Doctoral Degrees (Plant Pathology)

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The biology of Endophyllum osteospermi, and its use for the biological control of Chrysanthemoides monilifera ssp. monilifera
(Stellenbosch : Stellenbosch University, 2004-12) Wood, A. R. (Alan Robert); Crous, P. W.; Lennox, C. L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Chrysanthemoides monilifera ssp. monilifera is a shrub indigenous to South Africa, which has become a serious weed of native vegetation in Australia. Endophyllum osteospermi is a microcyclic, autoecious, rust fungus that induces witches' brooms on C. monilifera ssp. monilifera. This rust is considered as a candidate biocontrol agent for use against C. monilifera ssp. monilifera in Australia. The vegetative growth and reproductive output of healthy branches on bushes with different levels of E. osteospermi infections were measured at three sites. The growth of healthy branches on infected bushes was 26- 81% less than that of healthy branches on uninfected bushes. The number of buds, flowering capitulae, fruiting capitulae, and cypselas on healthy branches of infected bushes was 35-75%, 45-90%, 15-99%, and 15-90% less, respectively, than those on uninfected bushes. At five sites, the infection levels and number of witches' brooms were determined every two months. The increase in number of witches' brooms per bush ranged between o and 282 within one year, with an average increase per bush of28 (SE ± 4.8) and 39 (SE ± 9.2) during two years. The average simple interest rate (rs) increase of infection levels for all bushes was 0.015 month-I (s.e. ± 0.0041, n = 72) and 0.0098 month" (s.e. ± 0.0073, n = 43) during two years. Aecidioid teliospores germinated between 10 and 20oe, with 15°e as optimum. Light, and particularly near-uv light, stimulated germination. A period of 6 to 8 hours of light was needed to obtain optimum germination levels. The temperature requirements for basidiospore development differed from that of aecidioid teliospore germination. Optimum was at 15°e, but a rapid decrease in basidiospore production occurred at higher temperatures, few developed at 19°e. Two nuclear divisions occurred within 12 hours of germination to produce a metabasidium with three or four nuclei. A third nuclear division occurred in the basidiospores between 24 and 48 hours. Plants inoculated under controlled conditions took 5 to 24 months before witches' brooms began to develop. A Geographic Information System (GIS) approach was used to model the potential distribution of E. osteospermi in South Africa, based on monthly average climate surfaces with parameters derived from the above experiments. The same model was applied to Australia to suggest a potential distribution of the rust if released in Australia. This potential distribution was similar to one generated using the climate matching computer programme CLIMEX©, but gave greater spatial accuracy. Both approaches indicate that E. osteospermi should establish in temperate Australia. Chrysanthemoides species, as well as other South African asteraceaus plants, were monitored for E. osteospermi between 1992 and 2003. Endophyllum osteospermi was recorded on C. monilifera ssp. monilifera, C. monilifera ssp. pisifera, C. monilifera ssp. rotundata, C. monilifera ssp. canescens, C. monilifera ssp. subcanescens, C. incana, an undescribed taxon of Chrysanthemoides, Osteospermum ciliatum, 0. polygaloides and 0. potbergense. Endophyllum dimorphothecae sp. nov. is described on Dimorphotheca cuneata. Aecidium elytropappi, which was recorded on Elytropappus rhinocerostis and Stoebe plumose, is transferred to Endophyllum as E. elytropappi comb. nov. Germination of aecidioid teliospores and penetration by basidiospores were observed on the surface of excised leaves of 32 plant species at 4 days after inoculation. Germinating aecidioid teliospores aborted on 14 plant species, whilst no penetration was attempted on a further 12. Penetration only occurred on 9. Therefore only these 9 plant species need to undergo traditional host specificity testing. Pending these results, E. osteospermi could be safely released in Australia for the biological control of C. monilifera ssp. monilifera.
Botryosphaeria diseases of proteaceae
(Stellenbosch : Stellenbosch University, 2002-03) Denman, Sandra; Crous, P. W.; Wingfield, M. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Fungi belonging to the genus Botryosphaeria are heterotrophic micromycetes that can be pathogens on woody plants. They cause serious, and in some cases devastating losses to crops through leaf necrosis, stem cankers and plant death. The Proteaceae cut-flower industry in South Africa accounts for 70% of the national cut-flower enterprise. Botryosphaeria diseases are a major impediment to production and trade of Proteaceae and there is an urgent need to investigate the etiology, epidemiology and control of these diseases. Losses of one of the most important proteas, P. magnifica, amount to 50% or more, locally. The main aims of this study were therefore to establish the etiology and aspects of epidemiology of Botryosphaeria stem cankers on P. magnifica and other Proteaceae, and to investigate methods of disease control. Although there is a vast body of information pertaining to this fungus, which was reviewed in Chapter 1, there is relatively little information available on Botryosphaeria on Proteaceae. The taxonomy of Botryosphaeria requires thorough review, and molecular techniques need to be employed to resolve species identities. In Chapter 2, it was found that Phyllachora proteae, a leaf pathogen of proteas, produced a Fusicoccum anamorph, which is described as F. proteae. A sphaeropsis-like synanamorph was associated with F. proteae and a new combination for P. proteae is proposed in Botryosphaeria, as B. proteae. The taxonomy of Botryosphaeria is in disarray at both the generic and the specific level. In Chapter 3 the taxonomic history of Botryosphaeria is reviewed, and the genus circumscribed and distinguished from other morphologically similar genera. Although several anamorph genera have been linked to Botryosphaeria, based on morphological observations and phylogenetic analysis of lTS rDNA sequence data, two anamorph genera are now recognised, those with pigmented conidia (Diplodia), and those with hyaline conidia (Fusicoccum). Botryosphaeria proteae should thus be excluded from Botryosphaeria. Several pathogenic Botryosphaeria spp. have an endophytic phase within their hosts. They are therefore imported unwittingly into other countries where they may pose a risk to agriculture and indigenous vegetation. The current global distribution of Botryosphaeria spp. associated with Proteaceae is clarified and a key to these taxa associated with Proteaceae is provided in Chapter 4. Five Botryosphaeria spp. are associated with cut-flower Proteaceae worldwide viz. B. lute a, B. obtusa, B. protearum, B. proteae and B. rib is. B. protearum is described as a new species. A thorough understanding of disease epidemiology is essential to effect a reduction of losses. In Chapter 5, I show that on P. magnifica, lesions caused by Botryosphaeria protearum, which lead to the formation of stem cankers, are initiated in the mid-rib vein or margin of leaves. Koch's postulates were satisfied and it was found that the number of lesions that developed from artificial inoculations correlated with starch levels present in leaves at the time of inoculation. In Chapter 6 it is shown that B. protearum exists as an endophyte in leaves of P. magnifica in naturally occurring as well as cultivated plants. In natural stands of proteas stem cankers are rare, but in cultivated plantations the incidence is high. Nutritional analyses indicate that higher levels of nitrogen occur in leaves of cultivated plants in spring, which could enhance disease development. High levels of sodium in the leaves of wild plants may restrict disease development. The severe economic losses caused by B. protearum make the search for improved methods of disease control essential. Fungicide applications form an important component of an integrated approach to disease management. In Chapter 7, in vitro tests demonstrate that tebuconazole, benomyl, prochloraz me, iprodione and fenarimol reduce the mycelial growth of B. protearum effectively. In the field there was a 25-85% reduction in the occurrence of stem cankers by applying fungicides or sanitation pruning. The best control was achieved by using benomyl, bitertanol, fenarimol, iprodione, prochloraz manganese chloride alternated with mancozeb and tebuconazole prophylactically. If sanitation pruning is combined with regular applications of fungicides, disease can be combated.
