Doctoral Degrees (Plant Pathology)
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- ItemThe diversity and epidemiology of Botryosphaeriaceae species associated with grapevines and woody hosts surrounding vineyards in South Africa(Stellenbosch : Stellenbosch University, 2020-12) Du Plessis, Ihan Lambert; Halleen, Francois; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Botryosphaeriaceae species are reported globally as causal agents of grapevine trunk diseases which translate to yield losses as well as a reduction in the productive lifespan of affected vines. Growers rely on management practises to try and prevent vines from becoming infected. However, despite decades of implementation, current disease management strategies do not fully protect grapevines from becoming infected. This highlights a need for an improved understanding of the epidemiology of these pathogens as well as the development of improved disease management strategies. The aim of this study was to investigate the diversity of Botryosphaeriaceae species occurring on both grapevines as well as other woody hosts within the wine growing regions of the Western Cape Province. In addition, the potential threat that these other hosts pose to the grapevine industry by acting as sources of pathogen inoculum was investigated by characterizing and comparing different populations of the pathogen N. stellenboschiana isolated from both grapevines as well as non-grapevine hosts. The species diversity survey reported 20 different Botryosphaeriaceae species from 38 different host species which were located within 50 m of vineyards. These represented 114 different host/ fungi combinations which were not previously known in South Africa. This survey was dominated by three Botryosphaeriaceae species, Diplodia seriata, Neofusicoccum australe and N. stellenboschiana which constituted 85.1% of all the isolates obtained during this survey. These species are also known grapevine pathogens and were reported from 19, 11 and 24 different host species respectively which highlights the broad host range of these economically important pathogens. The species diversity survey also yielded six new Botryosphaeriaceae species which were formally described and their pathogenicity towards grapevines and olive trees, where relevant, were assessed through field pathogenicity trials. All of the new species were shown to form lesions on grapevine or olive shoots which were comparable to those caused by known Botryosphaeriaceae pathogens, demonstrating the capacity of these species to act as pathogens of these economically important hosts. The population genetics study was carried out based on seven microsatellite markers which were demonstrated to be polymorphic in this study. This study reported that N. stellenboschiana populations from grapevines and other hosts at three different locations in the Western Cape Province were genetically homogenous. This indicates that there are no barriers which prevent the movement of N. stellenboschiana between grapevines and other hosts. These results are disconcerting because they imply that woody hosts surrounding grapevines which are infected with Botryosphaeriaceae grapevine pathogens could be acting as disease reservoirs and sources of pathogen inoculum which threaten vineyards. To conclude, this study furthered our understanding of the diversity of Botryosphaeriaceae species occurring in woody hosts that commonly surround vineyards in the Western Cape Province of South Africa and described six new species. Furthermore, this study has contributed to our understanding of the epidemiology of these pathogens by demonstrating that the alternative hosts of Botryosphaeriaceae grapevine trunk disease pathogens represent a threat to grapevines by acting as sources of pathogen inoculum. This helps to lay the groundwork for future studies to address this threat by developing improved pathogen management strategies.
