Masters Degrees (Plant Pathology)

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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.
Characterisation and pathogenicity of soilborne fungi from Western Cape olive orchards
(Stellenbosch : Stellenbosch University, 2022-03) Bishop, Robyn; Spies, Chris, F. J.; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Soilborne diseases are of concern to the olive industry worldwide, causing significant financial losses. The most devastating of these diseases is Verticillium wilt caused by Verticillium dahliae, followed by Phytophthora root rot. Species of Cylindrocarpon-like fungi, Fusarium, Macrophomina, Pythium and Rhizoctonia have also been associated with these diseases on olive, resulting in symptoms of dieback, defoliation, wilting, root rot, stunted growth, and tree death. In 2019, a survey targeting Cylindrocarpon-like fungi, Phytophthora and Verticillium revealed a high incidence and wide distribution of Cylindrocarpon-like fungi (mainly the Dactylonectria macrodidyma species complex) and Pythium irregulare in Western Cape olive orchards. However, Phytophthora (three species) was recovered from only 8% of samples, and no Verticillium was found. The survey also uncovered other potential pathogenic genera such as Fusarium, Diaporthe, Macrophomina and Rhizoctonia, but these were not identified to species level. Consequently, the aims of this study were to identify the most frequently recovered fungi, refine the species identities of isolates previously identified as the Da. macrodidyma species complex, and evaluate the pathogenicity of widely distributed species and species suspected to be pathogens. Unidentified isolates form the 2019 survey were divided into morphological groups. Groups containing isolates recovered from five or more of the thirteen sampling sites were selected for identification to species level using PCR, sequencing and phylogenetic analyses. Phylogenetic analyses revealed four species of Diaporthe, fourteen species of Fusarium, one species of Macrophomina, eight species of Neocosmospora, and nine anastomosis groups (AGs) of Rhizoctonia. Morphological descriptions of two new Diaporthe species were compiled. Isolates previously identified as the Da. macrodidyma species complex were delineated into Da. macrodidyma, Da. novozelandica, Da. sp., Da. torresensis, and Da. pauciseptata. The most widely distributed species included Da. sp., Da. macrodidyma, Diaporthe sp RB-2019a., F. fabacearum, F. nirenbergiae, M. phaseolina, and Rhizoctonia AG-G. Species of Fusarium, Macrophomina, Neocosmospora, Phytophthora, Pythium and Rhizoctonia have been reported as olive pathogens in other countries, however, none of these reports identified phylogenetic species within Fusarium and Neocosmospora, and none reported AGs of Rhizoctonia. Species identified in this study that have been reported on olives previously include Da. ecuadoriensis, Da. macrodidyma, Da novozelandica, Da. sp., Da. torresensis, Da. valentina, Di. ambigua, F. oxysporum, M. phaseolina, N. solani, Pleurostoma richardsiae, and Rhizoctonia spp. In addition to the two new Diaporthe species, Cadophora constrictospora, Da. pauciseptata, F. clavus, F. udum, nine species from the Fusarium oxysporum species complex, seven species of Neocosmospora, as well as nine different AGs of Rhizoctonia are reported on olive for the first time worldwide. The pathogenicity of selected species was evaluated on three-month-old ‘Mission’ cuttings under wet, moderate and dry irrigation regimes in glasshouse trials. Twenty–nine isolates representing Da. macrodidyma, Da. novozelandica, Da. sp., Da. pauciseptata, Da. torresensis, Di. ambigua, Diaporthe. sp., F. fabacearum, F. nirenbergiae, M. phaseolina, N. solani, Ph. multivora, Ph. pseudocryptogea, Py. irregulare, Py. oligandrum, Rhizoctonia AG- G, and V. dahliae were included. Species of Da. macrodidyma, F. nirenbergiae, Ph. Multivora, Ph. pseudocryptogea, Rhizoctonia AG-G, and V. dahliae were found to be pathogenic towards olive trees. The different watering regimes had no consistent effect on pathogenicity. This study was the first to evaluate the pathogenicity of soilborne fungi on olive trees in South Africa and to report the pathogenicity of three species for the first time on olive, including F. nirenbergiae, Ph. pseudocryptogea, and Rhizoctonia AG-G. Future research should aim to address the management of these diseases on olive.
