Comparative analysis uncovers the limitations of current molecular detection methods for Fusarium oxysporum f. sp. cubense race 4 strains

Autoři: Freddy Magdama aff001;  Lorena Monserrate-Maggi aff002;  Lizette Serrano aff002;  Daynet Sosa aff002;  David M. Geiser aff001;  María del Mar Jiménez-Gasco aff001
Působiště autorů: Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, Pennsylvania, United States of America aff001;  Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador, Campus Gustavo Galindo, Guayaquil, Ecuador aff002;  Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ciencias de la Vida, Campus Gustavo Galindo, Guayaquil, Ecuador aff003
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222727


Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) is threatening banana production worldwide. Despite quarantine efforts, the pathogen continues to spread; thus, early diagnosis plays an essential role for the proper execution of contingency plans. Here, we assess the accuracy of four PCR-based molecular methods described in the literature for the identification and detection of race 4 strains, including Subtropical (Foc STR4) and Tropical Race 4 causing Fusarium wilt of banana. We screened a total of 302 isolates using these four markers, and performed phylogenetic analyses, Vegetative Compatibility Group (VCG) testing, sequence comparison, and pathogenicity tests for selected isolates. Our results show that three out of the four markers tested are not reliable for identification of Foc STR4 and TR4, as DNA from isolates from Ecuador, pathogenic and nonpathogenic to banana, obtained from different banana cultivars, displayed cross-reaction with these methods; that is, false positives can occur during the diagnostic process for race 4. Phylogenetic analyses, VCG testing, sequence comparison, and pathogenicity tests suggest the presence of non-target F. oxysporum isolates that share genomic regions with pathogenic strains but lack true pathogenicity to banana. The findings of this work are of foremost importance for international regulatory agencies performing surveillance tests in pathogen-free areas using the current diagnostic methods. We suggest the use of a genetic locus possibly related to virulence, previously identified by T-DNA, and amplified with primers W2987F/ W2987R, for diagnosis of Foc TR4 as the most reliable alternative. We urge the adoption of a more holistic view in the study of F. oxysporum as a plant pathogen that considers the biology and diversity of the species for the development of better diagnostic tools.

Klíčová slova:

DNA sequence analysis – Fusarium – Pathogenesis – Phylogenetic analysis – Polymerase chain reaction – Bananas – Fusarium oxysporum – DNA isolation


1. FAOSTAT. FAO statistical database. 2013.

2. D'Hont A, Denoeud F, Aury JM, Baurens FC, Carreel F, Garsmeur O, et al. The banana (Musa acuminata) genome and the evolution of monocotyledonous plants. Nature. 2012; 488: 213–17. doi: 10.1038/nature11241 22801500

3. Stover RH. Fusarial wilt (Panama Disease) of bananas and other Musa species. Common-wealth Mycological Institute UK. 1962. p. 117

4. Ploetz RC. Panama Disease: An old nemesis rears its ugly head. Part1: The beginnings of the banana export trades. Plant Health Progress. 2005

5. Moore NY, Pegg KG, Allen RN, Irwin JAG. Vegetative compatibility and distribution of Fusarium oxysporum f.sp. cubense in Australia. Aust. J. Exp. Agric. 1993; 33:797–802.

6. Su EJ, Hwang SC, Ko WH. Fusarial wilt of Cavendish bananas in Taiwan. Plant Dis. 1986; 70:814–818.

7. Waiter BH, Stover RH. Studies on Fusarium wilt of bananas: VI. Variability and cultivar concept in Fusarium oxysporum f. sp. cubense. Can. J. Bot. 1960; 38:985–994.

8. Ploetz RC. Panama disease: An old nemesis rears its ugly head. Part2. The Cavendish era and beyond. Plant Health Progress. 2006

9. Su HJ, Chuang TY, Kong WS. Physiological race of Fusarial wilt fungus attacking Cavendish banana in Taiwan. Taiwan Banana. 1977; p. 21.

10. Ploetz RC. Fusarium wilt of banana is caused by several pathogens referred to as Fusarium oxysporum f. sp. cubense. Phytopathology. 2006; 96:653–656. doi: 10.1094/PHYTO-96-0653 18943184

11. Stover RH, Malo RH. The occurrence of fusarial wilt in normally resistant 'Dwarf Cavendish' banana. Plant Dis. 1972; 56:1000–1003.