- ItemDistribution and genetic diversity of Pseudocercospora SPP. associated with banana Sigatoka diseases in East Africa(Stellenbosch : Stellenbosch University, 2020-12) Kimunye, Janet Njeri; Viljoen, Altus; Mahuku, George; Stellenbosch University. Faculty of Agrisciences. Dept. of Food Science.ENGLISH ABSTRACT: Sigatoka leaf diseases are a major constraint to banana production worldwide. They are caused by phylogenetically related pathogens belonging to the genus Pseudocercospora. Pseudocercospora fijiensis, the cause of black Sigatoka, is the most widespread and damaging species, causing yield losses of 20-50%. Pseudocercospora fijiensis is heterothallic, and produces infective conidia and ascospores that are dispersed by wind and rain splash. In commercial farms, black Sigatoka is managed by spraying fungicides weekly. This method is not suitable for smallholder farmers who represent most banana producers in Africa. Banana varieties with resistance to black Sigatoka is the most feasible control method for resource poor farmers. An understanding of pathogen distribution, genetic diversity and population dynamics is a prequisite for developing effective and sustainable disease management strategies. A survey was conducted in five banana-growing regions in Tanzania and Uganda to identify the Pseudocercospora spp. associated with Sigatoka leaf spots and determine disease severity. Sigatoka-like symptoms were present in all localities and on all cultivars. Species-specific primers revealed that P. fijiensis was the predominant species in all areas except Kilimanjaro, where Mycosphaerella musae was associated with Sigatoka- like leaf spots. Black Sigatoka was more severe in Uganda, with a mean disease severity index (DSI) of 37.5%, than in Tanzania (DSI=19.9%). Pseudocercospora fijiensis was detected at altitudes of up to 1877 m above sea level, which suggests a habitat range expansion from the previous threshold of <1350 m above sea level in East Africa. This expansion threatens sustainability of banana production in the region. Genetic diversity, population structure and mating type idiomorph distribution was assessed on 319 P. fijiensis single-spore isolates from seven regions, using 16 simple sequence repeat markers and mating type (MAT)-specific primers. The populations were characterised by a high genotypic diversity (296 multi-locus genotypes) and low clonality (7%), with MAT 1 and 2 occurring at a 1:1 ratio in Uganda, while MAT 1 was over- represented at a ratio of 4:1 in Tanzania. The index of association revealed that all populations were at linkage equilibrium (P>0.05), supporting the hypothesis of a random association of alleles. Sub-populations had a moderate level of genetic diversity (Hexp = 0.12-0.31; mean 0.29). These findings are consistent with a pathogen that reproduces both clonally and sexually. Isolates collected at the different locations did not show geographical differentiation, with 90% of the variation occurring among isolates within a subpopulation. This finding suggests a common origin for the isolates and supports the hypothesis of frequent recombination of genotypes. Multi-location evaluation of 21 newly developed East African Highland banana hybrids (NARITA) for resistance to P. fijiensis was conducted in five sites in Uganda and Tanzania. Significant differences in disease severity was observed between the hybrids, test locations, and their interaction (GEI). The environment had the greatest influence (39.1%) on genotypes’ response to P. fijiensis, with GEI accounting for 23.4%. Most of the hybrids exhibited broad adaptability in their response to black Sigatoka. Hybrids with low disease development and a stable response across locations were NARITA hybrids 2, 7, 8, 21 and 23. These can be provided to farmers for managing black Sigatoka in the region. NARITA hybrids 10 and 18 were identified as susceptible, and could be used as susceptible checks in future evaluations. The Mitarula site in Tanzania was identified as a representative test location to evaluate banana hybrids for their response to the black Sigatoka pathogen. To identify potential sources of P. fijiensis resistance, a collection of 95 banana accessions, including selected breeding parents, were evaluated in the field at Sendusu in Uganda. Out of these, 33% of the accessions; belonging to 22 subspecies of Musa acuminata ssp. malaccensis, M. acuminata ssp. zebrina and M. acuminata ssp. Burmannica; were either resistant or partially resistant to P. fijiensis. Symptom progression in these accessions stopped at early lesion development (Stages 2, 3, and 4). Symptom development in Long Tavoy, Pahang, Pisang KRA, 0074 Malaccencis, M.A Truncata, Tani and Balbisiana stopped at Stage 2, like that in the resistant Musa acuminata ssp. burmannicoides, var. Calcutta 4, and these varieties can thus be considered as potential sources of resistance.