The characterisation of basidiomycetes associated with esca disease in South African grapevines
(Stellenbosch : Stellenbosch University, 2015-03) Cloete, Mia; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Esca is a disease complex of grapevine that includes different foliar and vascular symptoms caused by various fungal pathogens. One of the distinguishing characteristics of the disease on mature vines is the white rot of the wood. Esca-related wood rot is caused by several lignicolous basidiomycetes from the order Hymenochaetales. The Hymenochaetales fungi associated with esca vary depending on geographic location. For example, in Europe and the Mediterranean grape-growing regions, Fomitiporia mediterranea is the prevalent species; in Argentina, Inocutis jamaicensis; in Chile “Fomitiporella vitis”, and in Australia Fomitiporia australiensis. In the United States, Fomitiporia polymorpha has been associated with esca, though not consistently. A previous study identified ten different taxa belonging to the genera Fomitiporella, Fomitiporia, Inocutis, Inonotus, and Phellinus associated with esca in South Africa. The current study was tasked with characterising these taxa and assessing their epidemiology and pathogenicity. The study has characterised three novel species, Fomitiporella viticola, Fomitiporia capensis and Phellinus resupinatus from Vitis vinifera and a first report of Inonotus setuloso-croceus occurring on Vitis vinifera and Salix spp. worldwide and in South Africa. The sporulation of F. viticola was surveyed over two seasons. The pathogenicity of all ten taxa was tested on mature field grown vines and enzymes secreted by all ten taxa were assayed. This study aimed to add in the understanding of the esca complex disease in South Africa and contributed towards the wider knowledge regarding the ecology of the Hymenochaetales. A novel Fomitiporia species, F. capensis, was described based on fruit body morphology and combined internal transcribed spacer rRNA ITS1-5.8S-ITS-2 (ITS) and large sub-unit (LSU) phylogeny, where it formed a clearly delineated and well-supported clade. Morphologically, F. capensis was similar to F. punctata in that both species essentially lack setae. Fomitiporia capensis, F. punctata and F. aethiopica produced similarly sized basidiospores, but differed in terms of host range, pore size and, possibly, fruiting body shape. Phylogenetically, F. capensis appeared to be related to F. tenuis, though morphologically the species differed significantly in that F. tenuis had smaller pores and smaller basidiospores. During all surveys conducted, Fomitiporia capensis was found to occur widely as throughout the Western Cape Province, though fruit bodies were scarce in comparison to mycelium isolated from symptomatic vines. Fruit bodies were also found in a vineyard in the Limpopo region in the north east part of the country. Phellinus resupinatus was described based on fruit body morphology, ITS and LSU phylogenies. It formed a well-supported clade closely related to Phellinus bicuspidatus, a species associated with white rot in oak trees in the United States. Morphologically, P. resupinatus was characterised by its resupinate fruit body shape, straight, ventricose hymenial setae, and broadly ellipsoid hyaline basidiospores. It was only found on diseased grapevines in the summer rainfall regions of South Africa, mainly in the Northern Cape and Limpopo provinces. Fomitiporella viticola was described from Vitis vinifera based on fruit body morphology and ITS phylogeny. It is characterised by a resupinate to effuse-reflexed fruit body with large, loosely spaced pores and fairly small yellowish-brown basidiospores. Inonotus setuloso–croceus was found occurring on Salix and Vitis vinifera and was identified based on fruit body morphology. The ITS region was sequenced from DNA isolated from cultures obtained from rotten wood or fruit bodies, and was matched to the Hymenochaetales species from Vitis previously classified as Taxon 7. The discovery of Inonotus setuloso-croceus on Salix validated the hypothesis that fruit bodies may occur on alternative hosts. Fomitiporella viticola was often isolated from white rot on vines affected by esca and fruit bodies were often found on vines in the Western Cape Province. Twelve fruit bodies of F. viticola were monitored for sporulation weekly over two seasons lasting between winter and early summer. Levels of sporulation had a weak positive correlation with rainfall and a weak negative correlation with average temperature. Sporulation was found to occur throughout the entire monitoring period. Little is known about the pathogenicity and aetiology of the Hymenochaetales taxa associated with esca in South Africa. All ten taxa were subjected to enzyme assays to determine which ligninolytic enzymes were secreted by each taxon. In addition, a field trial was undertaken to determine the pathogenicity of ten South African Hymenochaetales taxa associated with esca in grapevine. Twenty-seven fungal isolates and two negative controls were inoculated into mature grapevines and incubated for 24 months. The results of the enzyme assays indicated a difference in enzyme secretion between taxa and also among isolates of the same taxa. All isolates secreted cellulase and laccase, but there was a difference between isolates‟ ability to secrete manganese peroxidase and lignin peroxidase. The results of the pathogenicity trial showed that all of the isolates used were capable of causing the characteristic white rot symptom in the wood. There were also clear differences in susceptibility to white rot between the two cultivars tested. Cultivars also differed in which taxa proved pathogenic. On Shiraz, Taxon 6 (an Inonotus sp.), Phellinus resupinatus and Inonotus setuloso-croceus were significantly virulent. On Mourvédre, however, Taxon 3, an Inocutis sp. and Taxon 2, a Fomitiporella sp. were significantly virulent. Cultivar differences could be due to various factors, including differences in host response to colonisation and physical differences in wood structure, as well as the differences in enzyme secretion between taxa.
Characterisation of Cylindrocarpon spp. associated with black foot disease of grapevine
(Stellenbosch : Stellenbosch University, 2005-12) Halleen, Francois; Crous, Pedro W.; Fourie, Paul H.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: During the past few years a drastic reduction has been noted in the survival rate of grafted grapevines in nurseries, as well as in young vineyards in the Western Cape Province of South Africa. Circumstantial evidence suggested that Cylindrocarpon spp., which cause black foot disease of grapevine, were associated with this decline. Black foot disease of grapevine is a relatively new, and as yet poorly known disease affecting vines in various countries where grapevines are cultivated. Primary aims of this research have been (1) to conduct nursery surveys in order to determine which fungi are involved in the decline phenomenon, with special reference to the involvement of Cylindrocarpon spp., (2) to identify and characterise the organisms believed to be the causal organisms of black foot disease, and (3) the development of control and/or management strategies to prevent or eradicate Cylindrocarpon infections. Nursery grapevines were sampled at different stages from three commercial nurseries in the Wellington area of the Western Cape Province and were investigated during the 19992000 season by means of destructive sampling. The first samples were taken in September from callused cuttings prior to planting in nurseries. After planting, asymptomatic rooted cuttings were selected from nurseries after 3, 6 and 9 months. Isolation studies clearly demonstrated that different “Cylindrocarpon spp.” infected cuttings from nursery soils. These species rarely occurred in rootstock propagation material prior to planting. At the time of planting, the susceptible basal ends (especially the pith area) of most of the nursery cuttings are partly or even fully exposed. Callus roots also break during the planting process, resulting in small wounds susceptible to infection by soilborne pathogens. The isolation studies revealed that the first infections occurred in the roots, followed by infections of the rootstocks. These infections increased progressively during the course of the growing season. Substantial variation in cultural and morphological characters was observed among the Cylindrocarpon isolates obtained from the nursery survey, as well as from isolations that were made from diseased grapevines. Morphological and phylogenetic studies were conducted to identify these “Cylindrocarpon spp.” and to establish their association with black foot disease. Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial β-tubulin gene introns and exons were used for phylogenetic inference. Phylogenetic analyses confirmed the diversity observed among the isolates and four Cylindrocarpon-like species were identified. One of these species was initially identified as Cylindrocarpon destructans. However, further research revealed C. destructans to represent a species complex. Grapevine isolates of “C. destructans” proved to be identical to the ex-type strain of Cylindrocarpon liriodendri, which also produced a teleomorph, Neonectria liriodendri in culture. A second species was newly described in this study as Cylindrocarpon macrodidymum (Neonectria macrodidyma). The two remaining Cylindrocarpon-like species were placed in a new genus, Campylocarpon. The two species were named Campylocarpon fasciculare and Campylocarpon pseudofasciculare. Pathogenicity studies confirmed that all four species were able to reduce root and shoot mass significantly. Knowledge obtained pertaining to the disease cycle of black foot disease suggest that suitable management strategies should focus on prevention of primary infection in nurseries. However, at present, no fungicides are registered for control of this disease in South African vineyards or nurseries. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens. Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effective fungicides tested, and were subsequently included in semi-commercial field trials. Basal ends of grafted cuttings were dipped (1 min) in various chemical and biological treatments prior to planting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttings prior to planting in nurseries. Additional treatments involved soil amendments with Trichoderma formulations and hot water treatment (50°C for 30 min) of dormant nursery grapevines. Field trials were evaluated after a growing season of eight months. The incidence of black foot pathogens was not significantly and/or consistently reduced by the majority of chemical or biological treatments. However, these pathogens were not isolated from uprooted plants that were subjected to hot water treatment. It is therefore recommended that hot water treatment of dormant nursery plants be included in an integrated strategy for the proactive management of black foot disease in grapevine nurseries.
