Masters Degrees (Plant Pathology)
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- ItemCharacterisation 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.
- ItemAssessment of hot water treatment for control of grapevine trunk diseases in nurseries(2020-12) Webber, Matthew; Halleen, Francois; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Grapevine trunk diseases (GTDs) have been identified as a major factor contributing towards the decline of grapevines. The main GTDs in South Africa, and the pathogen species commonly associated with them, include Petri disease (Phaeomoniella chlamydospora, Phaeoacremonium minimum, Phaeoacremonium parasiticum, Cadophora luteo-olivacea and Pleurostoma richardsiae), Black foot disease (Campylocarpon fasciculare, Campylocarpon pseudofasciculare, Dactylonectria macrodidyma and Ilyonectria. liriodendri), Botryosphaeria canker and dieback (Neofusicoccum australe and Neofusicoccum parvum) and Phomopsis dieback (Diaporthe ampelina). Although GTDs are commonly associated with mature vines in established vineyards, it is of particular concern that planting material supplied by grapevine nurseries may already contain infections. Unfortunately, once infected, there are limited management strategies available to control GTD infections, with chemical and biological controls largely focusing on protection of pruning wounds. Hot water treatment (HWT) has shown to be effective in controlling a wide range of fungal pathogens from grapevines. Until recently, the HWT protocol recommended to South African nurseries was 50°C for 30 min. Although this protocol is already well studied and has shown to be effective against a wide range of GTD pathogens, it is also known that it does not completely eradicate all infections. A new HWT protocol of 50°C for 45 min has, however, been recommended to South African nurseries for the control of Aster Yellows. The effect of this HWT protocol on controlling fungal GTD pathogens has, however, yet to be determined under South African conditions. The aim of this study was, therefore, to determine the effect of HWT (50°C for 45 min) on fungal pathogens associated with GTDS found in South African grapevine nurseries, firstly in vitro, followed by in artificially inoculated rootstock cuttings of Ramsey, Richter 110, US 8- 7, Paulsen 1103 and 143B Mgt. Pathogen species evaluated in this study include Pa. chlamydospora, Pm. minimum, Pm. parasiticum, Ca. luteo-olivacea, Pl. richardsiae (Petri disease), Camp. fasciculare, Camp. pseudofasciculare, I. liriodendri, D. macrodidyma (Black foot disease), N. australe, N. parvum (Botryosphaeria canker and dieback) and D. ampelina (Phomopsis dieback). In vitro results concluded HWT (50°C for 45 min) was able to cause complete inhibition of conidial germination and mycelial growth of all pathogen species associated with Black foot disease, Botryosphaeria canker and dieback and Phomopsis dieback. Pathogens associated with Petri disease were, however, more tolerant of HWT (50°C for 45 min), with Pl. richardsiae identified as the most tolerant species within the disease complex. Of the Petri disease pathogens, Pa. chlamydospora was seen to be the most sensitive to HWT, followed by Ca. luteo-olivacea. A general trend observed for all pathogen species was that conidial germination was more sensitive to HWT than mycelial growth. Additionally, the effect of HWT using water temperatures greater than 50°C were also investigated using pathogen species able to tolerate 50°C. Pleurostoma richardsiae once again showed the highest tolerance to HWT with temperatures of up to 60°C not able to achieve complete control, suggesting HWT is not an effective means for controlling this pathogen. Results from the in vivo experiments concluded, with the exception to Pl. richardsiae, HWT (50°C for 45 min) was highly effective in reducing the presence of the inoculated pathogens, completely inhibiting the recovery of Pa. chlamydospora and Ca. luteo-olivacea from HWTed material. Although HWT did not completely remove the presence of Pm. minimum and Pm. parasiticum, the incidence and severity of which these species were able to be recovered from HWTed cuttings was significantly reduced. The effect of HWT on recovery of Pl. richardsiae was less consistent. The treatment was not able to significantly reduce the incidence of cuttings from which Pl. richardsiae was recovered from, however, was able to significantly reduce the severity of the infections, although inconsistently. Even though HWT (50°C for 45 min) may not eradicate all internal infections, it still provides nurseries a convenient and effective means of controlling a wide range of GTD pathogens in a single application. HWT of grapevine nursery material remains highly recommended and should be used in an integrated approach combined with all other available management strategies for optimal control of GTDs.
