Abstract
Rust fungi are generally considered low-risk pathogens in terms of resistance to fungicides. However, Puccinia horiana, which causes white rust in chrysanthemum, has developed resistance to various fungicides via mutations in genes encoding the target proteins of fungicides. We investigated cytochrome b haplotypes of 15 Japanese isolates of P. horiana. Among them, two were wild-type, and the others carried L299F, L275F + L299F, or N256S + L299F amino acid substitutions. To best of our knowledge, L299F and N256S + L299F haplotypes were found for the first time in this study and isolates of each haplotype showed 22- and 222-fold higher azoxystrobin EC50 values than their wild-type counterparts in the basidiospore germination test, respectively. Further tests confirmed cross-resistance among some quinone outside inhibitor (QoI) fungicides in N256S + L299F-harboring isolates whereas L275F-harboring isolates remained sensitive to other QoIs. Our in planta tests revealed that the performance of azoxystrobin against mutant isolates was reduced even at the labeled concentration. The homology model predicted the involvement of L275 and L299 in the interaction between azoxystrobin and cytochrome b, while it was unlikely for N256. In some model organisms such as Saccharomyces cerevisiae, asparagine 256 is known to interact with glycine 137. Considering that G137R substitution confers azoxystrobin resistance in other phytopathogenic fungi, we suggest that N256S might also be responsible for azoxystrobin resistance in P. horiana. This is the first record of a substitution at cytochrome b N256 in azoxystrobin-resistant phytopathogenic fungi.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Brasseur, G., Saribas, A. S., & Daldal, F. (1996). A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex. Biochimica et Biophysica Acta, 1275, 61–69.
Chechi, A., Ivey, M. L. L., Kaufman, R. R., Bryson, K. P., & Schnabel, G. (2020). Quinone outside inhibitor-resistant Colletotrichum nymphaeae isolates from strawberry lack mutations in cytb gene. Journal of Plant Pathology, 102, 681–683.
di Rago, J. P., Coppee, J. Y., & Colson, A. M. (1989). Molecular basis for resistance to myxothiazol, mucidin (strobilurin a), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. J Biol Chem, 264, 14543–14548.
di Rago, J. P., Hermann-Le Denmat, S., Paques, F., Risler, J. L., Netter, P., & Slonimski, P. P. (1995). Genetic analysis of the folded structure of yeast mitochondrial cytochrome b by selection of intragenic second-site revertants. Journal of Molecular Biology, 248, 804–811.
Fernández-Ortuño, D., Torés, D. A., de Vicente, A., & Pérez-García, A. (2008). Mechanisms of resistance to QoI fungicides in phytopathogenic fungi. International Microbiology, 11, 1–9.
Fisher, M., & Meunier, B. (2005). Re-examination of inhibitor resistance conferred by Qo-site mutations in cytochrome b using yeast as a model system. Pest Management Science, 61, 973–978.
Fisher, M., Meunier, B., & Biagini, G. A. (2020). The cytochrome bc1 complex as an antipathogenic target. FEBS Letters, 594, 2935–2952.
Fotoukkiaii, S. M., Tan, Z., Xue, W., Wybouw, N., & Leeuwen, T. V. (2020). Identification and characterization of new mutations in mitochondrial cytochrome b that confer resistance to bifenazate and acequinocyl in the spider mite Tetranychus urticae. Pest Management Science, 76, 1154–1163.
Giessler, A., Geier, B. M., de Rago, J. P., Slonimski, P. P., & von Jagow, G. (1994). Analysis of cytochrome-b amino acid residues forming the contact face with the iron-sulfur subunit of ubiquinol: Cytochrome-c reductase in Saccharomyces cerevisiae. European Journal of Biochemistry, 222, 147–154.
Kessl, J. J., Hill, P., Lange, B. B., Meshnick, S. R., Meunier, B., & Trumpower, B. L. (2004). Molecular basis for atovaquone resistance in Pneumocystis jirovecii modeled in the cytochrome bc1 complex of Saccharomyces cerevisiae. The Journal of Biological Chemistry, 279, 2817–2824.
Korsinczky, M., Che, N., Kotecka, B., Saul, A., Rieckmann, K., & Cheng, Q. (2000). Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrobial Agents and Chemotherapy, 44, 2100–2108.
Ma, D., Jiang, J., He, L., Cui, K., Mu, W., & Liu, F. (2018). Detection and characterization of QoI-resistant Phytophthora capsici causing pepper Phytophthora blight in China. Plant Disease, 102, 1725–1732.
Matsuura, S. (2019). Does QoI (strobilurin) resistance in isolates of Puccinia horiana, the causal agent of chrysanthemum white rust, occur in western Japan? J Plant Dis Prot, 126, 469–473.
Matsuzaki, Y., Harada, T., & Iwahashi, F. (2021). Amino acid substitutions responsible for different QoI and SDHI sensitivity patterns in Puccinia horiana, the causal agent of chrysanthemum white rust. Plant Pathology, 70, 377–386.
Matsuzaki, Y., Sakaguchi, H. (2011). Plant disease control composition and method of controlling plant disease. World Patent Application, WO2011162397A1.
Ortiz, A. R., Pisabarro, M. T., Gago, F., & Wade, R. C. (1995). Prediction of drug binding affinities by comparative binding energy analysis. Journal of Medicinal Chemistry, 38, 2681–2695.
Sierotzki, H. (2015). Respiration inhibitors: Complex III. In H. Ishii & D. W. Hollomon (Eds.), Fungicide resistance in plant pathogens (pp. 119–143). Springer Japan.
Sierotzki, H., Frey, R., Wullschleger, J., Palermo, S., Karlin, S., Godwin, J., & Gisi, U. (2007). Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance. Pest Management Science, 63, 225–233.
Standish, J. R., Brenneman, T. B., & Stevenson, K. L. (2019). Quantifying the effects of a G137S substitution in the cytochrome bc1 of Venturia effusa on azoxystrobin sensitivity using a detached leaf assay. Plant Disease, 103, 841–845.
Sugimoto, N., & Osakabe, M. (2019). Mechanism of acequinocyl resistance and cross-resistance to bifenazate in the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae). Applied Entomology and Zoology, 54, 421–427.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
Acknowledgments
This study was funded by Sumitomo Chemical Co., Ltd. All authors are employees of Sumitomo Chemical Co., Ltd. We would like to thank Dr. Akiho Harada for sharing the CWR1 isolate. We also would like to thank Dr. Yoshinao Sada for cooperation during the collection of P. horiana in Hyogo prefecture.
Availability of data and material
The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Yuichi Matsuzaki, Toshiyuki Harada, and Fukumatsu Iwahashi. The first draft of the manuscript was written by Yuichi Matsuzaki. Toshiyuki Harada and Fukumatsu Iwahashi commented on the previous versions of the manuscript. All authors have read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
All authors are employees of Sumitomo Chemical Co. Ltd.
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Code availability
Not applicable.
Rights and permissions
About this article
Cite this article
Matsuzaki, Y., Harada, T. & Iwahashi, F. New cytochrome b haplotypes, harboring L299F or N256S + L299F substitutions, were found in azoxystrobin-resistant Puccinia horiana, the causal agent of chrysanthemum white rust. Eur J Plant Pathol 160, 963–972 (2021). https://doi.org/10.1007/s10658-021-02299-4
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10658-021-02299-4