Pfcyp51 exclusively determines reduced sensitivity to 14α-demethylase inhibitor fungicides in the banana black Sigatoka pathogen Pseudocercospora fijiensis


Autoři: Pablo Chong aff001;  Aikaterini-Eleni Vichou aff002;  Henk J. Schouten aff002;  Harold J. G. Meijer aff002;  Rafael E. Arango Isaza aff003;  Gert H. J. Kema aff002
Působiště autorů: ESPOL Polythecnic University, Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador, Laboratorio de Fitopatología, Guayaquil, Ecuador aff001;  Laboratory of Phytopathology, Wageningen University and Research, The Netherlands, Wageningen, the Netherlands aff002;  Escuela de Biociencias, Faculta de Ciencias, Universidad Nacional de Colombia -Sede Medellín (UNALMED), Medellín, Colombia aff003;  Unidad de biotecnología (UNALMED-CIB), Corporación para Investigaciones Biológicas, Medellín, Colombia aff004
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223858

Souhrn

The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Klíčová slova:

Bananas – Fungal genetics – Fungicides – Gene mapping – Genetic linkage – Leaves – Linkage mapping – Physical mapping


Zdroje

1. Chong P, Carr C, Murillo G, Guzmán M, Sandoval J, Kema G. An historical treatise and critical review of black Sigatoka control in banana production. In: Chong P, editor. The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis. P.hD. Wageningen University: Wageningen 2016. p. 23–58.

2. Beaglehole R, Irwin A, Prentice T. The world health report 2003: shaping the future. Geneva, Switzerland: World Health Organization; 2003.

3. Marín DH, Romero RA, Guzmán M, Sutton TB. Black Sigatoka: An increasing threat to banana cultivation. Plant Disease. 2003;87 (3):208–22. doi: 10.1094/PDIS.2003.87.3.208 30812750

4. Guzmán M, Orozco M, Pérez L. Sigatoka leaf spot diseases of bananas: dispersion, impact and evolution of management strategies in Latin America-Caribbean region. XX Reunião Internacional da Associação para a Cooperação em Pesquisa e Desenvolvimento Integral das Musáceas (Bananas e Plátanos) 9 a 13 de setembro de 2013—Fortaleza, CE, Brasil. 2013.

5. Cañas GP, Velasquez MJA, Florez JMR, Rodríguez P, Moreno CX, Arango R. Analysis of the CYP51 gene and encoded protein in propiconazole-resistant isolates of Mycosphaerella fijiensis. Pest management science. 2009;65(8):892–9. Epub 2009/05/07. doi: 10.1002/ps.1770 19418481.

6. Lepesheva GI, Waterman MR. CYP51-the omnipotent P450. Molecular and cellular endocrinology. 2004;215(1–2):165–70. Epub 2004/03/18. doi: 10.1016/j.mce.2003.11.016 15026190.

7. Churchill AC. Mycosphaerella fijiensis, the black leaf streak pathogen of banana: progress towards understanding pathogen biology and detection, disease development, and the challenges of control. Molecular plant pathology. 2011;12(4):307–28. Epub 2011/04/02. doi: 10.1111/j.1364-3703.2010.00672.x 21453427.

8. Chong P, Arango R, Stergiopoulus I, Guzmán M, Crous P, Silva Gd, et al. Analysis of azole fungicide resistance in Mycosphaerella fijiensis, causal agent of black Sigatoka. Proceedings of the 16th International Reinhardsbrunn Symposium on Modern Fungicides and Antifungal Compounds 2010. p. 217–22.

9. Bolton MD, Ebert MK, Faino L, Rivera-Varas V, de Jonge R, Van de Peer Y, et al. RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction. Fungal genetics and biology: FG & B. 2016;92:1–13. doi: 10.1016/j.fgb.2016.04.003 27112724.

10. Diaz-Trujillo C, Chong P, Stergiopoulos I, Cordovez V, Guzman M, De Wit P, et al. A new mechanism for reduced sensitivity to demethylation-inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis. Mol Plant Pathol. 2018;19(6):1491–503. doi: 10.1111/mpp.12637 29105293.

11. Arango RE, Diaz C, Dhillon B, Aerts A, Carlier J, Crane CF, et al. Combating a global threat to a clonal crop: banana black Sigatoka pathogen Pseudocercospora fijiensis (synonym Mycosphaerella fijiensis) genomes reveal clues for disease control. PloS Genetics 2016;12(8):e1005876. doi: 10.1371/journal.pgen.1005876 27512984

12. Dyer PS, Hansen J, Delaney A, Lucas JA. Genetic control of resistance to the sterol 14a-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Applied and Enviromental Microbiology. 2000;66(11):4599–604.

13. Cowen LE. The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nature reviews Microbiology. 2008;6(3):187–98. Epub 2008/02/05. doi: 10.1038/nrmicro1835 18246082.

