Browsing by Author "Matthews, Megan Ceris"
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- ItemThe development of novel molecular diagnostic assays for fusarium oxysporum f. Sp. Cubense(Stellenbosch : Stellenbosch University, 2019-04) Matthews, Megan Ceris; Viljoen, Altus; Rose, Lindy J. ; Mostert, Diane; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Banana (Musa sp.) is an important crop for food security and income generation in developed and developing nations. Most bananas are grown for local consumption, with approximately only 14% exported to Europe, the US, Japan and Russia. The export industry is almost exclusively reliant on Cavendish bananas. Cavendish bananas also constitute approximately 47% of bananas grown globally. A major constraint to sustainable banana production is Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Race 1 and 2 Foc isolates cause disease of Gros Michel, other dessert varieties and some cooking banana like Bluggoe, but not Cavendish bananas. Subtropical (STR) and tropical (TR4) race 4 Foc isolates affect Cavendish and most banana cultivars susceptible to Foc races 1 and 2 in the subtropics and tropics, respectively. Fusarium wilt is difficult to manage as Foc chlamydospores can survive in the soil for decades. Phytosanitary regulation and clean planting material to exclude Foc, and the planting of resistant banana cultivars, are therefore required to manage the disease. Early detection and the geographic mapping of Foc can inform farmers and governing bodies about the distribution of the fungus and aid in containment strategies. DNA-based detection with PCR is favoured over phenotypic identification due to its speed and accuracy. PCR detection, however, is qualitative and lacks sensitivity when DNA is isolated from environmental samples. Quantitative (q)PCR has been developed to directly detect plant pathogens in environmental samples. Molecular markers are available to quantitatively detect Foc races 4 and TR4, but the Foc TR4 markers lack specificity. Molecular markers for Foc Lineage VI (race 1 and 2) and STR4 are also required. In this study, DNA markers and qPCR assays were developed to detect Foc Lineage VI, TR4 and STR4 in plant, water and soil samples. Markers were designed from the RNA polymerase III subunit, a hypothetical protein and the Foc supercontig 1.57 gene regions. Marker suitability and specificity was evaluated, and standard curves produced for Foc detection based on specificity, repeatability, reproducibility, limit of quantification (LOQ) and limit of detection (LOD). The Foc TR4 and Lineage VI markers were specific for qPCR detection, but the Foc STR4 markers amplified two non-target Fusarium members and two non-pathogenic F. oxysporum isolates. Quantitative PCR can detect Foc collected in the environment. DNA from non-viable cells, however, is then also amplified, which can lead to an overestimation of inoculum levels. In this study, propidium monoazide (PMA) and qPCR were combined to quantify Foc Lineage VI and TR4 spores that survive following sanitiser treatments. PMA applied at 20 μM, incubated in the dark for 1 min and activated with light for 5 min effectively separate viable from non-viable Foc spores. The PMA-qPCR results also correlate well with colony forming unit counts.