Sensitivity of citrus rootstocks to two citrus viroids and the influence on tree growth
(Stellenbosch : Stellenbosch University, 2022-03) Steyn, Chanel; Fourie, Paul; Cook, Glynnis; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Despite the supply of disease-free propagation material to the industry by the South African Citrus Improvement Scheme, citrus viroids, particularly citrus dwarfing viroid (CDVd) and hop stunt viroid (HSVd), are occasionally detected in commercial orchards. Locally, these viroids were associated with stunting of trees on sensitive trifoliate hybrid rootstocks. New rootstock cultivars are developed and, in addition to their horticultural performance, it is important to evaluate the effects of viroid infections. A field trial was established consisting of ‘Midknight’ Valencia on ten different rootstocks, including a trifoliate (‘Rich16-6’), two non-trifoliates (Rough Lemon ‘Cairn’ and ‘Rangpur’ lime) and seven trifoliate hybrids (‘US-812’, ‘MxT’, ‘C-22 Bitters’, ‘C-54 Carpenter’ and ‘C-57 Furr’ citrandarins, as well as ‘Carrizo’ and ‘C-35’ citranges). The scions were graft-inoculated with CDVd as a single infection, and in combination with HSVd to investigate potential viroid interactions. Transmission detection was notably erratic in scions on some rootstocks, particularly MxT and C-22, indicating that rootstocks may influence viroid distribution within scions. To study the effect of rootstocks on spatial distribution of CDVd and HSVd within scions on C-35, Carrizo, MxT and Rough Lemon, a relative RT-qPCR assay was developed, using GAPC2 (Glyceraldehyde-3- phosphate dehydrogenase C2) and UPL7 (Ubiquitin-protein ligase 7) reference genes for qPCR data normalization, to determine viroid concentration ratios from inner and outer scion canopy positions. Large variation in concentration ratios were seen for both viroids, demonstrating uneven spatial distribution, especially for CDVd in scions on C-35 and Rough Lemon, but also HSVd in scions on C-35. Despite this variation, the inner canopy proved to be a more reliable sampling position for detection as higher viroid titres were mostly determined from this position. Absolute CDVd and HSVd quantification was done from inner canopies of scions on the ten rootstocks. CDVd copies ranged from 1 to 831, and HSVd copies were determined in a broader range from 8 to 3963. Rootstock cultivar had a significant influence on viroid titres in the scion. Scions on C-22 and Rich16-6 had significantly lower CDVd titres than C-57, Carrizo, ‘Rangpur’ lime, Rough Lemon and US-812. However, scions on MxT and Rich16-6 accumulated significantly fewer HSVd copies than C-35, C-57, Carrizo and ‘Rangpur’ lime, indicating that viroid species accumulation differed for each rootstock. CDVd titres were not significantly influenced by co-infection with HSVd, but HSVd reached significantly higher copy numbers than CDVd in scions on all ten rootstocks, with as much as a 24-fold increase of HSVd compared to CDVd copies in scions on C-35. CDVd and HSVd genome sequences generated from a scion of each rootstock, 19 months post inoculation, were determined and mutations were detected within the pathogenic regions of both viroids. The significance of these mutations requires further investigation. After 2 years of field growth, no significant influence of viroid infections on either trunk circumference or canopy volume, were observed. However, impact on growth is expected only after 4 to 5 years in the field. This study indicated that rootstock cultivar influenced the spatial distribution, accumulation and genetic variability of two citrus viroids within ‘Midknight’ Valencia scions. The quantitative real-time RT-PCR assays developed in this study will be used in further analysis of this field trial to monitor viroid titres in both scions and rootstocks to unravel the complex interactions of the viroids with both scion and rootstocks.
