Skip to main content
Log in

Secreted in Xylem (SIX) genes in Fusarium oxysporum f.sp. cubense (Foc) unravels the potential biomarkers for early detection of Fusarium wilt disease

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript


Secreted in Xylem (SIX) are small effector proteins released by Fusarium oxysporum f.sp. cubense (Foc) into the plant’s xylem sap disrupting the host’s defence responses causing Fusarium wilt disease resulting in a significant decline in banana crop yields and economic losses. Notably, different races of Foc possess unique sets of SIX genes responsible for their virulence, however, these genes remain underutilized, despite their potential as biomarkers for early disease detection. Herein, we identified seven SIX genes i.e. SIX1, SIX2, SIX4, SIX6, SIX8a, SIX9a and SIX13 present in Foc Tropical Race 4 (FocTR4), while only SIX9b in Foc Race 1 (Foc1). Analysis of SIX gene expression in infected banana roots revealed differential patterns during infection providing valuable insights into host–pathogen interactions, virulence level, and early detection time points. Additionally, a comprehensive analysis of virulent Foc1_C2HIR and FocTR4_C1HIR isolates yielded informative genomic insights. Hence, these discoveries contribute to our comprehension of potential disease control targets in these plants, as well as enhancing plant diagnostics and breeding programs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.


Similar content being viewed by others

Data availability

The whole genome of Foc1_C2HIR and FocTR4_C1HIR has been submitted to NCBI Genbank database under the Biosample accession number SAMN37320067 and SAMN37320068.


  • Ahmad S, Fook CWK, Vadamalai G, Wahab MA, Saidi NB, Zulperi D (2020) Molecular characterization of Fusarium oxysporum f.sp. cubense Tropical race 4 (Foc-TR4) isolates from Malaysian banana using Secreted in Xylem (SIX) effector genes. Arch Phytopathol Plant Prot.

    Article  Google Scholar 

  • Alazem M, Lin NS (2017) Antiviral roles of abscisic acid in plants. Front Plant Sci 8(October):1–10.

    Article  Google Scholar 

  • Al-Obaidi JR, Yusuf YM, Chong TC, Noh NM, Othman RY (2010) Identification of a partial oil palm polygalacturonase-inhibiting protein (EgPGIP) gene and its expression during basal stem rot infection caused by Ganoderma boninense. Afr J Biotech 9(46):7788–7797

    Article  CAS  Google Scholar 

  • Amaral Carneiro G, Calì M, Cappelletti E, Baroncelli R, Prodi A (2023) Draft genome sequence of the apple pathogen Colletotrichum chrysophilum strain M932. J Plant Pathol, 105(3):1141-1143.

  • An B, Hou X, Guo Y, Zhao S, Luo H, He C, Wang Q (2019) The effector SIX8 is required for virulence of Fusarium oxysporum f.sp. cubense tropical race 4 to Cavendish banana. Fungal Biol.

    Article  PubMed  Google Scholar 

  • Armitage AD, Taylor A, Sobczyk MK, Baxter L, Greenfield BPJ, Bates HJ, Wilson F, Jackson AC, Ott S, Harrison RJ, Clarkson JP (2018) Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f.sp. cepae. Sci Rep.

    Article  PubMed  PubMed Central  Google Scholar 

  • Asai S, Ayukawa Y, Gan P, Masuda S, Komatsu K, Shirasu K, Arie T (2019) High-quality draft genome sequence of Fusarium oxysporum f.sp. cubense isolate 160527, a causal agent of Panama disease. Microbiol Res Announc 8(29):10–1128

    Google Scholar 

  • Boulesteix M, Weiss M, Biémont C (2006) Differences in genome size between closely related species: the Drosophila melanogaster species subgroup. Mol Biol Evol 23(1):162–167.

    Article  CAS  PubMed  Google Scholar 

  • Carvalhais LC, Henderson J, Rincon-Florez VA, O’Dwyer C, Czislowski E, Aitken EAB, Drenth A (2019) Molecular diagnostics of banana fusarium wilt targeting Secreted in Xylem genes. Front Plant Sci.

