Samenvatting
Pseudocercospora fijiensis is the causal agent of black Sigatoka which is the most serious leaf defoliation disease on Musa spp. (bananas and plantains). Many plantain and banana species are susceptible to black Sigatoka including the exporting Cavendish cultivars. Leaf defoliation results in significant yield loses and premature ripening of banana fruit, which is a serious problem for the banana exporting industry. The main control measure of black Sigatoka involves frequent fungicide application with a very high environmental and economic burden. Among these fungicides, the azole chemical family is one of the most frequently used fungicides for the control of the disease. Azole fungicides belong to the sterol demethylation-inhibitors (DMIs) that target the lanosterol 14α-demethylase enzyme (CYP51). One of the major problems in black Sigatokamcontrol has been the excessive and unplanned use of the DMI fungicide applications in many banana farms worldwide. This uncurbed use of the fungicide resulted in DMI resistance in pathogen population. Over time, resistance levels have increased to such an extent that the number of fungicide application cycles is now near maximum level. The reduction of sensitivity in P. fijiensis to currently used DMIs has been gradual in nature, suggesting a polygenic control (Cañas et al. 2009). Nevertheless, genetic evidence described in this thesis suggests that Pfcyp51 is the single major factor responsible for the sensitivity loss in the field. Our study is the first global analysis of DMI fungicide resistance in P. fijiensis, provides a lead to understand DMI sensitivity reduction, enables the development of better black Sigatoka management strategies, but also calls for more sustainable solutions of this unparalleled banana threat.
Chapter 1 describes the importance of the banana fruit as commodity and staple food worldwide and the impact of black Sigatoka on its cultivation. It introduces the subject of the thesis, the problem of the resistance to DMIs in the control of black Sigatoka and describes lifestyle features of the causal agent P. fijiensis, the history of fungicide control of the disease, the impact that DMI fungicides exerted in the population of this species and concludes with the set-up of the thesis.
Chapter 2 provides an historical treatise of black Sigatoka management – primarily in Costa Rica – including the strategies that were developed and applied. It concludes with a critical evaluation of the current practice and the required changes.
Chapter 3 describes an extensive worldwide phenotypic and genotypic survey of P. fijiensis resistance to DMI fungicides. The sensitivity of a set of 592 field isolates collected from various banana production zones in Colombia, Costa Rica, the Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon was tested. The sequence analyses of the 14α-demethylase enzyme CYP51 encoding the Pfcyp51 gene in 266 isolates showed a wide suite of modulations. Insertions of a 19 base pairs (bp) element found in the promoter region of the Pfcyp51 gene were described and the correlation between these changes in the Pfcyp51 gene and promoter and the increase in azole resistance was established. In addition, the contribution of the main CYP51 amino acid substitutions through the elucidation of seven in silico protein models was evaluated.
Chapter 4 describes the de-novo identification of a 19 bp element found in the promoter region of the Pfcyp51 gene. Evidence strongly suggested that insertion of this element in the promoter - up to 6 copies - of resistant strains causes over expression of the Pfcyp51 gene in comparison to strains that contain one element. PCR based assays were used to analyse the presence of the repeat element in four P. fijiensis populations of Costa Rica and some isolates from Ecuador, Africa and South East Asia. Promoter swap transformation experiments were used to analyse the role of the repeat element in the expression of the Pfcyp51 gene. This identified the repeat element as a novel component that, together with mutations in the Pfcyp51 open reading frame, are responsible for higher levels of resistance against azole fungicides.
Chapter 5 describes the generation of two F1 P. fijiensis progenies for the construction of two genetic maps that identifies the region encoding for DMI fungicide resistance using DArTseq technology. Full agreement was found between the genetic markers in either population, underlining the robustness of the approach. This genetic tool was essential to identify the genetic region that determines the resistant to DMI fungicides in the species and strongly supports the hypothesis that the Pfcyp51 gene is the single major determinant of resistance towards DMI fungicides in P. fijiensis. The mapped region comprises 250,660 bp and contains 53 putative genes, including the Pfcyp51 gene, which is the most plausible candidate as the driving molecular force for the resistance to DMI fungicides based on our and others’ findings.