Characterisation, 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.
Characterization and pathogenicity of South African isolates of Fusarium oxysporum f. sp. melonis
(Stellenbosch : Stellenbosch University, 2000-03) Schreuder, Wouter; Holz, G.; Lamprecht, Sandra, C. ; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: The purpose of this study was to characterize the race and vegetative compatibility of Fusarium oxysporum f. sp. melonis (FOM) isolates collected in the major melon producing areas, to report on their geographical distribution, and their possible relatedness to isolates from other countries. Seventy two FOM isolates obtained from 30 fields in 17 melon producing regions were race-typed using the differential cultivars Topmark (susceptible to all races), Doublon (Fomi), CM 17187 (Fom2) and Perlita (Fom3) and grouped by means of vegetative compatibility. All isolates belonged to vegetative compatibility group 0134, indicating a high degree of genetic homogeneity among the South African FOM population. Fifty four isolates were identified as race 0, eight as race 1, and 10 as race 2. Race 0 occurred in 15 of the regions whereas race 1 was sporadically recovered. Race 2, on the other hand, was obtained only from four fields located in one geographical region. Perlita plants (carrying the gene Fom3) inoculated with local isolates ofrace 0 and race 2 and reference isolates of race 0 became stunted, their leaves turned yellow, and became thickened and brittle. These results suggested that Fom3 in Perlita confers a tolerant reaction compared to the resistant reaction of gene FornI in Doublon. The disease reaction of cultivar Perlita to FOM was therefore reinvestigated. Twenty isolates, including the four FOM races (0, 1, 2, and 1,2) obtained from different countries, were used. The differential cultivars were included to verify virulence of the isolates. Perlita plants inoculated with three isolates of race 2 remained asymptomatic. The remaining race 2 and 0 isolates, induced severe stunting of Perlita plants, but mean percentage stunting values did not differ significantly (P = 0.05) and ranged between 25.1 and 50.0. Leaves of stunted plants were chlorotic, thickened and brittle. Disease reaction of Perlita was verified at a lower inoculum concentration with two race 2 (pipette method) and two race 0 isolates (root dip method). Results proved that Fom3 does not confer similar resistance towards race 0 and some race 2 isolates as FornI in Doublon. Cultivars possessing Fom3, should therefore be considered tolerant to FOM races 0 and 2. The ability of a nit mutant isolate, generated from FOM race 0 which belongs to VCG 0134, to change its virulence during infection of melon plants, was investigated under quarantine. Seedlings of melon cultivars Imperial 45 and Early Sweet (no resistance genes), Amber (Fom2) and Fiata (FomI, Fom2) were consecutively grown in two cement troughs in a gauzehouse. Each planting was terminated when plants had advanced Fusarium wilt or after the fruit were harvested. In the first planting, Imperial 45 seedlings were transplanted and artificially inoculated with the nil mutant isolate. In the consecutive plantings, seeds were sown in the infested soil to enable natural infection. For each crop, representative plants showing Fusarium wilt were selected for isolation. All F. oxysporum isolates recovered were single-spored and their nit mutant and VCG status verified. Virulence of the labelled isolates was determined using differential cultivars. In trough A, all plants of the susceptible cultivars Imperial 45 and Early Sweet crops showed Fusarium wilt. The labelled isolates recovered from the selected plants were all designated race O. In the first crop (planting No.5) of the resistant cultivar Amber, 6.7% of the plants developed Fusarium wilt. In the second Amber crop the disease incidence increased to 56.6%, and to 81.8% in the final crop. Contrary to the susceptible cultivars, only race 2 isolates were obtained from the symptomatic Amber plants. Similar data were found with the susceptible cultivar Imperial 45 and the resistant cultivar Amber in trough B. Planting of Fiata caused a dramatic reduction in Fusarium wilt incidence in trough B. However, 1.2% of plants were affected by Fusarium wilt in the first Fiata crop (planting No.6), whereas 4% of the plants were symptomatic in the final planting. From these symptomatic Fiata plants only race 1,2 isolates were obtained. These findings, and the fact that the symptomatic plants represented a substantial proportion of the first Amber (approximately 7-15%) and Fiata (approximately 2%) crops, provedthat changes in the race structure of this fungal pathogen occurred rapidly when confronted with a resistant cultivar. The potential of RAPD analysis to differentiate between the isolates displaying virulence changes was evaluated. Four F. oxysporum f. sp. niveum isolates were included as an outgroup. A histopathological study was conducted to verify whether these isolates retain their ability to behave as true vascular pathogens. The three primers used clearly distinguished the 12 FOM isolates from the four F. oxysporum f. sp. niveum isolates. However, the primers showed a highly conserved and characteristic banding pattern for the FOM isolates which represented three physiological races (race 0, race 2, race 1,2), indicating that RAPD analysis cannot detect race-specific groupings in FOM. Disease reactions on the three differential cultivars confirmed the virulence of FOM isolates. The histopathological data furthermore proved that the two FOM races (race 2, race 1,2), which derived from the race 0 parent isolate, retained their ability to behave as true vascular pathogens.
Distribution 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.
The 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.
