection, 125, 21-26.

Singh R, Sunder S. 2012. Foot rot and bakanae of rice: an overview. Rev. Plant Pathol, 5, 565-604.

Skinner W, Bailey A, Renwick A, Keon J, Gurr S, Hargreaves J. 1998. A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Current Genetics, 34, 393-398.

Stammler G, Rehfus A, Prochnow J, Bryson R, Strobel D. 2014. New findings on the development of insensitive isolates of Pyrenophora teres towards SDHI fungicides. Julius-Kühn-Archiv, 447, 568.

Steinhauer D, Salat M, Frey R, Mosbach A, Luksch T, Balmer D, Hansen R, Widdison S, Logan G, Dietrich R A. 2019. A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici. Plos Pathogens, 15, e1007780.

Studt L, Schmidt F, Jahn L, Sieber C, Connolly L, Niehaus E M, Freitag M, Humpf H U, Tudzynski B. 2013. Two histone deacetylases, FfHda₁ and FfHda₂, are important for Fusarium fujikuroi secondary metabolism and virulence. Applied and Environmental Microbiology, 79, 7719-7734.

Studt L, Wiemann P, Kleigrewe K, Humpf H U, Tudzynski B. 2012. Biosynthesis of fusarubins accounts for pigmentation of Fusarium fujikuroi perithecia. Applied and Environmental Microbiology, 78, 4468-4480.

Sultana S, Kitajima M, Kobayashi H, Nakagawa H, Shimizu M, Kageyama K, Suga H. 2019. A natural variation of fumonisin gene cluster associated with fumonisin production difference in Fusarium fujikuroi. Toxins, 11, 200.

Sun J, Pang C, Cheng X, Yang B, Jin B, Jin L, Qi Y, Sun Y, Chen X, Liu W. 2023. Investigation of the antifungal activity of the dicarboximide fungicide iprodione against Bipolaris maydis. Pesticide Biochemistry and Physiology, 190, 105319.

Sun Y, Shi H, Mao C, Wu J, Zhang C. 2021. Activity of a SDHI fungicide penflufen and the characterization of natural-resistance in Fusarium fujikuroi. Pesticide Biochemistry and Physiology, 179, 104960.

Sun Y, Wang Y, Xie Z, Guo E, Han L, Zhang X, Feng J. 2017. Activity and biochemical characteristics of plant extract cuminic acid against Sclerotinia sclerotiorum. Crop Protection, 101, 76-83.

Tsukamoto M, Nakamura T, Kimura H, Nakayama H. 2021. Synthesis and application of trifluoromethylpyridines as a key structural motif in active agrochemical and pharmaceutical ingredients. Journal of Pesticide Science, 46, 125-142.

Veloukas T, Karaoglanidis G S. 2012. Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity. Pest Management Science, 68, 858-864.

Wang H, Ke X, Jia R, Huang L, Liu Z, Zheng Y. 2023. Gibberellic acid overproduction in Fusarium fujikuroi using regulatory modification and transcription analysis. Applied Microbiology and Biotechnology, 107, 3071-3084.

Wang Q, Mao Y, Li S, Li T, Wang J, Zhou M, Duan Y. 2022. Molecular mechanism of Sclerotinia sclerotiorum resistance to succinate dehydrogenase inhibitor fungicides. Journal of Agricultural and Food Chemistry, 70, 7039-7048.

Wang Y, Duan Y, Wang J, Zhou M. 2015a. A new point mutation in the iron–sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum. Molecular Plant Pathology, 16, 653-661.

Wang Y, Duan Y B, Zhou M G. 2015b. Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum. Plant Pathology, 64.

Wu J, Xue Z, Miao J, Zhang F, Gao X, Liu X. 2020. Sensitivity of different developmental stages and resistance risk assessment of Phytophthora capsici to fluopicolide in China. Frontiers in Microbiology, 11, 185.

Wulff E, Sorensen J, Lubeck M, Nielsen K, Thrane U, Torp J. 2010. Fusarium spp. associated with rice Bakanae: ecology, genetic diversity, pathogenicity and toxigenicity. Environmental Microbiology, 12, 649-657.

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