12. Bentley S, Pegg KG, Moore NY, Davis RD, Buddenhagen IW. 1998. Genetic variation among vegetative compatibility groups of Fusarium oxysporum f. sp. cubense analyzed by DNA fingerprinting. Phytopathology. 1998; 88:1283–1293. doi: 10.1094/PHYTO.1998.88.12.1283 18944830

13. Pegg KG, Moore NY, Sorenson S. Variability in populations of Fusarium oxysporum f.sp. cubense from Asia/Pacific region. In: Jones DR, editor. The Improvement and Testing of Musa: A Global Partnership. Proceedings of the First Global Conference of the International Musa Testing program. INIBAP France; 1994. p. 70–82.

14. Pegg KG, Moore NY, Bentley S. Fusarium wilt of banana in Australia. Aust. J. Agric. Res. 1996; 47:637–650.

15. Karangwa P, Blomme G, Beed F, Niyongere C, Viljoen A. The distribution and incidence of banana Fusarium wilt in subsistence farming systems in east and central Africa. Crop Prot. 2016; 84:132–40.

16. Mostert D, Molina AB, Daniells J, Fourie G, Hermanto C, Chao CP, et al. The distribution and host range of the banana Fusarium wilt fungus, Fusarium oxysporum f.sp. cubense, in Asia. PLoS ONE. 2017; 12(7):e0181630. doi: 10.1371/journal.pone.0181630 28719631

17. O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci. 1998; 95:2044–49. doi: 10.1073/pnas.95.5.2044 9482835

18. Koenig RL, Ploetz RC, Kistler HC. Fusarium oxysporum f. sp. cubense consists of a small number of divergent and globally distributed clonal lineages. Phytopathology. 1997; 87:915–923. doi: 10.1094/PHYTO.1997.87.9.915 18945062

19. Fourie G, Steenkamp ET, Gordon TR, Viljoen A. Evolutionary relationships among the Fusarium oxysporum f. sp. cubense vegetative compatibility groups. Appl Environ Microbiol. 2009; 75:4770–81. doi: 10.1128/AEM.00370-09 19482953

20. Ordonez N, Seidl MF, Waalwijk C, Drenth A, Kilian A, Thomma BPHJ, et al. Worse comes to worst: Bananas and Panama disease—when plant and pathogen clones meet. PLoS Pathog. 2015; 11: 1–7. 26584184

21. Katan T. Current status of vegetative compatibility groups in Fusarium oxysporum. Phytoparasitica. 1999;27: 51–64.

22. Dita M, Barquero M, Heck D, Mizubuti ESG, Staver CP. Fusarium Wilt of Banana: Current Knowledge on Epidemiology and Research Needs Toward Sustainable Disease Management. Front Plant Sci. 2018; 9:1468. doi: 10.3389/fpls.2018.01468 30405651

23. Ploetz R, Freeman S, Konkol J, Al-Abed A, Naser Z, Shalan K, et al. Tropical race 4 of Panama disease in the Middle East. Phytoparasitica. 2015; 43:283–93.

24. Zheng S-J, García-Bastidas FA, Li X, Zeng L, Bai T, Xu S, et al. New Geographical Insights of the Latest Expansion of Fusarium oxysporum f.sp. cubense Tropical Race 4 Into the Greater Mekong Subregion. Front. Plant Sci. 2018; 9:457. doi: 10.3389/fpls.2018.00457 29686692

25. Daniells J, O’Neill W, Hermanto C, Ploetz R. Banana Varieties and Fusarium oxysporum f. sp. cubense in Indonesia—Observations from Fusarium Wilt Disease Databases. Acta Hort. 2011; 897:475–77.

26. Thangavelu R, Mustaffa MM. First report on the occurrence of a virulent strain of Fusarium wilt pathogen (Race-1) infecting Cavendish (AAA) group of Bananas in India. Plant Dis. 2010; 94:1379.

27. Groenewald S, Van Den Berg N, Marasas WF, Viljoen A. The application of high-throughput AFLP's in assessing genetic diversity in Fusarium oxysporum f. sp. cubense. Mycol Res. 2006;110: 297–05. doi: 10.1016/j.mycres.2005.10.004 16483757

28. Ploetz RC, Pegg KG. Fusarium wilt. In: Jones DR, editor. Diseases of Banana, Abacá and Enset. Wallingford: CABI Publishing; 2000. p. 143–159.

