- ItemFusarium verticillioides infection and fumonisin production during maize kernel maturation(Stellenbosch : Stellenbosch University, 2021-03) Shaikh, Ayesha; Rose, Lindy; 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.
- ItemDevelopment of a protocol for the rapid in vitro establishment of Eucalyptus clones(Stellenbosch : Stellenbosch University, 2020-03) Keret, Rafael; Hills, Paul N.; Nakhooda, Muhammad; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: The practice of cultivating Eucalyptus species using micropropagation has found favour in the development of successful forestry plantation programs due to its effectiveness in generating large numbers of juvenile plant stocks. Natural phytohormones and their synthetic analogues, called plant growth regulators (PGRs), are typically included in growth media during micropropagation. These compounds may be used to stimulate certain physiological processes in plants, allowing, for example, the in vitro manipulation of plant cell growth and differentiation, depending on the concentrations and combinations of specific PGRs in the medium. The development of micropropagation programs for Eucalyptus spp. are often clone-specific, necessitating optimisation for each clone. There is thus an inherent need for the development of a standardized protocol permitting the in vitro establishment and proliferation of multiple Eucalyptus clones. In the present study, the sensitivity and responsiveness of three Eucalyptus grandis × Eucalyptus nitens clones to various PGRs were investigated for the different in vitro growth stages. They include auxins such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA). Similarly, the cytokinins kinetin, 6-benzylaminopurine (BAP) and meta-topolin were tested. The effects of strigolactones on micropropagation were examined using the synthetic strigolactone analogue, GR24. Parameters such as explant multiplication, shoot elongation and rooting were examined for all clones. The explants were incubated on a reduced PGR maintenance medium, from which source material was derived for subsequent experimentation, enabling the growth stages to be investigated independently. The highest level of bud initiation and shoot growth was observed for all Eucalyptus clones when the growth medium was supplemented with 0.5 mg ℓ-1 of meta-topolin in combination with 0.1 mg ℓ-1 of IAA. Optimal elongation for all the clones was detected upon addition of meta-topolin at 0.05 mg ℓ-1 and IAA at 0.5 mg ℓ-1. The peak rooting response was obtained with 0.5 mg ℓ-1 of IBA for clones 2 and 3, whilst 0.029 mg ℓ-1 of GR24 in combination with 0.5 mg ℓ-1 of IAA elicited optimal rooting for clone 1. The most consistent rooting response across all clonal lines, however, was obtained with the GR24 treatment. Clone 2 was found to be the most responsive to in vitro stimulation by auxin and this observation was further probed via RT-qPCR. Expression of the genes encoding the auxin efflux and influx transporters PIN1 and AUX1, respectively, were analysed to assess whether clonal response could be linked to expression of a particular gene(s). Equal expression levels of PIN1:AUX1 were detected for clone 2. Clones 1 and 3, however, exhibited an expression profile whereby PIN1 transcript levels exceeded those of AUX1 when treated with 0.5 mg ℓ-1 of IAA. These expression profiles suggest that equivalent expression of PIN1:AUX1 correlated with greater responsiveness to exogenously supplied IAA for clone 2. The findings of this study thus suggest a novel approach for the rapid determination of in vitro responses of valuable Eucalyptus genotypes, by investigating culture growth stages and gene expression levels.
- ItemEvaluation 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.
- ItemPruning wound protection of rootstock mother vines(Stellenbosch : Stellenbosch University, 2019-12) Shannon, Strydom; Halleen, Francois; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: One of the factors contributing to the decline in grapevine production across the world is diseases commonly known as grapevine trunk diseases (GTD’s). These diseases are typically triggered by xylem-inhabiting pathogens which causes a variety of symptoms and an overall reduction in grapevine production worldwide. To date there is a great concern on the manifestation of these fungal trunk pathogens in rootstock mother vines and their accompanied propagation material. However, there are limited and inadequate information available of the vulnerability of South African rootstocks to these pathogens. Therefore, the objectives of this study were to determine the temporal susceptibility of pruning wounds against Phaeomoniella chlamydospora and to assess the protection of pruning wounds of commercial rootstocks with fungicides and biological agents against Pa. chlamydospora in South Africa. Pruning wound susceptibility was determined in certified mother vine blocks with two rootstock varieties most commonly grafted in South Africa, namely Ramsey and US 8-7. This was achieved by inoculations of spore suspensions directly after pruning, as well as 1, 7, 21, and 42 days after pruning. The trials were evaluated after nine months by determining Pa. chlamydospora incidences from inoculated wounds. A general decline in Pa. chlamydospora incidence was recorded up to 42 days after pruning with wounds challenged 24 hours after pruning being the most susceptible. In vitro mycelial and germination inhibition studies were conducted in order to determine whether selected chemicals which include chemical fungicide groups such as; benzimidazole, triazole, pyridine-carboxamide and strobilurin are effective against Pa. chlamydospora. Mycelial growth inhibition was assessed for six fungicides. The results obtained were used to determine the EC50 values for Pa. chlamydospora (LM310) and it was observed that all the tested fungicides were effective against Pa. chlamydospora. Furthermore, cell