Masters Degrees (Plant Pathology)

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Browsing Masters Degrees (Plant Pathology) by Author "Carelse, Gray-Lee"

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    Detection, quantification and pathogen interactions of citrus replant pathogens
    (Stellenbosch : Stellenbosch University, 2021-12) Carelse, Gray-Lee; Van Niekerk, Jan; Stempien, Elodie; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Citrus replant disease is a major problem in South African citrus orchards and has been reported in many other parts of the world. This disorder is prevalent in orchards where citrus trees have been grown previously for many years. Several fungi and oomycete species including Pythium irregulare, Phytophthora citrophthora, Phytophthora nicotianae, Neocosmospora solani, Neocosmospora ferruginea and Neocosmospora citricola have been implicated in citrus replant disease in South Africa. As these pathogens were found to occur together in the same soils, it could be expected that they would interact to cause typical citrus replant symptoms. However, studies of these pathogens causing replant disease alone or in combination are lacking. Previous studies have used several sets of primers for the detection of P. citrophthora and P. nicotianae in plant material; however, some of them showed sensitivity and specificity issues. Currently, there is no quantitative real-time PCR (qPCR) assays available for the newly described Neocosmospora species that have been isolated from citrus in South Africa. In this study, new species-specific qPCR primers based on Ypt1 (P. citrophthora and P. nicotianae), ITS (N. citricola), β-tubulin (N. solani) and RPB2 (N. ferruginea) genes were developed and validated for the detection of citrus replant pathogens in roots in South Africa. For Pythium irregulare, a previously published primer pair was optimised and employed in order to detect and quantify this species in citrus roots. The qPCR assays effectively detected and quantified Phytophthora, Neocosmospora and Pythium isolates in vitro. For the rapid and simultaneous detection of P. citrophthora and P. nicotianae in citrus roots, a multiplex PCR assay was developed. The multiplex PCR was highly effective in detecting the two pathogens and could be used for the simultaneous detection of P. citrophthora and P. nicotianae in citrus roots. This study investigated the role of P. irregulare, P. citrophthora, P. nicotianae, N. solani, N. ferruginea and N. citricola in citrus replant disease. Carrizo citrange rootstock seedlings were subjected to different treatments and an untreated control. Additionally, a combination of P. nicotianae and P. irregulare were included, along with N. solani combined with either of the two Phytophthora spp. Also included were a mixture of N. solani with P. nicotianae and P. irregulare. At trial termination, the shoot length, seedling fresh mass, root fresh mass and root volume were determined. The combination of N. solani + P. nicotianae + P. irregulare s.s. were the only inoculation that caused a significant reduction in seedling height. All the species caused a reduction in mean root mass, except for N. solani + P. citrophthora which were found to increase the mean root mass. Isolations were made from the roots of inoculated Carrizo citrange rootstock seedlings. Isolation studies showed that the Neocosmospora spp. were among the most frequently isolated species (range of isolation 62.1 to 67.5%). The combination of P. nicotianae + P. irregulare s.s. had the highest percentage of roots infected by P. irregulare s.s. (61.7%). Phytophthora citrophthora and P. nicotianae were also isolated from seedling roots. The percentage of roots infected with P. citrophthora ranged from 24.2 to 45.4%, whereas the percentage of roots infected with P. nicotianae ranged from 20.8 to 45.0%. Pathogen detection and quantification were determined by quantitative real-time PCR (qPCR) using the new and existing primers. The mean DNA concentrations of P. irregulare ranged from 1.6x10⁻⁴ to 2.7x10⁻³ ng/μL. Mean DNA concentrations of P. citrophthora were between 0 and 2.5x10⁻⁴ ng/μL, and for P. nicotianae were from 1.7x10⁻² to 6.4x10⁻² ng/μL. The Neocosmospora mean DNA concentrations ranged from 1.1x10⁻³ to 2.9x10⁻² ng/μL. The qPCR assays successfully detected and quantified P. irregulare, P. citrophthora, P. nicotianae, N. solani, N. ferruginea and N. citricola from citrus roots. This may be especially important since it provides a new opportunity for analysing citrus replant pathogen populations and their interactions.