Browsing by Author "Moller, Heike"

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    Characterisation and detection of mefenoxam sensitivity in phytophthora nicotianae and phytophthora citrophthora from citrus in South Africa
    (Stellenbosch : Stellenbosch University, 2024-03) Moller, Heike; Rose, Lindy J. ; Van Niekerk, Jan; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: In South Africa, citrus is of high agricultural and economic importance, representing one of the country's major fruit crops. This sector plays a pivotal role in the nation's economy by substantially contributing to export earnings and employment opportunities. Citrus production is, however, threatened by oomycete pathogens, particularly Phytophthora, that can cause citrus diseases resulting in significant economic losses. Phytophthora nicotianae and P. citrophthora have been reported in every citrus-producing province in South Africa including citrus nurseries. These soil-borne pathogens primarily target the roots and the lower parts of citrus trees, causing root rot, lesions, gummosis, and brown rot of citrus fruit. Infected trees experience a decline in vigour, leading to stunted growth, wilting, and death in severe cases. These diseases also compromise the tree's ability to translocate water and nutrients, resulting in reduced fruit production and poor fruit quality. Mefenoxam is routinely used in citrus nurseries and orchards to treat Phytophthora infections. This chemical inhibits RNA polymerase I, responsible for rRNA synthesis. Its action prevents mycelial growth, sporangia formation, and germ tube growth, but due to its site-specificity, there is a high risk of resistance development. Continuous use of mefenoxam by citrus growers has led to the detection of mefenoxam-resistant Phytophthora isolates globally, including in South African nurseries and orchards. The monitoring of resistance to mefenoxam is important to ensure the lasting efficacy of this highly effective chemical and is reliant on the rapid and accurate detection of mefenoxam sensitivity. In this study, mefenoxam-insensitive and -sensitive P. nicotianae and P. citrophthora isolates were identified by in vitro fungicide sensitivity testing using Ridomil Gold 480 SL. These isolates were subjected to whole genome sequencing (WGS) using an optimised DNA isolation protocol to obtain high-quality, intact DNA from Phytophthora mycelia. A complete genome assembly of P. citrophthora was generated, for the first time, using PacBio HiFi long-read sequencing and used as the reference genome for WGS obtained by Illumina sequencing. Single nucleotide polymorphisms (SNPs) were detected in ABC transporter and cytochrome P450 genes as well as in RNA polymerase III subunits for P. nicotianae isolates and in RNA polymerase II and III subunits for P. citrophthora isolates. A quantitative polymerase chain reaction (qPCR) assay was developed to differentiate between mefenoxam-sensitive and homozygous-resistant P. citrophthora isolates. The specificity of this assay for P. citrophthora was validated against various other citrus soil-borne pathogens. The low number of insensitive isolates significantly limited the design of qPCR assays for P. nicotianae. Additionally, we evaluated a multiplex assay to detect P. citrophthora and assess mefenoxam sensitivity, simultaneously, although the amplification products could not be differentiated from each other, necessitating further optimisation. Overall, this study offers important genetic insights into mefenoxam sensitivity in Phytophthora, setting a foundation for the development of diagnostic tools to monitor fungicide resistance and manage citrus diseases caused by oomycetes more effectively.