viability was assessed using three of the fungicides known to be effective against germination inhibition via a microtiter assay. EC50 values for Pa. chlamydospora was determined and found to be effective. Chemical fungicides that shown mycelial and germination inhibition, including other control agents, were evaluated in a detached shoot assay by pruning, treating and challenging these shoots (Ramsey, US 8-7 and 101-14 Mgt). After a 4-week incubation period, Pa. chlamydospora incidence was recorded and it was found that several of the chemicals were highly effective in lowering Pa. chlamydospora incidence, hence protecting these wounds from infection. Consequently, these control agents were further evaluated in field trials conducted in a rootstock mother vine nursery. Pruning wounds were treated with the selected control agents immediately after pruning and challenged with a Pa. chlamydospora spore suspension at 1 and 7 days after pruning. The trials were evaluated after nine months by determining the incidence of Pa. chlamydospora from inoculated wounds. The study concludes that integrated control treatments where the biological control agent Trichoderma atroviride are applied together with benzimidazole fungicides such as carbendazim or thiophanate-methyl showed to be the most effective in reducing Pa. chlamydospora incidence in rootstock mother vine pruning wounds. Results from this study have provided new information regarding the protection of pruning wounds via the integration of biological and chemical control techniques applied to rootstock pruning wounds at the most susceptible time period. Considering the results obtained in this study it is suggested to prune as early in the dormant season as possible, before the end of May, and the best fungicide chemical group to provide protection for rootstock pruning wounds was benzimidazole in combination with T. atroviride applied within 24 hours after pruning occurred. However, further research would be necessary to develop a better understanding to produce protocols and commercial products. The application of these fungicide together with Trichoderma spp. in the field holds promise to improve control but would require further trials for possible commercialisation. For successful grapevine propagation and subsequent longevity of new established vineyards, the quality of the planted material plays a crucial role. Therefore, integrating existing information of GTD management in the grapevine propagation process with the knowledge attained from this thesis, can the quality of grapevine material be improved ensuring the success of the South African grape industry.
- ItemThe treatment of Fusarium Oxysporum F. SP. cubense in soil and water(Stellenbosch : Stellenbosch University, 2019-12) Ullah, Sahabne; Viljoen, Altus; Mostert, G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Banana is an economically important food crop globally. It is, however, also severely affected by biotic and abiotic constraints, including banana Fusarium wilt (Panama disease). The causal agent of banana Fusarium wilt is the fungus Fusarium oxysporum f. sp cubense (Foc), which can survive as chlamydospores in soil for several years, making it difficult to control. The only effective way to manage Foc is by planting resistant cultivars and by preventing the fungus from being introduced into new areas. Resistant cultivars are often not acceptable to local and international markets, which makes disease prevention and reducing the spread of Foc to new areas an important disease management strategy. Foc can easily spread with contaminated soil and water. Contaminated soil attaches to boots, vehicles, plantation tools and machinery, and transfers the fungus over short or long distances. Heavy rainfall and typhoons may also carry infested soil with run-off water from infested to non-infested areas. The run-off water further contaminates irrigation sources such as rivers, dams and ponds. To prevent Foc from spreading, it is important to implement strict quarantine measures that prevent the movement of the pathogen on field equipment, shoes, vehicles and in contaminated water. Measures to do this include the use of footbaths and disinfestation areas, as well as the treatment of water used for irrigation. Limited studies, however, have been done on the survival and treatment of water contaminated with Foc. In this study, the sanitation products Sporekill, Saniwash, Farmcleanse, HTH and HyperCide were tested for efficacy against Foc race 1, STR4 and TR4 at their recommended dose and a 0.1 x dilution in the presence and absence of soil in water. The sanitisers were all tested at an exposure time of 0 s, 30 s and 2 min. All sanitisers were effective at both concentrations, but not in the presence of soil. Sporekill and Saniwash were the most effective sanitisers at the recommended dose upon contact (0 s) in the absence of soil and in the presence of soil after 2 min. This indicated that soil needs to be removed from shoes, field equipment and vehicles before being treated with Sporekill and Saniwash. The survival of Foc in 20-L buckets filled with water was determined in the presence and absence of soil. The water was either stirred or left stagnant. Water samples were extracted from the top, middle and bottom of the buckets 1, 7, 14, 30, 60 and 120 days after inoculation. The survival of Foc decreased over time, and the spores sunk to the bottom of water that remained stagnant. The contaminated water was also treated with ozone, UV radiation, HTH and the peracetic acid products (HyperCide and Tsunami 100) to determine the survival of Foc colony forming units in the presence and absence of soil. Ozone, HTH and HyperCide were only effective in the absence of soil, while UV radiation was ineffective. Thus, it is important that soil be removed for the treatment of irrigation water since Foc was able to survive for 4 months in water without agitation.