- ItemEtiology and control of Phomopsis cane and leaf spot of grapevines in the Western Cape(Stellenbosch : Stellenbosch University, 2020-12) Lebenya, Palesa; Halleen, Francois ; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Phomopsis cane and leaf spot (“streepvlek”) is an important grapevine disease caused by Diaporthe ampelina. An increase in “streepvlek” symptoms, including bleached canes, have been observed in Western Cape vineyards. This phenomenon has been observed on table as well as wine grapes. Several Diaporthe species have recently been isolated from internal wood necrosis associated with Phomopsis dieback in South Africa and raised the question whether other Diaporthe species are also associated with typical “streepvlek” symptoms, which might explain poor control observed in vineyards. A number of fungicides are registered in South Africa against Phomopsis, namely calcium polysulphide, copper oxychloride, copper oxychloride + sulphur, copper oxychloride+lime, folpet, mancozeb, maneb + zinc oxide, propineb and polysulphide sulphur. The fungicides used for the protective control of D. ampelina are aimed at the protection of young plant material during critical periods such as when conidia are released after prolonged rainy periods in spring. A detailed survey of vineyards in the Western Cape Province with typical “streepvlek” symptoms was performed to determine whether D. ampelina was the causative agent, or if other Diaporthe spp. are associated with these symptoms. In each vineyard, 100 grapevines were visually inspected for “streepvlek” symptoms. A total of 118 vineyards representing 32 grapevine cultivars from different regions in the Western Cape were surveyed for symptoms on green shoots and leaves. Forty-eight vineyards representing 19 cultivars were surveyed for bleached cane symptoms from the same regions. Symptomatic shoots were collected and taken to the laboratory where isolations were conducted. Diaporthe-like isolates were first identified using D. ampelina species-specific PCR primers (D. amp_F1/D. amp_ R2), and for a representative sub-sample of isolates, the internal transcribed spacer region (ITS1, 5.8S rRNA, and ITS2) and the beta-tubulin (tub2) were sequenced to confirm the results. Most symptoms were observed in the Durbanville, Stellenbosch and Somerset West areas, with Shiraz and Cabernet Sauvignon being the most susceptible cultivars. Diaporthe ampelina was associated with 100% of the typical cane and leaf spot symptoms as well as bleached canes and is undoubtedly the causal organism of Phomopsis cane and leaf spot in the Western Cape. Since there was, an increase in “streepvlek” symptoms, including bleached canes in the Western Cape during the survey, a study was conducted to determine if the fungicides used to control the disease was still effective. Firstly, in vitro sensitivity of D. ampelina against a selection of registered fungicides was assessed. Secondly, the effectiveness of mancozeb, folpet and maneb + zinc oxide in the laboratory on detached Bukkettraube shoots against artificial spray inoculation of D. ampelina conidia was evaluated, and thirdly, the effectiveness of mancozeb, folpet and maneb + zinc oxide in the field on Bukettraube and Cabernet Sauvignon vineyards against natural infection was evaluated. The field treatments were applied three times, the first at bud break, then after every seven days. The EC50 values for the seven fungicides tested in vitro indicated that mancozeb and folpet were more effective at lower concentrations in suppressing the growth of D. ampelina. In the detached shoots assay, folpet and mancozeb contained infection by D. ampelina to 23.08%, and maneb + zinc oxide to 34.62% on the leaves. On shoots, folpet contained infection to 5.77%, mancozeb to 11.54% and maneb +zinc oxide to 38.46%. In the field, on the Cabernet Sauvignon shoots, folpet and mancozeb contained infection to 8.3% and 5.5%, respectively, significantly better than maneb + zinc oxide (16.7%), although the maneb + zinc oxide treatment was significantly less than in the control treatment (77.8%). In the Bukettraube vineyard the three fungicide treatments resulted in similar disease incidences on the shoots, namely 0% for folpet and mancozeb and 8.3% for maneb +zinc oxide. These were significantly lower than in the water control (36.1%). Results from the in vitro assay, detached shoots, as well as field trials clearly showed that Phomopsis cane and leaf spot can be controlled by fungicides currently registered in South Africa. The perceived lack of control in industry or by farmers may be due to incorrect application and timing of fungicides.
