Molecular Diagnostics of Banana Fusarium Wilt Targeting Secreted-in-Xylem Genes.

Lilia C Carvalhais,Elizabeth Czislowski,Juliane Henderson,Vivian A Rincon-Florez,Cecilia O’Dwyer,Elizabeth A B Aitken,André Drenth

doi:10.3389/fpls.2019.00547

Abstract

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

Highlights

Commercial banana production is under serious threat worldwide
The alignment of different homologs of the genes SIX1, SIX13, SIX8, SIX6 allowed the identification of exclusive sequences for tropical race 4 (TR4), R4 (VCG 0122), subtropical race 4 (STR4) and R1, respectively
This assay was proven to be repeatable, This study supports the utilization of secreted in xylem (SIX) genes as targets for molecular diagnostics for races of Foc

Summary

Introduction

Commercial banana production is under serious threat worldwide. A destructive disease caused by the fungus Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) is spreading rapidly throughout the banana growing regions around the globe (Ordonez et al, 2015; Zheng et al, 2018). Cubense tropical race 4 (TR4) is spreading rapidly throughout the banana growing regions around the globe (Ordonez et al, 2015; Zheng et al, 2018). TR4 has decimated the Cavendish banana industry in Taiwan, but has been making its way through Southeast Asia in China, Indonesia, Malaysia, Philippines, Laos, Myanmar, Vietnam, and Pakistan (Ordonez et al, 2015, 2016; Chittarath et al, 2018; Hung et al, 2018; Zheng et al, 2018). Intercontinental spread has occurred with reports of the pathogen in Australia, Jordan, Lebanon, Oman, and Mozambique (Butler, 2013; García-Bastidas et al, 2014; Ploetz, 2015b; Dita et al, 2018)

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