- ItemCharacterisation, epidemiology and management of olive trunk disease pathogens in South Africa(Stellenbosch : Stellenbosch University, 2020-04) Van Dyk, Meagan; Halleen, Francois; Mostert, Lizel; Spies, Chris, F. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: The Olive Sector Development Plan of the Department of Trade and Industry identified low production and the lack of local research as weaknesses of the olive industry in South Africa. The management of trunk diseases forms an integral part of practices aimed at increasing olive production. A recent olive trunk disease survey performed in the Western Cape Province, South Africa, identified an undescribed Pseudophaeomoniella sp. as the most prevalent fungus associated with the trunk disease symptoms, with other fungal species occurring at much lower frequencies. In the current study, 40 of these isolates were selected for a pathogenicity study. The species forming lesions included several Botryosphaeriaceae, Phaeoacremonium and Phaeomoniellaceae species, as well as Biscogniauxia mediterranea, Coniochaeta velutina, Diaporthe foeniculina, Didymocyrtis banksiae, Eutypa lata, Pleurostoma richardsiae, Symbiotaphrina buchneri, isolates of the Cytospora pruinosa complex, and a Cytospora sp., Fomitiporella sp., Geosmithia sp. and Punctularia sp. The Pseudophaeomoniella sp. formed among the longest lesions, affirming its status as a potentially important trunk pathogen. Long distance dispersal of olive trunk pathogens is expected to occur via infected nursery material, similar to that found in other systems such as in grape and fruit trees. Nurseries as an inoculum source was investigated by making isolations from asymptomatic cuttings from mother blocks (Stage 1), rooted cuttings (Stage 2) and 1–2-year-old trees (Stage 3) of eight cultivars in two nurseries. Known olive trunk pathogens of the Botryosphaeriaceae, Diaporthaceae, Nectriaceae, Phaeomoniellaceae, Pleurostomataceae and Togniniaceae were recovered. Neofusicoccum australe was detected in a single Stage 1 cutting. Stage 3 material showed the highest incidence of fungi from these families, with P. richardsiae having the highest incidence in both nurseries (82.2% and 36.7% of the 1–2-year-old trees). Phaeoacremonium parasiticum was present in 28.9% of the trees from one nursery (Stage 3). The remaining pathogens occurred in 13.3% or less of the material. Pseudophaeomoniella sp. was present in the nurseries but at low frequencies. This suggests that alternative inoculum sources of this pathogen exists. A nested species-specific PCR was developed for the detection of Pseudophaeomoniella sp. from spore washes of pruning debris collected from established olive orchards. Pruning debris identified with a positive PCR was evaluated microscopically. Pycnidia of Pseudophaeomoniella sp. were observed on the pruning debris. Based on previous research, it is expected that the spore release coincides with rainfall and that the spores can be dispersed onto pruning wounds. The susceptibility of wounds from winter and spring pruning to Pseudophaeomoniella sp. was compared. Two-year-old olive branches of 16-year-old olive trees were pruned and inoculated with spore suspensions of Pseudophaeomoniella sp. at different time-points after pruning. The pruning wounds were susceptible for up to 42 days, with no difference between seasons (winter vs. spring). The wounds were the most susceptible within the first week after pruning. Eleven pruning wound protectants were evaluated and applied on pruning wounds made on 16–17-year-old trees directly after pruning. The treated wounds and positive (non-treated) controls were challenged with spore suspensions of Pseudophaeomoniella sp. at 1 or 7 days after pruning. Under low inoculum pressure (first season), Garrison, MT1, Neocil Plus and Tree Seal, reduced Pseudophaeomoniella sp. infections, while the Trichoderma-based protectant, MT1, was considered the most effective water-based protectant. Under higher inoculum pressure (during the second season), Tree Seal and Coprox Super/Bendazid consistently performed the best. In conclusion, several fungal species were identified as olive trunk pathogens, with Pseudophaeomoniella sp. being identified as one of the most important olive trunk pathogens. The propagation process was identified as a source of inoculum for some pathogens, including Pseudophaeomoniella sp. Inoculum sources of Pseudophaeomoniella sp. were also identified in established orchards. Olive pruning wounds are susceptible to Pseudophaeomoniella sp. for prolonged periods. MT1 was highly effective under lower inoculum pressure, while Tree Seal and Coprox/Bendazid were highly effective under high inoculum pressure. This study led to new knowledge with regards to olive trunk diseases, their pathogenicity, detection, epidemiology and control which can be used for the development of improved management strategies of olive trunk diseases in South Africa.