Identification and evaluation of biocontrol agents of citrus replant pathogens
(Stellenbosch : Stellenbosch University, 2021-12) Reens, Sone Veniese; Van Niekerk, Jan; Mostert, L.; Stempien, E.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Citrus is the second largest fruit crop in South Africa with a well-known position in the fruit industry due to its local consumption and exports to neighbouring countries. Citrus is susceptible to several pathogens that cause incalculable losses to the crop. One of these afflictions is replant disease which is associated with a complex of soilborne pathogens. Replant disease is observed where young, healthy nursery trees are planted on old orchard sites. The phenomenon is characterised by the newly planted trees being stunted with small leaves and showing low vigour. The major soilborne pathogens associated with replant soils are Phytophthora nicotianae, Phytophthora citrophthora, Pythium irregulare s.s. and Neocosmospora species. At present, no biological control measures are available to manage the complex of replant pathogens, as previous studies conducted in South Africa have only focused on the effect of different biocontrol agents (BCAs) on Phytophthora spp. related to citrus and did not consider other oomycete pathogens such as Pythium spp., fungal pathogens such as Neocosmospora spp., or instances where these pathogens occur together. Therefore, the aim of this study was to identify and evaluate potential biocontrol agents obtained from old citrus soils for the management of the citrus replant pathogen complex. Rhizospheric soil along with their roots were collected from two orchards, Citrusdal (Western Cape) and Kirkwood (Eastern Cape) that showed replant problems. Soil samples were subjected to serial dilution plating using mediums, NA, PDA and PDA+, while roots were also plated out on these mentioned mediums. The isolation of microorganisms from soil and roots was focused on Bacillus, Pseudomonas and Trichoderma spp., as these are known to have potential as BCAs. For the preliminary selection of potential candidates from all isolates found in the citrus orchards, a non-volatile test was performed for Trichoderma spp. and for the bacterial isolates, the dual culture test was used. From the eight Trichoderma isolates and 48 bacterial isolates obtained from the citrus rhizosphere soil and roots, two Trichoderma isolates and two bacterial isolates were selected and were molecularly identified as Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens. These selected candidates inhibited the growth of the pathogen complex by at least 50% compared to the control and it was assumed that it is satisfactory to possibly be a BCA. The selected BCAs were consequently subjected to further in vitro screening, where T. harzianum isolates, P4 and P16 were subjected to a volatile and dual culture test, while B. subtilis N19 and P. fluorescens N83 were subjected to a non-volatile and volatile test. Generally, the results of the non-volatile tests were satisfactory, as it showed that Trichoderma, Bacillus and Pseudomonas spp. has different inhibitory abilities against the replant pathogens and was therefore the best screening test. It has been shown that the T. harzianum isolates can be used against the whole complex of replant pathogens, while the bacterial isolates showed only good antagonistic activity against the oomycete pathogens. Based on this observation we hypothesize that the mode-of-action for the selected BCAs could be due to antibiosis (non-volatile test). The efficacy of the selected BCAs to colonize the roots of Troyer citrange seedlings and promote plant growth was also investigated. This was done by determining the effect of the interaction between seedlings treated with the pathogen alone, biocontrol alone and pathogen with biocontrol combination, by using specific growth parameters along with re-isolation of the pathogen and BCA. High levels of the pathogen were observed in the seedlings treated with the pathogen only. The BCAs were also able to colonize the roots at high levels. However, it was also clear that the BCA or pathogen population did not decrease when combined. Two exceptions were found, where P. fluorescens N83 had a higher colonization level when challenged with P. nicotianae, and T harzianum P16 colonization levels were lower in the presence of P. irregulare s.s. Regarding the growth parameters, it was found that none of the treatments had any significant effect on the plant growth. The relatively poor response of the plants to the BCAs may be because the BCAs may have needed more time to establish a symbiotic relationship with the plant before it could have any benefits. Therefore, it would be advisable to further investigate where optimal conditions can be established to obtain a clearer picture of the influence of the BCA on the replant pathogens.
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.

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