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

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

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.00547

DOI=10.3389/fpls.2019.00547

ISSN=1664-462X

ABSTRACT=<p>Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus <italic>Fusarium oxysporum</italic> f. sp. <italic>cubense</italic> (<italic>Foc</italic>), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to <italic>Foc</italic> 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, <italic>Foc</italic> 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 <italic>secreted in xylem</italic> (<italic>SIX</italic>) genes, which currently comprise the only known family of effectors in <italic>F. oxysporum</italic>, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of <italic>Foc</italic>. We present specific and reproducible diagnostic assays based on conventional PCR targeting <italic>SIX</italic> genes, using as templates DNA extracted from pure <italic>Foc</italic> cultures. Sets of primers specifically amplify regions of: <italic>SIX6</italic> in <italic>Foc</italic> race 1, <italic>SIX1</italic> gene in TR4, <italic>SIX8</italic> in subtropical race 4, <italic>SIX9</italic>/<italic>SIX10</italic> in <italic>Foc</italic> VCG 0121, and <italic>SIX13</italic> in <italic>Foc</italic> VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes <italic>SIX1</italic> and <italic>SIX13</italic>. Assay validations were conducted to a high international standard including the use of 250 <italic>Fusarium</italic> spp. isolates representing 16 distinct <italic>Fusarium</italic> species, 59 isolates of <italic>F. oxysporum</italic>, 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.</p>