- ItemEvaluation of East African bananas for resistance to Fusarium Oxysporum f. Sp. cubense race 1(Stellenbosch : Stellenbosch University, 2020-03) Ndayihanzamaso, Privat; Viljoen, Altus; Mostert, Diane; Mahuku, George; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Banana is a staple food and source of income for millions of smallholder farmers in East and Central Africa (ECA). Consumption per capita in countries such as Burundi, Rwanda and Uganda ranges from 120 kg to more than 400 kg per year, which is six to 20 times the global average consumption per capita. Bananas cultivated in ECA consist of cooking varieties, such as East African Highland bananas (EAHB), Bluggoe, and juice/sweet dessert varieties such as Pisang Awak, Sukari Ndizi, Gros Michel and Cavendish bananas. EAHB include diploid bananas such Mchare, Muraru, Mlali and Paka (mostly cooking types), whereas EAHB triploids include Matooke (a cooking type) and Mbidde (a juice/beer type). Fusarium wilt of banana, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is present in most banana-growing regions of ECA. Foc comprises three races based on their pathogenicity to a group of differential cultivars, with Foc race 1, race 2 and race 4 causing disease to Gros Michel, Bluggoe and Cavendish bananas, respectively. All three races are present in Africa, but only Foc races 1 and 2 occur in ECA. Foc races 1 and 2 strains in ECA group consist of six vegetative compatibility groups, which cluster together as Foc Lineage VI. In this study, molecular markers specific to Foc Lineage VI were developed from the DNA-directed RNA polymerase III subunit (RPC2) gene region. The primer set was combined in a multiplex PCR assay with the primer set FocLin6bF/R, which was developed from the translation elongation factor-1 alpha (TEF-1α) gene. The multiplex PCR assay was validated on a worldwide population of 623 known Foc isolates, other formae speciales and non-pathogenic isolates of Fusarium oxysporum. The multiplex PCR can be used as an accurate diagnostic tool for Foc Lineage VI strains. Effective management of banana Fusarium wilt can be achieved by planting banana varieties resistant to Foc. Resistant bananas, however, require many years of breeding and field-testing under multiple geographical conditions. Field evaluation is reliable but time consuming and expensive, and not feasible for quarantine strains. Small plant screening methods are, therefore, needed to speed up the evaluation of banana varieties for Foc resistance. To this end, a small plant screening method for resistance to banana Fusarium wilt was optimized by investigating the effect of inoculum concentration, inoculation method and plant age on disease development, and the value of phenolic compounds and Foc DNA as indicators of disease resistance. The method, which consisted of planting 2- to 3-month-old banana plants in soil amended with 2-10 g Foc-colonised millet seeds per kg of potting soil, was reliable, and qPCR and rhizome discoloration were suitable for evaluating and ranking the disease response of banana varieties. Phenolic compounds were, however, not consistent in differentiating cultivars’ resistance when the same genotypes were inoculated with Foc race 1 and subtropical race 4 (STR4), and cannot be considered a reliable indicator of resistance. The optimized millet seed technique is useful in mass screening of newly developed genotypes for resistance to Foc, and can be used in the screening for Fusarium wilt resistance against quarantine variants of Foc in quarantine facilities. EAHB triploid banana cultivars are resistant to Foc race 1 and 2 in ECA, but dessert varieties in the region are susceptible. Resistance of diploid Mchare, Muraru and Mlali bananas, as well as newly developed diploid and triploid EAHB hybrids, is largely unknown. Therefore, in this study eight Mchare cultivars and 19 NARITA hybrids were evaluated for resistance to Foc race 1 in the field and screen house in Tanzania and in Uganda. Eight Muraru cultivars, 23 Mchare hybrids and 60 Matooke hybrids were also screened in pot trials in a screen house. Mchare and Muraru cultivars were all susceptible to Foc race 1, whereas the response of Mchare, NARITA and Matooke hybrids ranged from susceptible to resistant. Triploid hybrids were not expected to be susceptible as their parents were resistant to Foc race 1. This suggest that resistance in banana is multi-gene controlled and heterozygous, and that the genes segregated during meiosis into gamete cells leading to a loss of resistance. This study generated valuable information towards the management of Fusarium wilt in the ECA region. Molecular markers that were developed are reliable and affordable to research centres and extension services in the region, and can speed up the diagnosis of Foc Lineage VI strains. The screening method developed in this study will improve the reliability of small plant testing, and will reduce time and cost associated with field evaluation of new varieties.
- ItemEvaluation of adjuvants in fungicide spray application for the control of alternaria brown spot in South African citrus orchards(Stellenbosch : Stellenbosch University, 2019-03) Van Zyl, Johannes Gideon; Fourie, Paul H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Citrus fruit and foliar diseases are mainly controlled through pre-harvest application of fungicides. Fungicides are only as effective as the application process and for effective disease control deposition of a uniformly distributed quantity of active ingredient(s) is required on the intended target(s). Adjuvants have the potential to improve fungicide deposition on a target surface. The influence of adjuvants on the deposition of fungicides, especially at the high spray volumes used in South African citrus production is unknown and was therefore investigated. A previously developed deposition assessment protocol, using a yellow fluorescent pigment as tracer for copper oxychloride (CuOCl) deposition, was improved through photomacrography and digital image analyses which proved accurate in determining the quantity and quality of deposition on citrus leaves. Spray deposition benchmarks indicative of the biologically efficacy of CuOCl against Alternaria alternata [causal agent of Alternaria brown spot (ABS) of mandarins] was developed. The deposition assessment protocol and deposition benchmarks was used to evaluate two organosilicone adjuvants (Break-Thru S240 and Break-Thru Union) at reduced spray volumes in dense and less dense citrus canopies in two separate orchard spray trials. Deposition quantity generally increased with increasing spray volume, but normalised values showed better spray efficiency at lower volumes. In pruned and less dense canopies, a beneficial effect of adjuvants was observed in terms of deposition quantity, efficiency and uniformity, especially at reduced volume applications. Some improvement in deposition quality was generally observed with the use of adjuvants. These benefits were not as evident in very dense canopies, illustrating the importance of canopy management when spraying at reduced volumes. Commercially available adjuvants [Break-Thru, Nu-Film-17, Citrole100, Villa51, Wetcit, Entrée and Exit] were evaluated in three orchard spray trials on different citrus types, cultivars and spray volumes. In trial one, adjuvants improved deposition quantity and canopy penetration. In trial 2 and 3, deposition quantity was generally higher at higher spray volumes, but spray efficiency was significantly better at lower spray volumes. Adjuvants generally improved deposition uniformity and deposition quality, but these benefits were significantly influenced by spray volume and the specific adjuvant treatment. Poor performance by adjuvants was ascribed to high spray volumes and/or too high adjuvant concentration used, which led to increased levels of run-off and poor deposition parameters. The effects of adjuvants on deposition quantity, quality and biological efficacy of CuOCl against ABS on mandarin leaves were determined in laboratory trials. Adjuvant treatments varied significantly in deposition quantity and quality and disease control achieved. Higher deposition quantity, beter quality and higher Cu residues was realized at pre- vs. post-run-off volumes. Adjuvants did not improve deposition parameters compared with the control treatment at both spray volumes. Leaf infection analysis indicated that CuOCl with adjuvant sprays (post-run-off volume) realized similar and in some cases slightly better control (although not significant) than copper oxychloride alone, but that deposition and Cu residue loading in some of these adjuvant treatments were markedly lower. This anomaly could be ascribed to direct or indirect effects of the adjuvant and was investigated further. In vivo and in vitro studies were done to identify possible direct adjuvant effects on pathogen development and potential synergistic effects between the adjuvants and CuOCl. Adjuvants alone did not influence conidial adhesion, appressorium formation, germ tube length and percent viable conidia. Adjuvant sprays together with CuOCl reduced conidial adhesion, germ tube length and percent viable conidia numerically; however, not significantly compared with CuOCl alone. Adjuvants also caused conidium/germ tube stress similar to CuOCl, but did not inhibit germination or growth. In the in vitro microtiter assay, adjuvants together with CuOCl improved germination or growth inhibition compared with the CuOCl treatment alone, although not at significant levels. The findings in Chapter 6 did not fully explain the anomalous findings in Chapter 5, and future studies should focus on developing methodology to support histopathology studies on sensitive leaf surfaces, as well as development of a more sensitive method of measuring deposition quality, especially on a microscopic scale.