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.
Fungal 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.
Evaluation 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.
Fusarium verticillioides infection and fumonisin production during maize kernel maturation
(Stellenbosch : Stellenbosch University, 2021-03) Shaikh, Ayesha; Rose, Lindy J. ; Viljoen, Altus; Van Zyl, Karlien; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Maize (Zea mays. L), a cereal crop readily consumed by humans and animals globally, is subject to infection by fungal pathogens such as Fusarium verticillioides. This pathogen is found wherever maize is grown, causes Fusarium ear rot (FER) and is also capable of producing harmful secondary metabolites known as mycotoxins. Fumonisins are the most important class of mycotoxins. Fumonisin contaminated maize has been associated with irreversible, nocuous effects in humans and animals. For this reason, fumonisins are of great concern to farmers and researchers. More recently, attention has been given to hidden fumonisins. These are fumonisin molecules trapped within the maize kernel that are not detected during mycotoxin screening and are potentially liberated upon ingestion, thus contributing to a greater risk of exposure. Fusarium verticillioides can be managed using cultural practices and controlling maize- feeding insects. However, once conditions that favour the growth of the pathogen occur, these practices are no longer adequate to prevent fungal infection in the field. Not only is the use of chemicals not feasible, but there are also no registered chemicals available for the control of F. verticillioides in South Africa. Incorporating host resistance into locally adapted maize serves as the most environmentally friendly means of managing F. verticillioides. This requires a better understanding of factors that may contribute to disease development and progression. As the physical and biochemical composition of maize contribute to resistance to FER, understanding the relationship between structural and physico-chemical factors and fungal infection as well as fumonisin contamination would provide pivotal knowledge for breeding resistant maize cultivars. In this study, we utilised locally adapted maize inbred lines with known response to FER and/or fumonisin contamination to investigate the role that structural traits, such as husk coverage, silk length, silk detachment and silk browning, may have on FER and/or fumonisin and hidden fumonisin contamination. We also investigated physico-chemical properties of the maize kernel, such as pH, moisture, total carbon and nitrogen, fatty acids and starch in the form of amylopectin. Maize ears were inoculated at 7 days after pollination (dap) while another independent set of maize plants were inoculated 35 dap and grain was subsequently harvested at 7, 28, 42 and 52 days after inoculation (dai). Infection indicators (FER disease severity, F. verticillioides target DNA and fumonisin contamination) as well as hidden fumonisins were correlated with the physico-chemical properties. Fusarium verticillioides growth and fumonisins increased progressively over time after inoculation, reaching a maximum at 52 dai for both inoculation events with significant differences between inoculated and control maize grain. Inoculated grain of resistant lines accumulated lower levels of F. verticillioides target DNA and fumonisins when inoculated 7 dap, however, when inoculated 35 dap resistant lines showed an increase in fumonisin contamination. The susceptible line accumulated high levels of fungal DNA and fumonisins in both inoculation events. Of the physico-chemical properties evaluated, pairwise correlations revealed that carbon and nitrogen had strong significant association with fungal DNA at both inoculation events. Silk browning, carbon, nitrogen and C/N were also significantly correlated with infection indicators. Moisture content had a significant negative association with fungal DNA at both inoculation events. Amylopectin increased over time in Inoculation Event 1 and remained constant in Inoculation Event 2 while no significant associations were observed between amylopectin and infection indicators. The fatty acid profile showed a synchronised increase and decrease over time, however, no significant associations with infection indicators or hidden fumonisins were noted. Hidden fumonisins extracted using alkaline hydrolysis was significantly higher when only free hydrolysed fumonisins was extractedusing a standard fumonisin extraction. Furthermore, hidden fumonisins followed a similar trend as free fumonisins, increasing over time after inoculation and peaking at the mature stages of kernel development. Inoculated resistant maize grain accumulated significantly less hidden fumonisins. Lastly, there were no significant correlations between hidden fumonisins and any of the physico-chemical factors evaluated. This study demonstrated that F. verticillioides growth and fumonisin accumulation in maize grain is dependent on the timing of infection and is not triggered by a specific kernel developmental stage. Response of maize lines should also be assessed by artificially inoculating early stages of kernel maturation to determine accurate plant response in matured grain. Physico-chemical factors, such as carbon, nitrogen and C/N, may serve as indicators of potential resistance to F. verticillioides and/or fumonisins. The maize inbred lines used in this study can now be further classified according to their ability to accumulate both free fumonisins and hidden fumonisins.
Evaluation of new postharvest fungicides for the control of Phytophthora brown rot
(Stellenbosch : Stellenbosch University, 2020-03) Van der Merwe, Elizabeth; Van Niekerk, Jan; Lennox, Cheryl L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Brown rot is a citrus postharvest disease caused by Phytophthora spp. during continuous wet conditions. Fruit closest to the soil surface in the orchard are usually infected when the infecting propagules present are splashed upward during irrigation or rain. When infections on fruit are still in early development, it may go unnoticed when fruit are harvested. Infections develop further after harvest and can infect other fruit while in storage or transit. In the current study, the efficacy of actives azoxystrobin, fludioxonil and potassium phosphite was tested for the control of postharvest Phytophthora brown rot on citrus, as there is currently nothing registered for the management of this disease in South Africa. The fungicide sensitivities of 121 Phytophthora nicotianae isolates belonging to either a previously unexposed population or previously possibly exposed population, were tested for against the strobilurin azoxystrobin, based on the growth of mycelium. The mycelial growth on corn meal agar (CMA) amended with azoxystrobin at 0-, 0.25-, 0.5-, 1-, 10-, 100-, and 2000 µg/ml with the addition of 100 µg/ml salicylhydroxamic acid (SHAM), to inhibit the alternative respiration route, was measured after 7 days. The addition of SHAM did not have a significant effect on the growth of mycelia. CMA was also amended with the phenylpyrrole fludioxonil at 0-, 1-, 100-, 1000-, and 10 000 µg/ml. The effective concentration for 50% reduction of mycelial growth (EC50) for azoxystrobin ranged from 0.01 to 0.46 µg/ml for both populations and the EC90 ranged from 4.28 to 84.85 µg/ml for both populations. Fludioxonil sensitivity had a much wider range and higher EC values. The EC50 for both populations ranged from 3.10 to 1613.52 µg/ml and the EC90 values were 1090.50 to 9929 µg/ml. Subsequently, curative and protective actions of aqueous postharvest dip treatments, were carried out with the use of azoxystrobin at 1125 µg/ml and fludioxonil at 598 µg/ml. With the in vivo trials, potassium phosphite was added as an additional treatment at 1500 µg/ml. These trials were repeated on three fruit types namely lemons, oranges and mandarins and at four time intervals. The data clearly showed that with all three fungicides, the curative efficacy is excellent when treatments occurred up to 12 hrs after inoculation. Azoxystrobin and potassium phosphite exhibited excellent protective activity up to 48 hrs between treatment and inoculation. An overall trend that was seen with all three fruit types, was that the longer the fungicide was present on the fruit, the better the protective activity. The nesting data clearly demonstrated that only azoxystrobin amended wax significantly reduced brown rot from spreading to the healthy fruit, when compared to the control. With this study, it could be seen that all three fungicides, with their different actives, have the potential to effectively manage postharvest brown rot. Considering that azoxystrobin and fludioxonil are already registered on citrus for the postharvest use of Penicillium management, and potassium phosphite that are registered as a preharvest Phytophthora treatment, this study indicate that they will also provide added protection against Phytophthora brown rot.

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