Chapter 6 discusses the experimental outcomes obtained in the thesis and describes them in a broader framework. It highlights the compelling evidence that modulation of the promoter and the coding gene sequence of Pfcyp51 correlate with the observed azole sensitivity. Finally, the impact and implications of these findings are discussed for future disease control strategies.
Chapter 1 describes the importance of the banana fruit as commodity and staple food worldwide and the impact of black Sigatoka on its cultivation. It introduces the subject of the thesis, the problem of the resistance to DMIs in the control of black Sigatoka and describes lifestyle features of the causal agent P. fijiensis, the history of fungicide control of the disease, the impact that DMI fungicides exerted in the population of this species and concludes with the set-up of the thesis.
Chapter 2 provides an historical treatise of black Sigatoka management – primarily in Costa Rica – including the strategies that were developed and applied. It concludes with a critical evaluation of the current practice and the required changes.
Chapter 3 describes an extensive worldwide phenotypic and genotypic survey of P. fijiensis resistance to DMI fungicides. The sensitivity of a set of 592 field isolates collected from various banana production zones in Colombia, Costa Rica, the Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon was tested. The sequence analyses of the 14α-demethylase enzyme CYP51 encoding the Pfcyp51 gene in 266 isolates showed a wide suite of modulations. Insertions of a 19 base pairs (bp) element found in the promoter region of the Pfcyp51 gene were described and the correlation between these changes in the Pfcyp51 gene and promoter and the increase in azole resistance was established. In addition, the contribution of the main CYP51 amino acid substitutions through the elucidation of seven in silico protein models was evaluated.
Chapter 4 describes the de-novo identification of a 19 bp element found in the promoter region of the Pfcyp51 gene. Evidence strongly suggested that insertion of this element in the promoter - up to 6 copies - of resistant strains causes over expression of the Pfcyp51 gene in comparison to strains that contain one element. PCR based assays were used to analyse the presence of the repeat element in four P. fijiensis populations of Costa Rica and some isolates from Ecuador, Africa and South East Asia. Promoter swap transformation experiments were used to analyse the role of the repeat element in the expression of the Pfcyp51 gene. This identified the repeat element as a novel component that, together with mutations in the Pfcyp51 open reading frame, are responsible for higher levels of resistance against azole fungicides.
Chapter 5 describes the generation of two F1 P. fijiensis progenies for the construction of two genetic maps that identifies the region encoding for DMI fungicide resistance using DArTseq technology. Full agreement was found between the genetic markers in either population, underlining the robustness of the approach. This genetic tool was essential to identify the genetic region that determines the resistant to DMI fungicides in the species and strongly supports the hypothesis that the Pfcyp51 gene is the single major determinant of resistance towards DMI fungicides in P. fijiensis. The mapped region comprises 250,660 bp and contains 53 putative genes, including the Pfcyp51 gene, which is the most plausible candidate as the driving molecular force for the resistance to DMI fungicides based on our and others’ findings.
Chapter 6 discusses the experimental outcomes obtained in the thesis and describes them in a broader framework. It highlights the compelling evidence that modulation of the promoter and the coding gene sequence of Pfcyp51 correlate with the observed azole sensitivity. Finally, the impact and implications of these findings are discussed for future disease control strategies.
| Originele taal-2 | Engels |
|---|---|
| Kwalificatie | Doctor of Philosophy |
| Toekennende instantie |
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| Begeleider(s)/adviseur |
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| Datum van toekenning | 31 okt. 2016 |
| Uitgever | |
| Gedrukte ISBN's | 978-94-6257-879-1 |
| DOI's | |
| Status | Gepubliceerd - 31 okt. 2016 |
| Extern gepubliceerd | Ja |