Ecology and characterization of Streptomyces species associated with common scab disease conducive and biofumigated soils in South Africa
(Stellenbosch : Stellenbosch University, 2013-03) Gouws, Reinette; McLeod, Adele; Mazzola, M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Common scab of potato is a serious cosmetic disease in South Africa as well as internationally. The disease affects the appearance and quality of potatoes resulting in major annual losses. Potato producers in South Africa, in the commercial, emerging and processing potato industries, struggle to manage the incidence of common scab, especially soilborne inoculum. Existing products and management programs against common scab are often insufficient. The two main aims of the study were to i) characterize and determine the pathogenic Streptomyces spp. occurring in potato production regions in South Africa and ii) investigate the mechanisms through which Brassica soil amendments can reduce common scab and ways in which it can be included in a sustainable management program. In South Africa, Streptomyces scabiei is still regarded as the main causal agent of common scab. However, world-wide, the disease is caused by a complex of Streptomyces species, with the dominant species varying in different regions. Therefore, a total of 132 Streptomyces isolates collected from six South African potato production regions were characterized. Potato pot trials showed that 53 % of the isolates were pathogenic. Analyses using species specific primers and phylogenetic analyses (16S rRNA phylogeny and multilocus phylogeny) showed that S. scabiei was the most prominent species in South Africa comprising 51.4 % of the pathogenic isolates, followed by S. europascabiei (30 %), S. cavisabies (5.7 %), and S. stelliscabies (1.45 %). The remaining 11.45 % of the pathogenic isolates comprised three taxa, which are related and fit within phylogenetic clades that do not contain common scab isolates from any country other than South Africa. The taxa are named here Streptomyces strains RSA1 (5.7 %), RSA2 (4.3 %) and RSA3 (1.45 %). Streptomyces strain RSA1, which occurred in two production regions, is of special concern since these isolates produce fissure scab symptoms that result in severe cosmetic tuber damage. Fissure scab has not been reported from any other region of the world and is of concern in South Africa since it occurs on the cultivar Mondial that is tolerant to typical common scab. PCR analyses targeting three marker pathogenicity island (PAI) genes (txtAB, nec1, tomA) showed that among the pathogenic isolates nec1 occurred in 89 % of the isolates, tomA in 81 % and txtAB in 89 % of the isolates. The isolates (11 %) that did not contain the txtAB gene and also did not produce thaxtomin, belonged to S. caviscabies and Streptomyces strains RSA2 and RSA3. The incorporation of Brassica tissue into soil has recently shown some potential for reducing common scab disease incidence. Brassica crop residues contain glucosinolates (GLN) that upon cell disruption are hydrolysed by the enzyme myrosinase to yield a diversity of biologically-active hydrolysis products that are toxic to soil microbes. This control mechanism is known as biofumigation. The current study showed that common scab was significantly reduced under field conditions through incorporation of fresh or air-dried residues of Brassica oleracea var. capitata (cabbage) in two consecutive potato plantings. The in-vitro effect of volatile emissions from various Brassica species towards Streptomyces was evaluated using two bioassay methods. An in-vitro agar plate bioassay showed that, in general, volatile emissions from water activated freeze-dried tissue of a B. juncea/S. alba mix and B. napus were superior to those from B. oleracea var italica and B. oleracea var capitata for suppression of growth and sporulation of Streptomyces. In a gas chamber bioassay that used freshly macerated Brassica tissue, B. oleracea var capitata and a B. juncea/S. alba mix suppressed sporulation but not hyphal growth of Streptomyces. The gas chamber bioassay showed that the biofumigation effect was bacteriostatic, i.e. isolates recovered after volatile exposure. Both bioassays showed that significant components of both the pathogenic (50 %) and non-pathogenic (20 %) Streptomyces population examined were unaffected by the Brassica tissue derived volatiles. Mechanisms of disease reduction through Brassica amendments are not limited to biofumigation, but changes in the structure of microbial communities involved in systemic induced resistance and/or general microbial suppression may also contribute to disease suppression. In the current study a potato split-root experiment that spatially separated the progeny tubers and roots of Brassica juncea/ Sinapus alba (mustard mix) and Brassica oleracea var oleracea (cabbage) amended soil sub-units from non-amended soil sub-units, showed that induced resistance induced in plants was involved in common scab suppression. The role of toxic GLN hydrolysis products was ruled out in the induced resistance mediated disease suppression, since volatiles were released from Brassica amended soil prior to initiating the experiment. Increased microbial activity in the Brassica amended units was evidenced by significant increases in ß-glucosidase and urease activities. Principal component analyses revealed some trends in the overall soil, tuber and root associated microbial genera (Trichoderma, Pseudomonas, Streptomyces, total bacteria and Fusarium) in the Brassica amended and non-amended units. The mustard amended treatment, and to a lesser extend the cabbage amended units, showed trends towards increases in soil Fusarium and Trichoderma and root Trichoderma populations, and decreases in total bacterial and Streptomyces populations in soil and tubers, and Streptomyces in roots. This study has contributed towards our knowledge of the Streptomyces species causing potato common scab in South Africa, and mechanisms through which Brassica soil amendments can reduce common scab. Several Streptomyces species, including novel pathogenic taxa, are involved in causing common scab and their differential virulence, and responses to being suppressed by Brassica amendments will require the implementation of an integrated management program. The planting of cabbage as a cash crop, with the subsequent incorporation of residues into soil shows promise as a management strategy for subsistence farmers. The mechanisms involved in common scab suppression through Brassica amendments were shown to involve systemic induced resistance in plants and general microbial suppression. Altogether, knowledge obtained in this study can be used to i) optimize management strategies for sustainable potato production, ii) further elucidate the mechanisms involved in disease suppression and iii) develop molecular techniques, such as quantitative real-time PCR for rapid identification and quantification of common scab-causing species in South Africa.
Epidemiology of Monilinia laxa on nectarine and plum : infection of fruits by conidia
(Stellenbosch : Stellenbosch University, 2001-03) Fourie, Paul H. (Paul Hendrik); Holz, G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Postharvest decay of stone fruit in the Western Cape province of South Africa is caused primarily by Botrytis cinerea (grey mould) and Monilinia laxa (brown rot). Little is known about the relative importance and seasonal occurrence of the two pathogens in nectarine and plum orchards, the mode of penetration of fruits by M laxa, latency and subsequent disease expression by the latter pathogen. These aspects were investigated in this study. By sampling from the Unifruco Quality Evaluation Scheme and from 11 stone fruit orchards, observations were made over a 3-year period of the occurrence of grey mould and brown rot in the major stone fruit regions. Botrytis cinerea was found to be the most important pathogen causing blossom blight and postharvest decay on stone fruit. The pathogen was most prominent on early- and mid-season culti~ars. Brown rot was exclusively caused by M laxa and no evidence was found that M fructicoZa had been introduced into the region. Monilina laxa was most prominent on the later maturing cultivars. Botrytis cinerea blossom infection did not contribute directly to postharvest decay. Both surface inoculum and latent infection consistently occurred on fruit in each orchard, although at fluctuating levels. Disease expression on developing fruit was not governed by the amount of B. cinerea occurring on fruit surfaces, but by the ability of fruit to resist disease expression. The amount of B. cinerea on fruits was generally higher during spring than during summer. Monilinia laxa occurred sporadically on the blossoms of late-maturing cultivars. Immature fruit were generally pathogen-free and disease expression occurred on maturing fruit only. These findings suggest that conidia of M laxa are generally produced in orchards when fruits are approaching maturity and can penetrate and infect maturing fruit only. The behaviour of airborne M laxa conidia was subsequently studied on nectarine (cultivar Flamekist) and plum (cultivar Laetitia) fruit. For these studies, an inoculation method that simulates natural infection by airborne conidia was used. Fruit at pit hardening, 2 wk before harvest, harvest stage and after cold storage (nectarines 4 wk at -o.soC followed by 1 wk at 23°C at ±56% RH; plums 10 days at .....().5°C,18 days at 7.5°C followed by 1 wk at 23°C at ±56% RH) were dusted with dry conidia of M laxa in a settling tower. The fruits were incubated for periods ranging from 3 to 48 h at high relative humidity (2':93%, humid fruit) or covered with a film of water (wet fruit). Behaviour of the solitary conidia was examined with an epifluorescence microscope on skin segments stained in a differential stain containing fluorescein diacetate, aniline blue and blankophor. The ability of solitary conidia to colonise the fruit surface, penetrate fruit skins and to induce disease expression was determined by using a differential set of tests. For these tests, fruit were surface-sterilised (30 s in 70% ethanol) or left Unsterile. From each group, fruit were selected for isolation (skin segment test), immersed in a 3% paraquat solution (paraquat-treated fruit test) or left untreated (sound fruit test). 1be findings demonstrated that solitary conidia of M laxa behaved consistently on plum and nectarine fruit surfaces: appressorium formation and direct penetration was not observed on any of the fruit surfaces and germ tubes penetrated fruit predominantly through stomata, lenticels and microfissures in the fruit skin. The monitoring of airborne conidia revealed subtle effects of the fruits on the behaviour of solitary germlings, which could not be seen when using conidial suspensions. On both fruit types, no deleterious effect was seen on conidial and germling survival when fruit were kept humid at pit hardening, 2 wk before harvest and harvest. However, conidial and germling survival were drastically reduced by prolonged wet incubation of fruits. The findings on disease expression in the skin segment, paraquat-treated fruit and sound fruit tests clearly showed that the skin of both nectarine and plum fruits were not penetrated at the pit hardening stage, latent infections were not established and fruitsreacted resistant to disease expression. These facets on both fruit types were furthermore unaffected by wetness. The barrier capacity of the fruit skin of the two stone fruit types however differed drastically later in the season. On nectarine, fruit skins were more readily penetrated and disease expression became more pronounced when fruit approached maturity. Penetration and disease expression on ripening nectarine fruit were furthermore greatly influenced by wetness. Maturing plum fruit, on the other hand, did not display the drastic change in the barrier capacity of fruit skins as observed on nectarine. The influence of wetness on infection and disease expression was also less pronounced than on nectarine. In fact, plum fruit remained asymptomatic in the sound fruit test after inoculation and humid incubation at the 2 wk before harvest stage, harvest stage and after cold storage. Plum fruit at these stages only developed disease after a prolonged period (~12 h) of wet incubation. The paraquat fruit test revealed that these fruits became more susceptible to latent infection, but they were not as susceptible as nectarine. Collectively, these findings indicate that M. laxa fruit rot epidemics on plum and nectarine are driven by inoculum levels on fruit approaching maturity and by weather conditions prevailing during the preharvest and harvest period. However, the barrier capacity of plum skins is considerably more effective than that of nectarine fruit. Wounds would therefore play an important role in the epidemiology of M. laxa on plum fruit. Infection of fresh wounds by airborne M. laxa conidia, and by conidia and germlings that have established on fruits, was therefore investigated. Plum fruit (cultivar Laetitia) at pit hardening, 2 wk before harvest, harvest stage and after cold storage were dusted with dry conidia of M. laxa in a settling tower.- Infection of rionwounded fruit and of fresh wounds by \ the airborne conidia on dry, humid and wet plum fruit surfaces, and by conidia and germlings that have been established on fruits under the wetness regimes was then investigated. Nonwounded immature and mature fruit remained mostly asymptomatic, whereas nonwounded cold stored fruit decayed readily. Wounding drastically increased infection by airborne conidia. Immature fruits were less susceptible to wound infection by the airborne conidia than mature fruits. Conidia dispersed freshly were more successful in infecting fresh wounds than conidia that were deposited, or germlings that established, on fruit surfaces 4 days prior to wounding. This decrease in infectivity was especially pronounced on humid and even more on wet incubated fruit. This study clearly showed that in order to reduce. the incidence of brown rot, inoculum levels on fruit approaching maturity should be reduced by sanitation practices and fungicide applications. Furthermore, it is essential to protect fruits, especially. near-mature fruits, from being wounded.
Evaluation 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.
Evaluation 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.
Evolution and detection of Fusarium oxysporum f. sp. cepae in onion in South Africa
(Stellenbosch : Stellenbosch University, 2010-03) Southwood, Michael J.; McLeod, Adele; Viljoen, Altus; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs.
Genetics of fumonisin biosynthesis and resistance to fusarium verticillioides in maize
(Stellenbosch : Stellenbosch University, 2018-12) Van Zyl, Karlien; Viljoen, Altus; Rose, Lindy J. ; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Fusarium ear rot (FER) is a serious disease of maize (Zea mays L.) caused by the hemi- biotrophic fungus Fusarium verticillioides. The fungus also produces toxic secondary metabolites, known as fumonisins, in the grain that pose serious animal and human health risks. The role that fumonisins play during F. verticillioides infection is unclear, but they are believed to suppress host basal defence and facilitate necrotrophic proliferation. Disease severity and fumonisin contamination of maize are influenced by host and fungal genetics, and by plant stresses imposed by warm and dry climates. Resistance to F. verticillioides is quantitatively controlled and strongly influenced by the environment. No maize cultivar exists that is immune to FER and fumonisin contamination. The availability of the full genome sequences of both maize and F. verticillioides, however, makes it possible to investigate plant and fungal genetic responses during infection. A cluster of genes responsible for fumonisin biosynthesis in F. verticillioides has been identified, but its regulation is not yet well understood. Random insertional mutagenesis was thus performed to potentially identify genes governing fumonisin production. A mutant was obtained that produced significantly more fumonisins than its wild-type strain. Functional annotation of the single insertion site in the mutant strain showed that it was in a non-protein- coding area of F. verticillioides chromosome 10. The integration potentially causes transcriptional interference of the downstream gene encoding a F. verticillioides 7600 hypothetical protein (FVEG_08564). In future this gene needs to be inactivated by targeted mutagenesis to confirm its function, and the expression of fumonisin in maize grain infected by the mutant determined. Information on the expression of F. verticillioides genes in maize kernels during infection is limited. This is due to the small amount of fungal RNA produced in maize, which makes transcriptome sequencing unreliable. A targeted approach to study the expression of genes involved in fumonisin production in planta was, therefore, employed. A reverse-transcription quantitative PCR (RT-qPCR) assay was first optimized, and then used to study the relative expression of two fumonisin biosynthesis (FUM) genes in maize kernels. The expression of these genes was also correlated with fumonisins levels. A positive but non-significant correlation was obtained between FUM1 and FUM19 gene expression and fumonisin concentration. This finding was in conflict to a significantly positive correlation between FUM1 and FUM19, and fumonisin production, in vitro. The disparity could be attributed to factors affecting FUM gene expression and mycotoxin production in planta, such as host and pathogen genotype, the climate and kernel maturation. The RT-qPCR used can be a valuable tool to further investigate fungal genes expressed in maize kernels. Resistance to F. verticillioides in maize is controlled by many genes that are expressed to protect the plant from early infection, through colonization, to fumonisin production. These defence-related genes are present in both resistant and susceptible genotypes, but their induction is more rapid and stronger in resistant than susceptible plants. When transcript profiles of resistant and susceptible South African maize inbred lines were studied over a 52- day time period, genes associated with pathogen recognition and redox homeostasis were most strongly induced in the resistant than in the susceptible inbred line. During the necrotrophic phase of infection the plant responded by activating jasmonic acid/ethylene signalling and genes that modulate programmed cell death. The study provides novel insights into the upstream host recognition processes over the course of F. verticillioides infection and gene expression during the latter stages of infection.