14. Cools HJ, Hawkins NJ, Fraaije BA. Constraints on the evolution of azole resistance in plant pathogenic fungi. Plant Pathology. 2013;62:36–42. doi: 10.1111/ppa.12128

15. Chong P, Ngando JE, Arango Isaza RE, Keizer P, Stergiopoulos I, Seidl MF, et al. Global analysis of reduced sensitivity to azole fungicides in the banana black Sigatoka pathogen Pseudocercospora fijiensis. In: Chong P, editor. The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis Ph.D. Wageningen University: Wageningen 2016. p. 75.

16. Conde-Ferraez L, Waalwijk C, Canto-Canche BB, Kema GH, Crous PW, James AC, et al. Isolation and characterization of the mating type locus of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana. Molecular plant pathology. 2007;8(1):111–20. Epub 2007/01/01. doi: 10.1111/j.1364-3703.2006.00376.x 20507483.

17. Kema GHJ. Sequencing the major Mycosphaerella pathogens of wheat and banana. Acta Hort. 2009;828:147–52.

18. Arango Isaza RE, Diaz-Trujillo C, Dhillon B, Aerts A, Carlier J, Crane CF, et al. Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control. PLoS Genet. 2016;12(8):e1005876. doi: 10.1371/journal.pgen.1005876 27512984; PubMed Central PMCID: PMC4981457.

19. Leroux P, Walker AS. Multiple mechanisms account for resistance to sterol 14a-demethylation inhibitors in field isolates of Mycosphaerella graminicola. Pest management science. 2011;67(1):44–59. doi: 10.1002/ps.2028 20949586.

20. Becher R, Wirsel SG. Fungal cytochrome P450 sterol 14a-demethylase (CYP51) and azole resistance in plant and human pathogens. Applied Microbiology and Biotechnology. 2012;95(4):825–40. Epub 2012/06/12. doi: 10.1007/s00253-012-4195-9 22684327.

21. Hollomon DW. Fungicide resistance: Facing the challenge. Plant Protection Science. 2015;51(4):170–6. doi: 10.17221/42/2015-PPS

22. Villani SM, Hulvey J, Hily JM, Cox KD. Overexpression of the CYP51A1 Gene and Repeated Elements are Associated with Differential Sensitivity to DMI Fungicides in Venturia inaequalis. Phytopathology. 2016;106(6):562–71. doi: 10.1094/PHYTO-10-15-0254-R 26863444.

23. Verweij PE, Kema GHJ, B BZ, Melchers WJ. Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus. Pest management science. 2013;69(2):165–70. Epub 2012/10/31. doi: 10.1002/ps.3390 23109245.

24. Eddouzi J, Parker JE, Vale-Silva LA, Coste A, Ischer F, Kelly S, et al. Molecular mechanisms of drug resistance in clinical Candida species isolated from Tunisian hospitals. Antimicrobial agents and chemotherapy. 2013;57(7):3182–93. Epub 2013/05/01. doi: 10.1128/AAC.00555-13 23629718; PubMed Central PMCID: PMC3697321.

25. Cools HJ, Fraaije BA. Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control. Pest management science. 2013;69(2):150–5. doi: 10.1002/ps.3348 22730104

26. Chowdhary A, Kathuria S, Xu J, Meis JF. Emergence of Azole-Resistant Aspergillus fumigatus Strains due to Agricultural Azole Use Creates an Increasing Threat to Human Health. PLoS Pathog. 2013;9(10). doi: 10.1371/journal.ppat.1003633 24204249

27. Sun X, Xu Q, Ruan R, Zhang T, Zhu C, Li H. PdMLE1, a specific and active transposon acts as a promoter and confers Penicillium digitatum with DMI resistance. Environmental microbiology reports. 2013;5(1):135–42. Epub 2013/06/13. doi: 10.1111/1758-2229.12012 23757142.

28. Peláez JE, Vásquez LE, Díaz TJ, Castañeda DA, Rodríguez E, Arango R. Use of a micro title plate dilution assay to measure activity of antifungal compounds against Mycosphaerella Fijiensis, Morelet. Revista Facultad Nacional de Agronomía Medellín 2006;59(2):3425–33.

29. Ramsay JO. Monotone regressions splines in action. Statistical Science. 1988;3(4):425–41.

30. Arzanlou M, Crous PW, Zwiers LH. Evolutionary dynamics of mating-type loci of Mycosphaerella spp. occurring on banana. Eukaryot Cell. 2010;9(1):164–72. Epub 2009/11/17. doi: 10.1128/EC.00194-09 19915079; PubMed Central PMCID: PMC2805284.

31. Kilian A, Wenzl P, Huttner E, Carling J, Xia L, Blois H, et al. Diversity arrays technology: a generic genome profiling technology on open platforms. Methods in Molecular Biology 2012;888:67–89. Epub 2012/06/06. doi: 10.1007/978-1-61779-870-2_5 22665276.

32. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, et al. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PloS one. 2011;6(5):e19379. Epub 2011/05/17. doi: 10.1371/journal.pone.0019379 21573248; PubMed Central PMCID: PMC3087801.

33. Stam P. Construction of integrated genetic linkage maps by means of a new computer package: JOINMAP. The Plant Journal 1993;3(5):739–44.

34. Manzo-Sanchez G, Zapater MF, Luna-Martinez F, Conde-Ferraez L, Carlier J, James-Kay A, et al. Construction of a genetic linkage map of the fungal pathogen of banana Mycosphaerella fijiensis, causal agent of black leaf streak disease. Current genetics. 2008;53(5):299–311. Epub 2008/03/28. doi: 10.1007/s00294-008-0186-x 18365202.

35. Kema GHJ, Verstappen ECP, Todorova M, Waalwijk C. Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola. Current genetics. 1996;30:251–8. doi: 10.1007/s002940050129 8753655

36. Goodwin SB, M'Barek SB, Dhillon B, Wittenberg AHJ, Crane CF, Lee TAJVd, et al. Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plastici-ty and stealth pathogenesis PLoS Genet. 2011;7(6). e1002070

37. Wittenberg AHJ, Lee TAJVd, M'Barek SB, Ware SB, Goodwin SB, Kilian A, et al. Meiosis drives extraordinary genome plasticity in the haploid fungal plant pathogen Mycosphaerella graminicola. PloS one. 2009;4 (6):1–10.

38. Rodríguez CM, Raigosa N, Conde L, Peraza L, Canto B, James A. Variation in electrophoretic karyotype among Mexican isolates of Mycosphaerella fijiensis. Canadian Journal of Plant Pathology. 2006;28:236–41.

39. Johnson L, Johnson R, Akamatsu H, Salamiah A, Otani H, Kohmoto K, et al. Spontaneous loss of a conditionally dispensable chromosome from the Alternaria alternata apple pathotype leads to loss of toxin production and pathogenicity. Current genetics. 2001;40:65–72. doi: 10.1007/s002940100233 11570518

40. Saleh D, Milazzo J, Adreit H, Tharreau D, Fournier E. Asexual reproduction induces a rapid and permanent loss of sexual reproduction capacity in the rice fungal pathogen Magnaporthe oryzae: results of in vitro experimental evolution assays. BMC Evol Biol. 2012;12:42. doi: 10.1186/1471-2148-12-42 22458778; PubMed Central PMCID: PMC3379926.

41. Krokene P, Solheim H. Loss of pathogenicity in the blue-stain fungus Ceratocystis polonica. Plant Pathology 2001;(50):497–502.

42. Kashino SS, Singer-Vermes LM, Calich VLG, Burger E. Alterations in the pathogenicity of one Paracoccidioides brasiliensis isolate do not correlative with its in vitro growth. Mycopathologia 1990;111 173–80. doi: 10.1007/bf02282801 2233986

43. Zwiers L-H. ABC transporters of the wheat pathogen Mycosphaerella graminicola. Wageningen, The Netherlands Wageningen University & Research; 2002.

44. Stergiopoulos I, Zwiers L-H, Waard MAD. Secretion of natural and synthetic toxic compounds from filamentous fungi by membrane transporters of the ATP-binding cassette and major facilitator superfamily. European Journal of Plant Pathology. 2002;108:719–34.

45. Leroux P, Gredt M, Leroch M, Walker AS. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Appl Environ Microbiol. 2010;76(19):6615–30. Epub 2010/08/10. doi: 10.1128/AEM.00931-10 20693447; PubMed Central PMCID: PMC2950445.

46. Cools HJ, Bayon C, Atkins S, Lucas JA, Fraaije BA. Overexpression of the sterol 14a-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype. Pest management science. 2012;68(7):1034–40. Epub 2012/03/14. doi: 10.1002/ps.3263 22411894.

47. Akins RA, Sobel JD. Antifungal targets, mechanisms of action, and resistance in Candida albicans. 2009:347–407. doi: 10.1007/978-1-59745-180-2_29.

48. Aouini L, Mirzadi Gohari A, Ware SB, Van den Bosch F, Helps J, Alonso-Chavez V, et al. A paradigm shift: sex deals with biotic and abiotic threats in the fungal plant pathogen Zymoseptoria tritici. Submitted to Nature Genetics. 2016.

49. Lendenmann MH, Croll D, McDonald BA. QTL mapping of fungicide sensitivity reveals novel genes and pleiotropy with melanization in the pathogen Zymoseptoria tritici. Fungal genetics and biology: FG & B. 2015. Epub 2015/05/17. doi: 10.1016/j.fgb.2015.05.001 25979163.

50. Latin R. A practical guide to turfgrass fungicides. USA: American Phytopathological Society; 2011. 270 p.


Článek vyšel v časopise

PLOS One


2019 Číslo 10