    Article  PubMed  PubMed Central  Google Scholar 

  • Chakrabarti A, Rep M, Wang B, Ashton A, Dodds P, Ellis J (2011) Variation in potential effector genes distinguishing Australian and non-Australian isolates of the cotton wilt pathogen Fusarium oxysporum f.sp.vasinfectum. Plant Pathol, 60(2):232–243.

  • Cheng C, Liu F, Sun X, Tian N, Mensah RA, Li D, Lai Z (2019) Identification of Fusarium oxysporum f.sp. cubense tropical race 4 (Foc TR4) responsive miRNAs in banana root. Sci Rep.

    Article  PubMed  PubMed Central  Google Scholar 

  • Czislowski E, Fraser-Smith S, Zander M, O’Neill WT, Meldrum RA, Tran-Nguyen LTT, Batley J, Aitken EAB (2018) Investigation of the diversity of effector genes in the banana pathogen, Fusarium oxysporum f.sp. cubense, reveals evidence of horizontal gene transfer. Mol Plant Pathol.

    Article  PubMed  Google Scholar 

  • Czislowski E, Zeil-rolfe I, Aitken EAB (2021) Effector profiles of endophytic Fusarium associated with asymptomatic banana (Musa sp.) hosts. Int J Mol Sci.

    Article  PubMed  PubMed Central  Google Scholar 

  • Daayf F, El Hadrami A, El-Bebany AF, Henriquez MA, Yao Z, Derksen H, El-Hadrami I, Adam LR (2012) Phenolic compounds in plant defense and pathogen counter defense mechanisms. Recent Adv Polyphen Res 3(3):191–208.

    Article  CAS  Google Scholar 

  • Fang Y, Ramasamy RP (2015) Current and prospective methods for plant disease detection. Biosensors 5(3):537–561.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • FAO 2020 Banana market review In: Intergovernmental group on bananas and tropical fruits. Rome: FAO publications.

  • Fraser-Smith S, Czislowski E, Meldrum RA, Zander M, O’Neill W, Balali GR, Aitken EAB (2014) Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f.sp. cubense. Plant Pathol.

    Article  Google Scholar 

  • Gawehns F, Houterman PM, Ichou FA, Michielse CB, Hijdra M, Cornelissen BJC, Rep M, Takken FLW (2014) The Fusarium oxysporum effector SIX6 contributes to virulence and suppresses I-2-mediated cell death. Mol Plant Microbe Interact 27(4):336–348.

    Article  CAS  PubMed  Google Scholar 

  • Grützmann K, Szafranski K, Pohl M, Voigt K, Petzold A, Schuster S (2014) Fungal alternative splicing is associated with multicellular complexity and virulence: a genome-wide multi-species study. DNA Res 21(1):27–39.

    Article  CAS  PubMed  Google Scholar 

  • Guo L, Han L, Yang L, Zeng H, Fan D, Zhu Y, Feng Y, Wang G, Peng C, Jiang X, Zhou D, Ni P, Liang C, Liu L, Wang J, Mao C, Fang X, Peng M, Huang J (2014) Genome and transcriptome analysis of the fungal pathogen Fusarium oxysporum f.sp. cubense causing banana vascular wilt disease. PLoS ONE.

    Article  PubMed  PubMed Central  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Houterman PM, Speijer D, Dekker HL, De Koster CG, Cornelissen BJC, Rep M (2007) The mixed xylem sap proteome of Fusarium oxysporum-infected tomato plants: short communication. Mol Plant Pathol 8(2):215–221.

    Article  CAS  PubMed  Google Scholar 

  • Huerta-Cepas J, Forslund K, Coelho LP, Szklarczyk D, Jensen LJ, Von Mering C, Bork P (2017) Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol Biol Evol, 34(8):2115–2122.

  • Jangir P, Mehra N, Sharma K, Singh N, Rani M, Kapoor R (2021) Secreted in Xylem Genes: Drivers of Host Adaptation in Fusarium oxysporum. Front Plant Sci, 12(April), 1–17.

  • Jenkins S, Taylor A, Jackson AC, Armitage AD, Bates HJ, Mead A, Harrison RJ, Clarkson JP (2021) Identification and expression of secreted In Xylem pathogenicity genes in Fusarium oxysporum f.sp. pisi. Front Microbiol.

    Article  PubMed  PubMed Central  Google Scholar 

  • Kabbage M, Yarden O, Dickman MB (2015) Pathogenic attributes of Sclerotinia sclerotiorum: switching from a biotrophic to necrotrophic lifestyle. Plant Sci 233:53–60

    Article  CAS  PubMed  Google Scholar 

  • Karmawan LU, Dwivany FM, Esyanti RR, Aryantha INP (2018) Improved in vitro bioassay for Musa Acuminata cv. Pisang Ambon Kuning (AAA group) based on quantitative analysis of necrosis area and biomass changes during Foc4 infection. Arch Phytopathol Plant Prot.

    Article  Google Scholar 

  • Khairi MHF, Nor Muhammad NA, Bunawan H, Abdul Murad AM, Ramzi AB (2022) Unveiling the Core effector proteins of oil palm pathogen ganoderma boninense via Pan-Secretome analysis. J Fungi.

    Article  Google Scholar 

  • Li C yu, Deng G ming, Yang J., Viljoen A., Jin Y., Kuang R bin, Zuo C wu, Lv Z cheng, Yang Q song, Sheng O., Wei Y rong, Hu, C hua, Dong, T., & Yi, G jun. (2012). Transcriptome profiling of resistant and susceptible Cavendish banana roots following inoculation with Fusarium oxysporum fsp cubense tropical race 4. BMC Genomics,

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2− ΔΔCT method, Methods 25:402–408

  • Lievens B, Rep M, Thomma BP (2008) Recent developments in the molecular discrimination of formae speciales of Fusarium oxysporum. Pest Manag Sci, 64(8):781-788.

  • Maia M, Ferreira AEN, Nascimento R, Monteiro F, Traquete F, Marques AP, Cunha J, Eiras-Dias JE, Cordeiro C, Figueiredo A, Sousa Silva M (2020) Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine. Sci Rep 10(1):1–15.

    Article  CAS  Google Scholar 

  • Mak, C., Mohamed, A. A., Liew, K. W., & Ho, Y. W. (2004). Early screening technique for Fusarium wilt resistance in banana micro propagated plants. In Banana improvement: cellular, molecular biology, and induced mutations. Proceedings of a meeting held in Leuven, Belgium, 24–28 September 2001 (pp. 219–227). Science Publishers, Inc.

  • Maldonado BLD, Villarruel OJL, Calderón OMA, Sánchez EAC (2018) Secreted in Xylem (SIX) genes in Fusarium oxysporum f. sp. cubense and their potential acquisition by horizontal transfer. Adv Biotech & Micro.

    Article  Google Scholar 

  • Mandal S, Mallick N, Mitra A (2009) Salicylic acid-induced resistance to Fusarium oxysporum f. sp. lycopersici in tomato. Plant Physiol Biochem.

    Article  PubMed  Google Scholar 

  • Masheva S, Todorova V (2013) Response of pepper varities F1 hybrids and breeding lines to Verticillium dahlia Kleb in two methods of infestation. Bulgarian J Agric Sci 19(1):133–138

    Google Scholar 

  • Mohd-Yusuf Y, Khalid N, Al-Obaidi JR, Baharum NA, Zaidi K, Salleh B, Azuddin NF, Aziz FAA, Munusamy U, Othman RY (2019) Standardized bioassays: An improved method for studying Fusarium Oxysporum f. sp. cubense race 4 (FocR4) pathogen stress response in Musa acuminata cv. ‘Berangan.’ Asia-Pac J Mol Biol Biotechnol.

    Article  Google Scholar 

  • Monforte J, McPhail S (2005) Strategy for gene expression-based biomarker discovery. Biotechniques.

    Article  PubMed  Google Scholar 

  • O’Connell RJ, Thon MR, Hacquard S, Amyotte SG, Kleemann J, Torres MF, Damm U, Buiate EA, Epstein L, Alkan N, Altmüller J, Alvarado-Balderrama L, Bauser CA, Becker C, Birren BW, Chen Z, Choi J, Crouch JA, Duvick JP, Vaillancourt LJ (2012) Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nat Genet.

    Article  PubMed  PubMed Central  Google Scholar 

  • Paul JY, Becker DK, Dickman MB, Robert MH, Khanna HK, Dale JL (2011) Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas. Plant Biotechnol J 9(9):1141–1148

    Article  CAS  PubMed  Google Scholar 

  • Pegg KG, Coates LM, O’Neill WT, Turner DW (2019) The Epidemiology of fusarium wilt of banana. Front Plant Sci 10:1395.

    Article  PubMed  PubMed Central  Google Scholar 

  • Pérez Vicente, L.F., Dita, M. and Martinez De La Parte, E. 2014. Technical Manual: Prevention and diagnostic of Fusarium Wilt (Panama disease) of banana caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (TR4). Proceedings of Regional Workshop on the Diagnosis of Fusarium Wilt (Panama disease) caused by Fusarium oxysporum f.sp. cubense Tropical Race 4: Mitigating the threat and preventing its spread in the Caribbean, St. Augustine, Trinidad and Tobago, 05–09/05/2014.FAO, Rome, Italy. 74p.

  • Ploetz RC (2015) Fusarium wilt of banana. Phytopathology 105(12):1512–1521.

    Article  PubMed  Google Scholar 

  • Ploetz RC, Kema GH, Ma LJ (2015) Impact of diseases on export and smallholder production of banana. Ann review phytopathology, 53:269-288.

  • Poon NK, Teo CH, Othman RY (2019) Differential gene expression analysis of secreted in Xylem (SIX) genes from Fusarium oxysporum f.sp. cubense tropical race 4 in Musa acuminata cv. Berangan and potential application for early detection of infection. J Genl Plant Pathol.

    Article  Google Scholar 

  • Pradhan A, Ghosh S, Sahoo D, Jha G (2021) Fungal effectors, the double edge sword of phytopathogens. Curr Genet 67(1):27–40.

    Article  CAS  PubMed  Google Scholar 

  • Raman T, Raj EE, Muthukathan G, Loganathan M, Periyasamy P, Natesh M, Manivasakan P, Kotteeswaran S, Rajendran S, Subbaraya U (2021) Comparative whole-genome sequence analyses of fusarium wilt pathogen (Foc R1, STR4 and TR4) infecting cavendish (AAA) bananas in India, with a special emphasis on pathogenicity mechanisms. J Fungi 7(9):1–19.

    Article  CAS  Google Scholar 

  • Rep M, Dekker HL, Vossen JH, De Boer AD, Houterman PM, Speijer D, Back JW, De Koster CG, Cornelissen BJC (2002) Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol 130(2):904–917.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rodríguez-Kessler M, Baeza-Montañez L, García-Pedrajas MD, Tapia-Moreno A, Gold S, Jiménez-Bremont JF, Ruiz-Herrera J (2012) Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis. Microbiol Res 167(5):270–282

    Article  PubMed  Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101

    Article  CAS  PubMed  Google Scholar 

  • Staib P, Kretschmar M, Nichterlein T, Köhler G, Michel S, Hof H, Morschhäuser J (1999) Host-induced, stage-specific virulence gene activation in Candida albicans during infection. Mol Microbiol.

    Article  PubMed  Google Scholar 

  • Taylor A, Vágány V, Jackson AC, Harrison RJ, Rainoni A, Clarkson JP (2016) Identification of pathogenicity-related genes in Fusarium oxysporum f.sp. cepae. Mol Plant Pathol.

