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.
In the last century, Fusarium wilt of banana (FWB) destroyed the banana cultivar Gros Michel. The Cavendish cultivars saved the global banana industry, and currently they dominate global production (~50%) and the export trade (~95%). However, a new strain called Tropical Race 4 (TR4) surfaced in the late 1960’s, spread globally and greatly damages Cavendish plantations as well as manifold local varieties that are primarily grown by small holders. Presently, there is no commercially available replacement for Cavendish and hence control strategies must be developed and implemented to manage FWB. Here, we studied whether it is possible to induce resistance to TR4 by pre-inoculations with different Fusarium spp. Only pre-treatments with an avirulent Race 1 strain significantly reduced disease development of TR4 in a Cavendish genotype and this effect was stable at various nutritional and pH conditions. We then used transcriptome analysis to study the molecular basis of this response. Several genes involved in plant defence responses were up-regulated during the initial stages of individual infections with TR4 and Race 1, as well as in combined treatments. In addition, a number of genes in the ethylene and jasmonate response pathways as well as several gibberellin synthesis associated genes were induced. We observed upregulation of RGA2 like genes in all treatments. Hence, RGA2 could be a key factor involved in both R1 and TR4 resistance. The data support the hypothesis that activating resistance to Race 1 in Cavendish bananas affects TR4 development and provide a first insight of gene expression during the interaction between various Fusarium spp. and banana.
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Fusarium wilt is one of the most damaging diseases of banana that currently puts the global production and food security at risk. The causal agents belong to the genus Fusarium and the soilborne species that is currently threatening Cavendish varieties and many locally important cultivars is F. odoratissimum, colloquially called Tropical Race 4 (TR4), which spreads easily and rapidly within and between farms. Even though limited information exists on the efficacy of disinfectants, they are promoted as a front-line defense against the spread of TR4 in the banana production areas by equipment and human activity. In this study, 13 disinfectants, the majority marketed in the Philippines, were tested for efficacy on TR4 mycelium, conidia, chlamydospores suspensions and chlamydospore-infested soil. They encompass five chemical groups, quaternary ammonium, halogens, alcohols, diamidines and aldehydes, and were tested at a range of concentrations and exposure times. Conidiospores were sensitive to all tested disinfectants at the manufacturer’s suggested rates. Ten disinfectants controlled mycelial growth with the exception of the quaternary ammonium-based disinfectant GUAA. Eleven disinfectants showed adequate efficacy at all tested concentrations towards chlamydospores in suspension, whereas a calcium hypochlorite-based disinfectant lacked efficacy at all concentrations tested. Despite in vitro efficacy, all disinfectants, except the diamidine-based disinfectant Formo, were largely ineffective in treating chlamydospores present in soil (in situ). However, Formo is corrosive to metals, making it less suitable for various applications. The in vitro results illustrate that overall efficacy of disinfectants depends on the fungal propagule, exposure time, and the environment in which they are exposed. We also determined the exposure times of footwear and vehicles in sanitation baths under field conditions and conclude that those significantly deviate from the minimal required contact time under in vitro conditions. Suboptimal quarantine measures, therefore, are likely to contribute to continued local, regional, and international TR4 dissemination.
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