Genetics of pathogenicity in Pyrenophora leaf diseases of barley
(Stellenbosch : Stellenbosch University, 2001-12) Campbell, Graham F. (Graham Findlay); Crous, P. W.; Lucas, J. A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most important diseases of this cereal in the south Western Cape Province of South Africa. This fungus exists as two different types (forms), namely a nettype and a spot-type that are distinguished by differential symptom expression on barley leaves. Based on this specific plant pathological difference a series of studies of agricultural importance were executed to investigate the effects of sexual recombination between these two types. In addition, studies were done to determine the difference between local net- and spot-type populations with regards to population structure and fungicide sensitivity. This dissertation therefore, consists of a collection of separate publications and as a result a certain degree of redundancy has been unavoidable. Recombination is one of the most important evolutionary forces involved with sexual reproduction. In plant-fungal agricultural ecosystems this may result in pathogenic fungal populations adapting more rapidly to control programs such as fungicide applications. The first section of the review in part 1 of this dissertation covers different aspects of sexual reproduction in ascomycetes, specifically focussing on mating-type genes, vegetative incompatibility and recombination. The major part of the review is then dedicated to various plant pathological aspects of P.teres, specifically addressing the differences between the two types, and in various cases highlighting the significance of sexual recombination within and between the net- and spot-type. Using morphological criteria for identification purposes there have been many conflicting reports concerning the identity of leaf spot isolates in the Western Cape Province of South Africa. In part 2, the correct identity was eventually achieved employing mating studies and molecular markers .: This was accomplished after single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on the differential cultivars susceptible to the net-blotch and leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. Fifteen of the net-spot hybrid progeny (F1) produced from the mating study in Part 2 were screened in Part 3 to assess their viability and genetic stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and Clipper (both differentially susceptible to spot-type isolates) produced intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a healthy set of seedlings produced similar intermediate symptoms. RAPDs conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny revealed profiles similar to those obtained for F1 isolates. RAPD molecular data, therefore, indicated that hybrid progeny of this net x spot mating were genetically stable after having been subjected to two repetitive inoculation and reisolation cycles. Phylogenetic analysis of DNA sequences of the internal transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA gene and the 5' end partial histone-3 gene confirmed the genetic stability of the hybrid progeny. These results also indicated that the hybrid progeny produced consistent symptoms throughout the series of experiments, and maintained their virulence to the differential cultivars screened. Both types of P. teres are prevalent in the south Western Cape Province of South Africa, found on susceptible cultivars often grown within close proximity of each other. In Part 4, a net- and spot-type population were characterised in terms of their population structure using RAPD markers. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. A total of 65 loci were produced of which 54 were polymorphic. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spottype populations. A coefficient of genetic differentiation (Gs) of 0.0149 was obtained between sites within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis done on the two populations together with six progeny from the mating between a netand spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the nettype population also contained a unique spot-type RAPD fragment. This suggested that sexual recombination may be taking place between isolates of the net- and spot-type under field conditions. Fungicide application is the most important method used in the control of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values) of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol demethylation inhibiting fungicides were determined, based on the inhibitory effect on radial mycelial growth. The fungicides evaluated were triadimenol, bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and spot-type isolates revealed strong resistance to triadimenol while flusilazole was shown to be the strongest inhibitor of fungal growth. Spot-type isolates showed a higher resistance than net-type isolates to all five fungicides screened. The ICsD values indicated significant differences between four of the fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The ICsD values between propiconazole and bromuconazole were not significant. This study suggested that spot-type isolates showed a higher degree of resistance to commercially used fungicides than net-type isolates. The overall conclusion of this study is that the spot-type of P. teres is the pathogen associated with leaf spots of barley in the south western Cape province of South Africa and not P. japonica as earlier reported. Together with the net-type, both types exist as genetically variable populations in this barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to one another in which either net- or spot-type susceptible cultivars are being cultivated may lead to sexual progeny being produced. This in turn may lead to an increased rate at which fungal populations may become resistant to commercially used fungicides. It is furthermore suggested that an alternative fungicide seed treatment is used instead of triadimenol due to high resistance of P. teres to this fungicide.
Identification and characterisation of diatrypaceae species associeated with declining grapevines and alternative hosts in South Africa
(Stellenbosch : Stellenbosch University, 2017-03) Moyo, Providence; Halleen, Francois; Lizel Mostert; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Grapevine trunk diseases have devastating impacts on the sustainability of viticulture, worldwide. Eutypa dieback, in particular, has caused large economic losses and premature mortality of vines. This disease has, for many years, been associated with the Diatrypaceae fungus, Eutypa (E.) lata. Several species of Diatrypaceae were, however, recently discovered to be associated with Eutypa dieback-affected grapevines in different grape growing areas including Australia, Chile, Spain and United States of America. No extensive study has been conducted to identify and characterise the species of Diatrypaceae in South Africa. Surveys were conducted in vineyards located in different grape growing regions of the Western Cape and Diatrypaceae fungi were isolated from grapevines with dying spurs or wood with wedge-shaped necrosis in cross section, as well as from perithecia on dead wood. Isolates were studied using phylogenetic analyses of combined DNA sequences of the internal transcribed spacer regions (ITS1 and ITS2) and 5.8S rRNA gene as well as partial β-tubulin gene. Morphological characteristics of perithecia were also studied. Morphological and phylogenetic analyses revealed the presence of seven Diatrypaceae species to occur on grapevine in South Africa, namely Cryptovalsa (C.) ampelina, C. rabenhorstii, E. consobrina, E. lata, Eutypella (Eu.) citricola, Eu. microtheca and E. cremea, which was described as a new species. The most common species isolated from dying spurs, in order of abundance, were C. ampelina (46.4% of total number of isolates), Eu. citricola (26.8%), E. lata (20.1%), E. cremea (4.3%), Eu. microtheca (1.2%), E. consobrina (0.6%) and C. rabenhorstii (0.6%). On the other hand, from wedge-shaped necrosis, E. lata represented the most frequent species (89.2% of all isolates obtained) followed by Eu. citricola (8.5%), E. cremea (1.4%) and C. ampelina (0.9%). Five species namely, E. lata, C. ampelina, E. cremea, Eu. citricola and Eu. microtheca were found to produce perithecia on dead grapevine wood. These results suggest that Eutypa dieback in South Africa can be associated with several Diatrypaceae species. Different fruit and ornamental trees occurring near vineyards were investigated to determine whether they are colonised by Diatrypaceae species, which are associated with Eutypa dieback of grapevine. Isolates of Diatrypaceae were collected from these trees showing symptoms of dieback, cankers and perithecia. Isolates were analysed by morphological and phylogenetic analyses as described above. Fourteen species namely, C. ampelina, E. consobrina, E. lata, Eu. citricola, Eu. microtheca, E. cremea, Cryptosphaeria (Cr.) multicontinentalis, Cr. ligniota, Diatrypella sp., Eu. leprosa, Eu. australiensis and three undescribed Eutypella species were identified from 29 different fruit and ornamental trees, occurring in close proximity to vineyards. The five most prevalent species were E. lata, C. ampelina, E. cremea, Eu. citricola and Eu. microtheca, which were also the most prevalent on grapevine. These findings suggest that cross infections are possibly occurring between grapevine and other woody hosts growing near vineyards in South Africa. These five species were also the only Diatrypaceae species isolated from stone fruit trees. Pathogenicity of these five Diatrypaceae species on stone fruit trees (apricot and plum) was also determined. In these pathogenicity studies, all five species were pathogenic on both apricot and plum, producing brown-red discolouration, typical of Eutypa dieback of apricot. Finally, pathogenicity of Diatrypaceae species identified from grapevine and other woody hosts in South Africa was evaluated on grapevine, under field conditions. Artificial inoculations of these fungal species were conducted on fresh pruning wounds and lignified shoots of Cabernet Sauvignon as well as green shoots of Cabernet Sauvignon and Sauvignon blanc. After 10 months, all the species caused disease symptoms (brown discolouration) on pruning wounds and lignified shoots of Cabernet Sauvignon. Disease symptoms were also observed on green shoots of both cultivars. Pathogenicity results revealed that several species including C. ampelina, Eu. microtheca, Eu. leprosa, and Eu. citricola were equally virulent as the well-known pathogen, E. lata. Quantitative real-time PCR (qPCR) assays were also developed for the detection and quantification of E. lata and C. ampelina in grapevine wood. The qPCR assays were specific and successfully quantified target taxa in artificially inoculated wood samples. The present study provides knowledge on the identity of Diatrypaceae species associated with declining grapevines and other woody hosts occurring adjacent to vineyards in South Africa. This knowledge, together with qPCR assays can be useful in early diagnosis of infection caused by Diatrypaceae species in vineyards. Furthermore, pathogenicity studies have shown that many Diatrypaceae species, including those obtained from other woody hosts, are pathogenic to grapevine. As such, this study forms the platform for further studies aimed at managing Diatrypaceae species causing disease on grapevine in South Africa.