    Article  PubMed  PubMed Central  Google Scholar 

  • van Dam P, Fokkens L, Schmidt SM, Linmans JH, Kistler HC, Ma LJ, Rep M (2016) Effector profiles distinguish formae speciales of Fusarium oxysporum. Environ Microbiol 18(11):4087–4102.

    Article  CAS  PubMed  Google Scholar 

  • Vignolle GA, Mach RL, Mach-Aigner AR, Derntl C (2021) Genome sequence of the black yeast-like isolate Aureobasidium pullulans var. aubasidani CBS 100524. Microbiol Resour Announc.

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang X, El Hadrami A, Adam LR, Daayf F (2008) Differential activation and suppression of potato defence responses by Phytophthora infestans isolates representing US-1 and US-8 genotypes. Plant Pathol 57:1026–1037

    Article  CAS  Google Scholar 

  • Wang Z, Jia C, Li J, Huang S, Xu B, Jin Z (2015) Activation of salicylic acid metabolism and signal transduction can enhance resistance to Fusarium wilt in banana (Musa acuminata L. AAA group, cv. Cavendish). Funct Integr Genomics 15:47–62

    Article  CAS  PubMed  Google Scholar 

  • Widinugraheni S, Niño-Sánchez J, Van Der Does HC, Van Dam P, García-Bastidas FA, Subandiyah S, Meijer HJG, Kistler HC, Kema GHJ, Rep M (2018) A SIX1 homolog in Fusarium oxysporum f.sp. cubense tropical race 4 contributes to virulence towards Cavendish banana. PLoS ONE.

    Article  PubMed  PubMed Central  Google Scholar 

  • Wong GR, Latif FBA, S. N., & Mazumdar, P. (2022) Genome-wide investigation and comparative expression profiling reveal R2R3-MYB genes involved in Sclerotinia sclerotiorum defence in tomato. Physiol Mol Plant Pathol 121(April):101873.

    Article  CAS  Google Scholar 

  • Yun Y, Song A, Bao JD, Chen S, Lu S, Cheng C, Zheng W, Wang Z, Zhang L (2019) Genome data of Fusarium oxysporum f.sp. cubense race 1 and tropical race 4 isolates using long-read sequencing. Mol Plant-Microbe Interact.

    Article  PubMed  Google Scholar 

  • Zaidi.K. (2019). Molecular analysis of Fusarium oxysporum f.sp. cubense isolates and defence gene expression in banana infected with Fusarium wilt. Universiti Malaya.

Download references


The authors jointly acknowledge the Institute of Biological Sciences, Faculty of Science and the Centre for Research in Biotechnology for Agriculture (CEBAR), Universiti of Malaya, Malaysia. We also acknowledge the Universiti Malaya’s research council for the publication support.


This study was supported by the Fundamental Research Grant Scheme, Ministry of Higher Education, Malaysia (Grant No.: FP058-2018A), Geran Penyelidikan Fakulti by the Faculty of Science, Universiti Malaya (Grant No.: GPF042B-2020) and Research University grant from Universiti Malaya (Grant No.: RU004A-2020).

Author information

Authors and Affiliations



Conceptualization: KK, SNAM and YMY. Writing and original draft preparation: KK. Writing-Review & Editing: KK, SNAM, NAR, CKO and YMY. Visualization: KK, SNAM and YMY. Supervision: NAR, CKO and YMY. Funding acquisition: SNAM and YMY. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yusmin Mohd-Yusuf.

Ethics declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Additional information

Communicated by Yusuf Akhter.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 143 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kaliapan, K., Mazlin, S.N.A., Chua, K. et al. Secreted in Xylem (SIX) genes in Fusarium oxysporum f.sp. cubense (Foc) unravels the potential biomarkers for early detection of Fusarium wilt disease. Arch Microbiol 206, 271 (2024).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: