Fusarium wilt of bananas, popularly known as Panama disease, is one of the most threatening fungal diseases of banana production. Bananas are an essential staple food and a significant income for agricultural-based economies in developing countries. Fusarium oxysporum f.sp. cubense (Foc) is the causal fungal agent of this disease. The first outbreak of the disease caused by Foc race 1 strains transformed the banana industry in Latin America in the early 1960s. Farmers rejected their preferred banana cultivar ‘Gros Michel’ as race 1 strains caused havoc in plantations that relied on this cultivar. To save the banana industry, farmers adopted the race 1-resistant Cavendish banana cultivars. Unfortunately shortly after, pioneer banana plantations using Cavendish clones in Taiwan succumbed to Fusarium wilt. This emerging strain is known as tropical race 4 (TR4), and has nowadays spread to multiple countries in Southeast Asia, Australia, the Middle East, the Indian subcontinent and Africa. Currently, there is no effective management strategy available to control this disease. This PhD thesis describes genetic diversity of Foc and its global dispersion as well as tools for rapid diagnosis of TR4 and vegetative compatible group (VCG) characterization of large Foc populations. This contributes to developing effective management strategies.
Chapter 1 is an introduction outlining the background of this thesis research: the importance of bananas, the Fusarium wilt disease cycle and the genetic diversity, classification, evolution and distribution of Foc strains. This chapter summarizes scientific publications available prior to the start of this project and our research objectives, including unraveling the genetic diversity of this pathogen, its current dissemination, available molecular diagnostic tools and pathogenicity.
Chapter 2 elaborates on the genetic diversity of Foc as captured by a suite of major genetic lineages and/or VCGs, with a focus on TR4. The data enabled comparison of re-sequenced and Diversity Array Technology sequencing (DArTseq) data of geographically diverse TR4 isolates, which suggests that a single clone is temporally and spatially dispersed. This finding underscores the need for global awareness and quarantine campaigns to protect banana producers from another pandemic that particularly hits vulnerable small-holder farmers and agricultural based countries.
Chapter 3 describes the development of a molecular detection tool to monitor the spread of TR4. In this study, we generated and used a genotyping by sequencing database to discover unique genomic regions for primer design. DNA was extracted from Foc strains representing the global genetic diversity of Foc. Alignments of the sequences with the reference genome of Foc TR4 II-5 enabled the design of Loop-Mediated Isothermal Amplification primers for TR4. We successfully used the developed assay to detect TR4 in artificially inoculated and naturally infected Cavendish banana rhizomes and pseudostems. This assay enables rapid, routine and unambiguous detection of TR4 in the field and is therefore of immense value for charting the progression of its spread.
Chapter 4 reveals the genetic diversity of a global Foc collection that includes Foc isolates from all banana-growing areas. In this study, we explore the genetic variation of Foc isolates by genotyping-by-sequencing using DArTseq. The generated 25,282 DArTseq in-silico markers grouped all isolates into three clades within the Fusarium oxysporum species complex (FOSC), showing a robust genetic resolution of genetic variation among Foc isolates within VCGs. The 24 known VCGs were associated with either clade 1 or 2, but we also report a new third clade for Foc isolates. The analyses show that DArTseq is an excellent approach to efficiently assign VCGs to Foc isolates without the need for laborious microbiological experimentation. For instance, we show for the first time that VCG0120/15 and 0126 are present in Peru and Papua New Guinea, respectively. More importantly, we detected 20 new VCGs, particularly linked with Latin America, which may point to a secondary center of diversity. Our results provide a high resolution picture of the genetic proximity of Foc strains and its global dispersal as well as highlight the immense versatility of Foc.
Chapter 5 describes the pathogenicity of the widest panel of Foc VCGs towards the iconic banana varieties ‘Gros Michel’ and ‘Grand Naine’ that dominated the international trade for over 100 years. In this study, we explore the pathogenicity of 22 Foc VCGs in addition to multiple TR4 isolates under greenhouse conditions. The results show that ‘Gros Michel’ was generally susceptible to more strains than ‘Grand Naine’. All TR4 isolates, regardless of the year of isolation and country of origin, were highly infectious on both varieties, underpinning the risk for banana plantations that only rely on ‘Gros Michel’ and ‘Grand Naine’ cultivars. The responses of ‘Gros Michel’ and ‘Grand Naine’ were indistinctively associated with Foc VCGs from clade 1 and 2 of the FOSC and did not frequently correspond with the traditionally associated Foc races. These findings show that the current race concept does not accurately reflect the pathogenic diversity of Foc.
Chapter 6 is a summarizing discussion of the PhD thesis. The generated data are examined and contextualized with overall scientific information on the genetic diversity and virulence of Foc. The setbacks of the current classification system are debated and suggestions for improvement and future directions on diversity analyses are discussed. The genetic diversity of Foc will severely impact the management of Fusarium wilt. Therefore, it is urgently required to elucidate the banana–Foc interaction and to develop molecular tools for disease monitoring and containment.
|Qualification||Doctor of Philosophy|
|Award date||16 Oct 2018|
|Place of Publication||Wageningen|
|Publication status||Published - 2018|