29. Gordon TR. Fusarium oxysporum and the Fusarium wilt Syndrome. Annu Rev Phytopathol. 2017;55: 1.1–1.17. doi: 10.1146/annurev-phyto-080615-095919 28489498

30. Lin Y-H, Chang J-Y, Liu E-T, Chao C-P, Huang J-W, Chang P-F. Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol. 2009; 123(3):353–65.

31. Dita MA, Waalwijk C, Buddenhagen IW, Souza JT, Kema GHJ. A molecular diagnostic for tropical race 4 of the banana fusarium wilt pathogen. Plant Pathol. 2010; 59: 348–57.

32. Li B, Du J, Lan C, Liu P, Weng Q, Chen Q. Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol. 2013; 135(4): 903–11.

33. Li M, Shi J, Xie X, Leng Y, Wang H, Xi P, et al. Identification and application of a unique genetic locus in diagnosis of Fusarium oxysporum f. sp. cubense tropical race 4. Can J Plant Pathol. 2013; 35(4):482–93.

34. Lin Y-H, Su C-C, Chao C-P, Chen C-Y, Chang C-J, Huang J-W, et al. A molecular diagnosis method using real-time PCR for quantification and detection of Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol. 2013; 135(2): 395–05.

35. Zhang X, Zhang H, Pu J, Qi Y, Yu Q, Xie Y, et al. Development of a Real-Time Fluorescence Loop- Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil. PloS One. 2013;8(12): e82841. doi: 10.1371/journal.pone.0082841 24376590

36. Peng J, Zhang H, Chen F, Zhang X, Xie Y, Hou X, et al. Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification. J Appl Microbiol. 2014;117(6): 1740–9. doi: 10.1111/jam.12645 25200557

37. Yang L-L, Sun L-X, Ruan X-L, Qiu D-Y, Chen D-H, Cai X-Q, et al. Development of a single-tube duplex real-time fluorescence method for the rapid quantitative detection of Fusarium oxysporum f. sp. cubense race 1 (FOC1) and race 4 (FOC4) using TaqMan probes. Crop Protection. 2015; 68(0): 27–35.

38. Lin Y-H, Lin Y-J, Chang TD, Hong L, Chen T. Development of a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay for detection of Fusarium oxysporum f.sp. cubense race 4. PLoS One. 2016; 11:1–13.

39. Dita MA, Waalwijk C, Buddenhagen IW, Souza JT, Kema GHJ. A molecular diagnostic for tropical race 4 of the banana fusarium wilt pathogen. Erratum in: Plant Pathol. 2011; 60:384.

40. Dita MA, Echegoyén PE, Pérez LF. Plan de contingencia ante un brote de la raza 4 tropical de Fusarium oxysporum f.sp. cubense en un país de la OIRSA. 2013. San Salvador, El Salvador: OIRSA, 2013.

41. Pérez-Vicente L, Dita MA, Martínez-de la Parte E. Technical Manual. Prevention and diagnostic of Fusarium Wilt (Panama disease) of banana caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4). Rome, Italy: FAO, 2014.

42. Dita MA, Waalwijk C, Mutua P, Daly A, Chang PFL, Corcolon BM, et al. Detecting Fusarium oxysporum f. sp. cubense tropical race 4 in soil and symptomless banana tissues. Acta hortic. 2013; (986):127–30.

43. Gordon TR, Martyn RD. The evolutionary biology of Fusarium oxysporum. Annu Rev Phytopathol. 1997;35: 111–28. doi: 10.1146/annurev.phyto.35.1.111 15012517

44. Balmas V, Migheli Q, Scherm B, Garau P, O’Donnell K, Ceccherelli G, et al. Multilocus phylogenetics show high levels of endemic fusaria inhabiting Sardinian soils (Tyrrhenian Islands). Mycologia. 2010;102: 803–12. 20648748

45. Laurence MH, Burgess LW, Summerell BA, Liew EC. High levels of diversity in Fusarium oxysporum from non-cultivated ecosystems in Australia. Fungal Biol. 2012;116: 289–97. doi: 10.1016/j.funbio.2011.11.011 22289774

46. Demers JE, Gugino BK, Jiménez-Gasco MM. Highly diverse endophytic and soil Fusarium oxysporum populations associated with field-grown tomato plants. Appl Environ Microbiol. 2015; 81(1):81–90. doi: 10.1128/AEM.02590-14 25304514

47. Geiser DM, Jiménez-Gasco MM, Kang S, Makalowska I, Veeraraghavan N, Ward TJ, et al. FUSARIUM-ID v.1.0: A DNA sequence database for identifying Fusarium. Eur J Plant Pathol. 2004; 110:473–79.

48. Appel DJ, Gordon TR. Relationships among pathogenic and nonpathogenic isolates of Fusarium oxysporum based on the partial sequence of the intergenic spacer region of the ribosomal DNA. Mol Plant Microbe Interact. 1996;9: 125–38. 8820752

49. Edel V, Steinberg C, Gautheron N, Recorbet G, Alabouvette C. Genetic diversity of Fusarium oxysporum populations isolated from different soils in France. FEMS Microbiol Ecol. 2001; 36(1): 61–71. doi: 10.1111/j.1574-6941.2001.tb00826.x 11377774

50. O’Donnell K, Gueidan C, Sink S, Johnston PR, Crous PW, Glenn A. et al. A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex. Fungal Genet Biol. 2009;46: 936–48. doi: 10.1016/j.fgb.2009.08.006 19715767

51. Edel V, Steinberg C, Avelange I, Laguerre G, Alabouvette C. Comparison of three molecular methods for the characterization of Fusarium oxysporum strains. Phytopathology. 1995; 85: 579–85.

52. Swofford DL. “PAUP*. Phylogenetic Analysis Using Parsimony and Other Methods,” Version 4, Sinauer associates, Sunderland, Massachusetts, 2003.

53. Silvestro D, Michalak I. raxmlGUI: a graphical front-end for RAxML. Org Divers Evol. 2012; 12: 335–37.

54. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinforma. 2003; 19(12): 1572–74.

55. Puhalla J. Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility. Can J Bot.1985; 63:179–83.

56. Correll JC, Klittich CJR, Leslie JF. Nitrate non-utilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology. 1987; 77: 1640–46.

57. Ssali RT, Kiggundu A, Lorenzen J, Karamura E, Tushemereirwe W, Viljoen A. Inheritance of resistance to Fusarium oxysporum f. sp. cubense race 1 in bananas. Euphytica. 2013; 194: 425–30.

58. Nash SM, Snyder WC. Quantitative estimations by plate counts of propagules of bean root rot Fusarium in fields soils. Phytopathology.1962; 52: 567–72.

59. Ploetz RC. Management of Fusarium wilt of banana: A review with special reference to tropical race 4. Crop Prot. 2015;73: 7–15.

60. Ma LJ. Horizontal chromosome transfer and rational strategies to manage Fusarium vascular wilt diseases. Mol Plant Pathol.2014;15: 763–66. doi: 10.1111/mpp.12171 25040107

61. Ma LJ, van der Does HC, Borkovich KA, Coleman JJ, Daboussi MJ, Di Pietro A, et al. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature.2010;464: 367–73. doi: 10.1038/nature08850 20237561

62. Visser M, Gordon T, Fourie G, Viljoen A. Characterisation of South African isolates of Fusarium oxysporum f.sp. cubense from Cavendish bananas. S Afr J Sci.2010; 106: 1–6.

63. Aguayo J, Mostert D, Fourrier-Jeandel C, Cerf-Wendling I, Hostachy B, Viljoen A, et al. Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4. PLoS ONE.2017; 12(2): e0171767. doi: 10.1371/journal.pone.0171767 28178348

64. Nel B, Steinberg C, Labuschagne N, Viljoen A. Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the Roots of healthy banana. Plant Pathol.2006; 55:207–16.

65. McKillop C. Independent review blames unreliable test, not Biosecurity Queensland, for false panama diagnosis. ABC Rural. 14 Feb 2016.

66. McKillop C, Zonca C. Quarantine lifted on Mareeba banana farm suspected of having Panama disease. ABC Rural. 5 Mar 2015.

67. Australian Banana News. August 2015.

68. Martin RR, Constable F, Tzanetakis IE. Quarantine regulations and the impact of modern detection methods. Annu Rev Phytopathol.2016; 54: 189–205. doi: 10.1146/annurev-phyto-080615-100105 27491434

69. Buddenhagen I. Understanding strain diversity in Fusarium oxysporum f.sp. cubense and history of introduction of Tropical race 4 to better manage banana production. Acta Hortic.2009; 828:193–204.

70. McTaggart AR, van der Nest MA, Steenkamp ET, Roux J, Slippers B, Shuey LS, et al. Fungal genomics challenges the dogma of name-based biosecurity. PLOS Pathog.2016; 12(5):12:e1005475. doi: 10.1371/journal.ppat.1005475 27149511

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