Development of species specific SCAR based molecular marker for the detection of Pseudocercospora eumusae, the causal agent of eumusae leaf spot disease in banana

  • Perumal Ganga Devi
  • Raman ThangaveluEmail author
Original Article


Eumusae leaf spot disease caused by Pseudocercopra eumusae is one of the destructive leaf spot diseases in banana. A diagnostic method based on conventional and quantitative real-time PCR (qPCR) assay was developed in the present study for the specific detection of P. eumusae from pure cultures and leaf spot tissues. The sequence characterized amplified region (SCAR) based molecular marker and TaqMan probe was designed and validated using the genomic DNA of different Pseudocercopra spp. and 11 other leaf spot pathogens of banana. The primers and TaqMan probe proved to be highly specific to the target pathogen P. eumusae and no cross reactivity was encountered with any of the other leaf spot pathogens tested. Both conventional and qPCR assay developed detected the target pathogen P. eumusae directly from single leaf spot tissue and thus simplifies the identification by eliminating the need for culturing of pathogen. However, the sensitivity of qPCR (1 pg/μL) is higher compared to conventional PCR (100 pg/μL). To the best of our knowledge, this is the first report on the use of SCAR based molecular marker for the specific and early detection of P. eumusae in banana.


Pseudocercospora eumusae SCAR primers Conventional PCR qPCR assay TaqMan probe 



The authors are grateful to the Director, NRC for Banana and Tiruchirappalli for providing facilities to carry out this research work and also grateful to CIRAD, France for the supply of DNA of Mycosphaerella spp. for the study. The authors are also grateful to Dr. Jeff Daniells, Agri-Science Queensland, Australia, for carrying out English editing of this manuscript.


  1. Arzanlou M, Abeln ECA, Kema GHJ, Waalwijk C, Carlier J, de Vries I, Guzman M, Crous PW (2007) Molecular diagnostics for the Sigatoka disease complex of banana. Phytopathology 97:1112–1118CrossRefGoogle Scholar
  2. Arzanlou M, Groenewald JZ, Fullerton RA, Abeln ECA, Carlier J, Zapater MF, Buddenhagen IW, Viljoen A, Crous PW (2008) Multiple gene genealogies and phenotypic characters differentiate several novel species of Mycosphaerella and related anamorphs on banana. Persoonia 20:19–37CrossRefGoogle Scholar
  3. Arzanlou M, Crous PW, Lute-Harm Z (2010) Evolutionary dynamics of mating-type loci of Mycosphaerella spp. occurring on Banana. Eukaryot Cell 9:164–172CrossRefGoogle Scholar
  4. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523CrossRefGoogle Scholar
  5. Bonants PJM, George Carroll C, de Weerdt M, van Brouwershaven R, Robert Baayen P (2003) Development and validation of a fast PCR-based detection method for pathogenic isolates of the citrus black spot fungus, Guignardia citricarpa. Eur J Plant Pathol 109:503–513CrossRefGoogle Scholar
  6. Brouwer M, Lievens B, Van Hemelrijck W, Van den Ackerveken G, Cammue B, Bart Thomma PHJ (2003) Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR. FEMS Microbiol Lett 228:241–248CrossRefGoogle Scholar
  7. Carlier J, Mourichon X, Gonzales de León D, Zapater MF, Lebrun MH (1994) DNA restriction fragment length polymorphisms in Mycosphaerella species causing banana leaf spot diseases. Phytopathology 84:751–756CrossRefGoogle Scholar
  8. Churchill A (2010) Mycosphaerella fijiensis, the black leaf streak pathogen of banana: progress towards understanding pathogen biology and detection, disease development, and the challenges of control. Mol Plant Pathol 14:307–328Google Scholar
  9. CORBANA, 1996. Combat black Sigatoka. Research Department, National Banana Corporation, Bulletin No. 4. CORBANA, San José, Costa RicaGoogle Scholar
  10. Cortinas MN, Crous PW, Wingfield BD, Wingfield MJ (2006) Multi-gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complex associated with eucalyptus stem cankers. Stud Mycol 55:133–146CrossRefGoogle Scholar
  11. Crous PW, Mourichon X (2002) Mycosphaerella eumusae and its anamorph Pseudocercospora eumusae spp. Nov.: causal agent of eumusae leaf spot disease of banana. Sydowia 54:35–43Google Scholar
  12. Crous PW, Aptroot A, Kang JC, Braun U, Wingfield MJ (2000) The genus Mycosphaerella and its anamorphs. Stud Mycol 45:107–121Google Scholar
  13. Crous P.W., Groenewald J.Z., Aptroot A., Braun U., Mourichon X., Carlier, J., 2002. Integrating morphological and molecular data sets on Mycosphaerella, with specific reference to species occurring on Musa. In: Proceedings of the 2nd International Workshop on Mycosphaerella Leaf Spot Disease of Bananas: 34–57. INIBAP, Montpellier, FranceGoogle Scholar
  14. Crous PW, Wingfield MJ, Mansilla JP, Alfenas AC, Groenewald JZ (2006) Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on eucalyptus II. Stud Mycol 55:99–131CrossRefGoogle Scholar
  15. Crous PW, Braun U, Groenewald JZ (2007) Mycosphaerella is polyphyletic. Stud Mycol 58:1–32CrossRefGoogle Scholar
  16. Cullen DW, Lees AK, Ian Toth K, James Duncan M (2001) Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers. Eur J Plant Pathol 107:387–398CrossRefGoogle Scholar
  17. Filion M, St-Arnaud M, Jabaji-Hare SH (2003) Direct quantification of fungal DNA from soil substrate using real-time PCR. J Microbiol Methods 53:67–76CrossRefGoogle Scholar
  18. Ganga Devi P, Thangavelu R (2014) Genetic diversity analyses of Mycosphaerella eumusae causing Septoria leaf spot disease of banana in India. Indian Phytopath 67:388–395Google Scholar
  19. Groppe K, Boller T (1997) PCR assay based on a microsatellite containing locus for detection and quantification of Epichloe endophytes in grass tissue. Appl Environ Microbiol 63:1543–1550Google Scholar
  20. Henderson J, Pattemore JA, Prochim SC, Hayden HL, Brunschot SV, Grice KRE, Peterson RQ, Thomas-Hall SR, Aitken EAB (2006) Black Sigatoka disease: new technologies to strengthen eradication strategies in Australia. Australas Plant Pathol 35:181–193CrossRefGoogle Scholar
  21. Hidayat I (2012) A species-specific PCR assay based on the internal transcribed spacer (ITS) regions for the identification of Mycosphaerella eumusae, M. fijiensis and M. musicola on banana. Biotropia 19:19–29Google Scholar
  22. Hussain T, Singh BP, Anwar F (2014) A quantitative real time PCR-based method for the detection of Phytophthora infestans causing late blight of potato in infested soil. Saudi J Biol Sci 21:380–386CrossRefGoogle Scholar
  23. Jaufeerally-Fakim Y, Dheema S, Benimadhu S (2009) Molecular differentiation of Mycosphaerella spp. from Musa. Australas Plant Pathol 38:461–466CrossRefGoogle Scholar
  24. Johanson A., Jeger M.J., 1993. Use of PCR for detection of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka leaf spots in banana and plantain. Natural Resources Institute, Chatham, UKGoogle Scholar
  25. Johanson A, Crowhurst RN, Rikkerink EHA, Fullerton RA, Templeton MD (1994) The use of species specific DNA probes for the identification of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka disease of banana. Plant Pathol 43:701–707CrossRefGoogle Scholar
  26. Jones DR (2000) Diseases of banana, abaca, and Enset. CABI Publishing, Wallingford, pp 37–97Google Scholar
  27. Kishore Babu B, Mesapogu S, Sharma A, Somasani SR, Arora DK (2011) Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples. Mycologia 103:466–473CrossRefGoogle Scholar
  28. Lievens B, Thomma BPHJ (2005) Recent developments in pathogen detection arrays: implications for fungal plant pathogens and use in practice. Phytopathology 95:1374–1380CrossRefGoogle Scholar
  29. Lopez-Mondejar R, Anton A, Raidl S, Rose M, Pascual JA (2010) Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass. Bioresour Technol 101:2888–2891CrossRefGoogle Scholar
  30. Maxwell A, Hardy GEJ, Wingfield MJ, Dell B (2000) First record of Mycosphaerella lateralis on eucalyptus in Australia. Australas Plant Pathol 29:279CrossRefGoogle Scholar
  31. Maxwell A, Jackson SL, Dell B, Hardy GE (2005) PCR-identification of Mycosphaerella species associated with leaf diseases of eucalyptus. Mycol Res 109:992–1004CrossRefGoogle Scholar
  32. McCartney FJ, Foster SJ, Fraaije A, Ward E (2003) Molecular diagnostics for fungal pathogens. Pest manag Sci 59: 129–142 Google Scholar
  33. Ngongo PMK (2002) Integrated crop management strategies for plant production and control of Black Leaf Streak (Black Sigatoka) disease in Democratic Republic of Congo. Disease control of Black Sigatoka. Infomusa 11:3–6Google Scholar
  34. Roe JD, Haugland RA, Vesper SJ, Wymer LJ (2001) Quantification of Stachybotrys chartarum conidia in indoor dust using real time, fluorescent probe-based detection of PCR products. J Expo Anal Environ Epidemiol 11:12–20CrossRefGoogle Scholar
  35. Schena L, Nigro F, Ippolito A (2004) Real-time PCR detection and quantification of soil borne fungal pathogens: the case of Rosellinia necatrix, Phytophthora nicotianae, P. citrophthora and Verticillium dahlia. Phytopathol Mediterr 43:273–280Google Scholar
  36. Waalwijk C, van der Heide R, de Vries I, van der Lee T, Schoen C, Costrel-de Corainville G, Häuser-Hahn I, Kastelein P, Köhl J, Lonnet P, Demarquet T, Kema GHJ (2004) Quantitative detection of Fusarium species in wheat using TaqMan. Eur J Plant Pathol 110:481–494CrossRefGoogle Scholar

Copyright information

© Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2019

Authors and Affiliations

  1. 1.Division of plant pathologyICAR – National Research Centre for BananaTiruchirappalliIndia

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