The identification and characterization of resistance musa to Fusarium Oxysporum F.SP cubense race 1
(Stellenbosch : Stellenbosch University, 2016-03) Ssali, Reuben Tendo; Viljoen, Altus; Kiggundu, A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Fusarium oxysporum f. sp. cubense (Foc), a soil-borne fungus affecting bananas (Musa spp.), is considered one of the most devastating pathogens in agricultural history. The fungus infects banana roots, colonises the rhizome and pseudo stem, and causes a lethal wilting disease called Fusarium wilt. Fusarium wilt can cause losses of up to 100% in banana fields planted with susceptible genotypes, without any known cure. Host plant resistance to Foc, which has been identified in the Musa gene pool, is widely considered the only feasible method to control the disease. However, conventional breeding to improve susceptible banana varieties is hampered by male and female sterility and the long generation period of the crop. The inheritance of resistance in Musa to Foc race 1 in the ‘SN8075F2’ population, derived from the cross of cultivar ‘Sukali Ndiizi’ and the diploid banana ‘TMB2X8075-7’, was investigated in this study. One hundred and sixty three F2 progenies were evaluated for their response to Fusarium wilt in a screen house experiment. The test plants were inoculated by mixing loam soil with millet grains, colonized by Foc race 1, in polythene pots. One hundred and fifteen genotypes were categorized as susceptible and 48 as resistant based on rhizome discolouration. Mendelian segregation analysis for susceptible vs. resistant fitted the segregation ratio of 3:1 (X2 =1.72, P=0.81), suggesting that resistance to Fusarium wilt in the diploid line ‘TMB2X8075-7’ is provided by a single recessive gene. The name pd1 (Panama disease 1) has been proposed for the recessive gene responsible for resistance to Fusarium wilt in the diploid line ‘TMB2X8075-7’. DArT markers were identified in a segregating population following a cross between the susceptible banana cultivar ‘Sukali Ndiizi’ and a resistant diploid banana ‘TMB2X8075-7’. The markers were in qualitative linkage disequilibrium, with 13 markers linked to resistance and 88 markers associated with susceptibility to Foc race 1. Putative functions have been assigned to candidate genes through in-silico database analysis including Laccase-25 (LAC25), Homeobox-leucine zipper protein (HOX32), SWIM zinc finger family protein, Transcription factor MYB3, GDSL esterase/lipase EXL3 among others. The candidate markers and genes closely associated with resistance/susceptibility could also be used in genetic engineering or for marker-assisted selection (MAS) in breeding for Fusarium wilt resistance. The Foc race 1-banana binomial interaction of three genotypes (‘Sukali Ndiizi’ AAB, ‘Mbwazirume’ AAA and ‘TMB2X8075-7 AA) was investigated by deep sequencing of the root transcriptome to study Fusarium wilt resistance in bananas. A total of 299 million raw reads, each about 100-nucleotides long, were derived from cDNA libraries constructed at four time points: 0, 48, 96 and 192 hrs after inoculation with Foc race1. From the 10136 differentially expressed genes (DEGs), 5640 (55.7%) were uniquely up-regulated and 4496 (44.4%) uniquely down-regulated in the libraries of ‘Mbwazirume’, ‘TMB2X28075-7’ and ‘Sukali Ndiizi at 48, 96 and 192 hrs post inoculation. The DEGs were annotated with Gene Ontology (GO) terms and pathway enrichment analysis, and significant pathway categories identified included the ‘Metabolic’, ‘Ribosome’, ‘Plant–pathogen interaction’ and ‘Plant hormone signal transduction’ pathways. Salicylic acid and ethylene were stimulated in the ‘Plant hormone signal transduction’ pathways in all the three genotypes. Fifteen defence-related genes were identified as candidate genes contributing to Fusarium wilt resistance in banana. These candidate genes could be used to improve susceptible banana genotypes to enhance levels of fungal disease resistance to Foc race 1.
Identification and management of toxigenic fusarium species associated with fusarium head blight and fusarium crown rot of wheat in South Africa
(Stellenbosch : Stellenbosch University, 2018-03) Van Coller, Gerhardus Johannes; Viljoen, Altus; Lamprecht, Sandra, C. ; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Fusarium head blight (FHB) and Fusarium crown rot (FCR) are important diseases of wheat. FHB, which is caused by members of the Fusarium graminearum species complex (FGSC), reduces yields and grain quality, and contaminates grain with mycotoxins (toxic secondary fungal metabolites) like deoxynivalenol (DON), nivalenol, and zearalenone (ZEA). FCR, caused by Fusarium pseudograminearum, infects the lower stem and causes stem rotting, which also results in yield losses. Fusarium pseudograminearum can also cause FHB. Management of FHB involves integrating agronomical practices (crop rotation, tillage), host resistance and chemical control. In the irrigation regions of South Africa, however, producers rotate wheat with summer crops like maize, which also hosts the FGSC, while crop rotation and minimum/no-till is common in the Western Cape. These practices necessitates the use of host resistance and chemical control to manage FHB in South Africa. Accurate identification of pathogens causing FHB and FCR in South Africa is needed to determine their distribution and to assist breeders in developing resistant wheat cultivars. Comprehensive surveys were thus conducted over 2 years to identify Fusarium species and their chemotypes in the country. The FGSC with the 15-cetyldeoxynivalenol chemotype predominated in the irrigation regions of South Africa, while F. pseudograminearum was the dominant species in the Western Cape region. Commercial irrigation and dryland wheat cultivars, as well as test lines with different quantitative trait loci (QTL), were evaluated for resistance to FHB by field-inoculation with F. graminearum s.s. and F. pseudograminearum. Among the irrigation cultivars, Krokodil, SST843, SST866, and SST884 performed the best. The Fhb1 QTL had a marked effect on the percentage Fusarium-damaged kernels, and reduced DON content in most test lines. One test line, BFUS2011-17, with no QTLs from Sumai 3, grouped among the top performers. Disease levels were low in dryland trials, but DON and ZEA levels were high in the first year, with the resistant control and SST027, SST056 and SST087 containing low mycotoxin levels. The efficacy of three commercial fungicides (Abacus, Amistar Xtra and Prosaro) and two chemical seed treatments (Galmano Plus and Vitavax Plus) to manage FHB and increase grain quality was determined in field trials over 2 years. All foliar treatments reduced disease and DON levels, while significantly improving grain quality and yield. Seed treatments had little effect on FHB, but Abacus combined with seed treatments reduced disease incidence more than Abacus alone. Prosaro combined with Galmano Plus reduced yields compared to Prosaro alone. This study provided the first comprehensive report of Fusarium species and their type B trichothecene chemotype associated with FHB and FCR in South Africa. Irrigation cultivars, test lines and dryland cultivars with improved resistance to FHB and DON contamination were identified, while fungicides reduced FHB and DON, and improved grain quality. Future studies should determine the natural occurrence of mycotoxins in wheat fields throughout South Africa, develop resistant irrigation and dryland cultivars, determine the efficacy of chemical control of FHB in different environments over multiple years, and optimise chemical control of FHB through timing of application, different nozzles and adjuvants.
Improving pruning wound protection against grapevine trunk disease pathogens
(Stellenbosch : Stellenbosch University, 2014-03) Mutawila, Cheusi; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Grapevine trunk diseases are a cause of decline and loss of productivity in grapevines at all stages of growth. These diseases are caused by a complex of wood-inhabiting fungi that infect mainly through pruning wounds. The management of these diseases relies on wound protection to prevent infection since there are no eradicative control measures to cure infected vines. There are few or no fungicides registered for grapevine pruning wound protection in most countries, while Trichoderma biocontrol agents are often available. This study aimed at improving grapevine wound protection by Trichoderma (T.) spp. and to gain a better understanding of the factors and mechanisms involved in biocontrol. The effect of pruning time (early or late) and five timings of application of the biocontrol agent after pruning on pruning wound colonisation by T. atroviride and T. harzianum were determined. Chenin blanc and Cabernet Sauvignon vineyards were pruned in July (early) and August (late) of 2011 and 2012, and pruning wounds were treated with suspensions of the Trichoderma spp. at various times (0, 6, 24, 48 and 96 hours) after pruning. Wound colonisation was depended on the physiological state of the vine at pruning for both cultivars. However, for the 2012 season in Chenin blanc, wound colonisation was similarly high for both pruning times, which was attributed to high rainfall and humidity. Application of the biocontrol agents 6 hours after pruning consistently resulted in high wound colonisation by the Trichoderma spp. in both cultivars and pruning times. In both cultivars, pruning wound infection due to natural inoculum was higher in wounds made in late winter than those made earlier. The effect of conidial formulation in nutritional (glucose, yeast extract and urea) and bio-enhancing (chitin and cell free culture filtrates) additives, on pruning wound colonisation by T. atroviride was also investigated. Nutritional additives increased the extent of pruning wound colonisation by T. atroviride compared to the un-amended conidial suspensions in a glass house study. The additives as well as Garrison, a fungicide containing pruning wound paint, and Eco77®, a registered T. harzianum biocontrol product, were tested in field trials for wound protection from infection by Phaeomoniella (Pa.) chlamydospora. In 2011, the pathogen was inoculated a day after pruning and all the Trichoderma spp. treatments similarly reduced Pa. chlamydospora infection by 75% to 90% in Thompson Seedless, while control was less in Chenin blanc and ranged from 40% to 74%. In 2012, the trial was carried out on Chenin blanc only and the pathogen was inoculated at intervals of 1, 3 and 7 days after pruning. Wound protection by the Trichoderma treatments was highest when wounds were inoculated with Pa. chlamydospora seven days after pruning. Two conidial formulations, a culture filtrate made from a chitin based medium and a combination of yeast extract, urea and glucose, consistently enhanced biocontrol efficacy. These formulations reduced Pa. chlamydospora infection to levels similar to those of Garrison. The integration of chemical and biological wound protection could provide both immediate and long term wound protection, but is limited by the sensitivity of the biocontrol agent to fungicides. Benzimidazole resistant Trichoderma strains were generated by gamma irradiation from the wild type isolates of T. atroviride (UST1 and UST2) and T. harzianum (T77). Mutants from UST1 and UST2 were of similar biological fitness as the wild type isolates and retained their in vitro antagonistic activity against grapevine trunk pathogens, while the mutant from T77 had reduced fitness and was not antagonistic to the pathogens. The wild type, UST1, and its mutant were tested alone and in combination with thiophanate methyl and carbendazim, respectively, for their ability to prevent pruning wound infection by Pa. chlamydospora. The combination of the UST1 mutant and carbendazim was the most effective treatment and gave the highest reduction in Pa. chlamydospora infection (70% to 93% control). Grapevine cell cultures were used to compare the response of grapevines to T. atroviride and Eutypa (E.) lata as a first step to determining the importance of Trichoderma-grapevine interactions in pruning wound bio-protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis related (PR) proteins was profiled over a 48-hour period using quantitative reverse transcriptase PCR. The cell cultures responded to fungal elicitors in a hypersensitive-like response that lead to a decrease in cell viability. Fungal elicitors from both fungi triggered the same genes and caused up-regulation of phenylalanine ammonia-lyase (PAL), 4 coumaroyl Co-A ligase (CCo-A), stilbene synthase (STS), chitinase class IV (CHIT IV), PR 3 and PR 4, and a down regulation of chalcone synthase (CHS) genes. Higher expression of PAL and CHIT IV in cell cultures treated with the T. atroviride elicitor led to a significantly higher (P < 0.05) total phenolic content and chitinolytic enzyme activity of the cell cultures compared to cell cultures treated with the E. lata elicitor. The response of the cell cultures to the T. atroviride elicitor signifies that the induction of grapevine resistance may be involved in wound bio-protection. The role of secondary metabolites produced by Trichoderma spp. used in pruning wound protection was also investigated. A volatile antimicrobial compound, 6-pentyl α-pyrone (6PP), was isolated and found to be the major secondary metabolite from the T. atroviride (UST1 and UST2) and T. harzianum (T77) isolates. This metabolite was found to inhibit mycelial growth, spore and conidia germination of E. lata, Neofussicocum (N.) australe, N. parvum and Pa. chlamydospora. The production of 6PP was induced when the T. atroviride isolates were grown in a grapevine wood extract medium while for UST1, the 6PP concentration was further doubled when it was co-cultured with N. parvum. Results therefore, indicate that 6PP is involved in the Trichoderma-pathogen interactions on pruning wounds. The results of this study have provided new information in regards to the application of Trichoderma-based pruning wound products. The best time of application proved to be 6 hours post pruning. The formulation of conidial suspensions of Trichoderma spp. with nutritional additives and in protein extracts of the biocontrol agent showed potential in reducing variability of wound bio-protection. However, further research would be necessary to develop commercial products. The application of a fungicide together with Trichoderma spp. in the field holds promise to improve control, but would require further trials for possible commercialisation. This study is the first to report on grapevine host defence genes that are activated by the Trichoderma spp. used in pruning wound protection. Together with the characterisation of the major secondary metabolite produced by these Trichoderma spp., this information aids in understanding the mechanisms involved in the complex interaction between the biocontrol agent, the host and the pathogen.
The influence of varying ratios of potassium, calcium and magnesium nutrition on quality and storage of potatoes (Solanum tuberosum L.)
(Stellenbosch : Stellenbosch University, 1993-12) Bester, Gabriel Gerhardus; Maree, P. C. J.; De Villiers, O. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: The low calcium concentration of the soils and nutritional imbalances between potassium. calcium and nitrogen have been Identified by the potato industry of South Africa as some of the reasons for Insufficient quality and keeping quality of potato tubers A nutrient fertigation study was conducted under controlled environmental conditions to Investigate the Influence of nutritional Imbalances between potassium. calcium and magnesium (varying cation ratios) on quality parameters One experiment included a boron foliar spray at tuber initiation. Cultivars Hertha. Pimpernel, Up-to-date, Sandvelder and BPI were included in the experiments. Total tuber yield was not influenced to any great extent by different cation ratios. The calcium magnesium mllliequivalent ratio of 26 5 significantly decreased the tuber yield and the calcium: magnesium ratio of 3 6 produced the highest yield under investigation. Potato tuber size grading was improved by an increase in calcium concentration in the different treatments Internal brown spot decreased with an increase in calcium concentration in the different cation ratio treatments. Although the specific gravity, chip colour, fructose, glucose and sucrose concentration of the potato tubers were influenced by the cation ratios, the difference, were not marked. The boron foliar spray applied at tuber initiation increased nitrogen, potassium, calcium and boron, and decreased the copper, zinc and manganese concentrations of potato plants The varying cation ratios influenced the mineral concentration of the potato plants and interacted with cultivars. Haulms and leaves showed a different response to treatments. The varying cation ratios and boron foliar spray resulted in a difference in mineral concentrations in the periderm, cortex and medulla of freshly harvested potato tubers Cultivars differed in mineral concentration in each treatment. