A loop-mediated isothermal amplification (LAMP) assay based on unique markers derived from genotyping by sequencing data for rapid in planta diagnosis of Panama disease caused by Tropical Race 4 in banana

N. Ordóñez, M. Salacinas, O. Mendes, M. F. Seidl, H. J.G. Meijer, C. D. Schoen, G. H.J. Kema*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL−1 pure TR4 DNA or 102 copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.

Original languageEnglish
Pages (from-to)1682-1693
Number of pages12
JournalPlant Pathology
Volume68
Issue number9
DOIs
Publication statusPublished - 1 Dec 2019

Bibliographical note

Funding Information:
This research is funded by the Interdisciplinary Research and Education Fund (INREF) of Wageningen University & Research (WUR), Wageningen, Netherlands, comprising private and public partners, see www.panamadisease.org . Banana research at WUR is supported by the Dutch Dioraphte Foundation endowed chair in Tropical Phytopathology of G.H.J.K. at the WUR‐Laboratory of Phytopathology. Research in the laboratory of M.F.S. is supported by the Research Council Earth and Life Science (ALW) of The Netherlands Organization of Scientific Research (NWO). NEH‐Philippines generously supported field experimentation and Tadeco‐Philippines provided moko isolates from Cavendish banana. The authors thank Rahan Meristem, Israel, for providing all greenhouse plants for experimentation and Nani Maryani for making infected plants and Indonesian Fusarium isolates available for this project. Finally, the authors acknowledge the support of Iris Visscher for assisting on LAMP testing of Indonesian isolates. UNIFARM, WUR, Netherlands is greatly acknowledged for greenhouse maintenance and plant care.

Publisher Copyright:
© 2019 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.

Funding

This research is funded by the Interdisciplinary Research and Education Fund (INREF) of Wageningen University & Research (WUR), Wageningen, Netherlands, comprising private and public partners, see www.panamadisease.org . Banana research at WUR is supported by the Dutch Dioraphte Foundation endowed chair in Tropical Phytopathology of G.H.J.K. at the WUR‐Laboratory of Phytopathology. Research in the laboratory of M.F.S. is supported by the Research Council Earth and Life Science (ALW) of The Netherlands Organization of Scientific Research (NWO). NEH‐Philippines generously supported field experimentation and Tadeco‐Philippines provided moko isolates from Cavendish banana. The authors thank Rahan Meristem, Israel, for providing all greenhouse plants for experimentation and Nani Maryani for making infected plants and Indonesian Fusarium isolates available for this project. Finally, the authors acknowledge the support of Iris Visscher for assisting on LAMP testing of Indonesian isolates. UNIFARM, WUR, Netherlands is greatly acknowledged for greenhouse maintenance and plant care.

Keywords

  • DArTseq
  • field diagnostic
  • Fusarium odoratissimum
  • LAMP
  • Musa
  • Tropical Race 4

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