- ItemFungal composition and mycotoxin contamination of commercial wheat in South Africa in association with climate and agronomic practices(Stellenbosch : Stellenbosch University, 2021-03) Schreuder, Huibrecht M.; Rose, Lindy J. ; Viljoen, Altus; Van Coller, Gert J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Mycotoxigenic fungi play an important role in wheat production. They produce toxic secondary metabolites that are detrimental to human and animal health and some of these fungi are also phytopathogens. Fusarium spp. are also responsible for Fusarium head blight which is one of the most destructive diseases of wheat, globally while Fusarium mycotoxins deoxynivalenol (DON), nivalenol and zearalenone (ZEA) are most frequently detected in wheat grain. Alternaria spp. are ubiquitously associated with wheat in most regions and can cause black point, leaf blight and leaf spot on wheat. Mycotoxins produced by this genus, such as alternariol, alternariol monomethyl ether and tenuazonic acid, frequently contaminate wheat grain. Other mycotoxigenic fungi present in wheat grain include Penicillium spp. that can produce ochratoxins, Aspergillus spp. that can produce ochratoxin and aflatoxin and Claviceps spp. that produces ergot alkaloids. Disease and mycotoxin contamination caused by mycotoxigenic fungi can be managed with the integrated use of tillage practices, crop rotation, fungicides, host resistance and disease forecasting systems. To determine the fungal composition and mycotoxin contamination in commercial wheat grain in South Africa, wheat was sampled over two seasons at 49 locations across all major wheat production regions. A total of 4 223 fungal isolates were obtained with Alternaria as the predominant genus (87%) followed by Fusarium and Epicoccum (4%), respectively. Fusarium graminearum (25%) and F. poae (15%) were the Fusarium spp. with the highest abundance and incidence in samples. The biggest difference in fungal composition was found between the production regions of the Western Cape and those isolated from the rest of South Africa. Samples from the Western Cape had a higher abundance of Alternaria spp., but the fungal diversity in these samples were lower than samples from other provinces. DON was detected in 12 samples and 3-acetyldeoxynivalenol in three samples, while 15-acetyldeoxinivalenol, ZEA and sterigmatocystin were only detected in one sample each. To determine the influence of agronomic practices and climate on fungal composition and mycotoxin contamination, information on agronomic practices was obtained from growers and weather data (humidity and temperature) was measured with data loggers at each location. Associations were found between the incidence of DON and rotations with F. graminearum host crops (maize, wheat, barley and soybeans) and also between irrigation and the incidence of Cladosporium, Epicoccum, Penicillium, Nigrospora, F. brachygibbosum, the Fusarium incarnatum-equiseti species complex, F. poae and F. oxysporum. A positive correlation was found between F. graminearum and DON contamination. Correlations were also found between weather conditions before anthesis and the abundance of Alternaria, Epicoccum, Nigrospora and F. brachygibbosum. This study reports on the fungal composition and natural mycotoxin contamination of commercial wheat in South Africa in association with weather and agronomic practices. It revealed the distribution of fungal genera in the different wheat production areas and showed that mycotoxin contamination is relatively low in South African wheat grain. It further highlights certain relationships between climate, agronomic practices, fungal composition and mycotoxin contamination in commercial wheat. Future studies should use polymerase chain reaction- based methods to determine fungal biomass in wheat grain to allow for the accurate determination of correlations between weather variables and fungi in grain.
- ItemEvaluation of foliar fungicides for the control of mycotoxigenic fungi associated with Fusarium head blight of wheat in South Africa(Stellenbosch : Stellenbosch University, 2021-03) Jacobs, Carlynn Melissa; Rose, Lindy J. ; Viljoen, Altus; Van Coller, Gert J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Wheat (Triticum aestivum L.) is considered one of the most important cereal grains constrained by the fungal genus Fusarium, globally as well as in South Africa. Fusarium species are the causal organisms of Fusarium head blight (FHB), an economically important disease that results in significant yield and grain quality losses. These plant pathogens are also known to produce secondary metabolites called mycotoxins that further reduce grain quality, but also induce foodborne intoxication in humans and animals when contaminated grain is ingested. Trichothecene type B mycotoxins, especially deoxynivalenol (DON), is important in South Africa as it is predominantly associated with commercially produced wheat. Management practices to reduce FHB and mycotoxin accumulation depends on factors like agronomical practices, resistant cultivars, and the use of fungicides. To date, there are no fungicides registered for the control of FHB in South Africa, yet fungicides are extensively used in wheat production. Therefore, this study aimed to determine the potential of foliar fungicides, currently used in South Africa’s wheat production, to reduce FHB and mycotoxin accumulation. Fungicides evaluated in this study included Folicur (tebuconazole), Prosaro (prothioconazole + tebuconazole) and Abacus (pyraclostrobin + epoxiconazole). Initially the effect of these fungicides was tested in vitro at different concentrations while three agar-based assays were used to assess the sensitivity of Fusarium and Alternaria pathogens. Following this optimisation, the fungicide sensitivity of three Fusarium species was determined on a population of 25 isolates per species. The sensitivity of isolates, based on the effective concentration (EC50 and EC90) values, differed significantly (P<0.05) for each species and fungicide tested. Fusarium graminearum was the least sensitive to prothioconazole + tebuconazole with EC50 and EC90 values ranging from 0.51 ppm to 2.35 ppm while F. pseudograminearum was the least sensitive to both tebuconazole [EC50:0.96 ppm and EC90: 3.07 ppm] and pyraclostrobin + epoxiconazole [EC50:1.82 ppm and EC90:51.3 ppm]. Overall, prothioconazole + tebuconazole showed the best efficacy in reducing fungal growth of FHB pathogens. Significant differences of fungicides on mycotoxin production were also obtained. A significant cultivar x fungicide interaction was determined for all FHB disease parameters measured in the greenhouse trial while a significant fungicide x cultivar x treatment interaction was determined for the field trial. Generally, the application of fungicides significantly decreased FHB incidence, decreased the percentage Fusarium-damaged kernels and increased the thousand kernel weight of most wheat cultivars evaluated. The results of the study provide support for the use of commercial foliar fungicides that can additively contribute to the management of FHB and mycotoxin contamination.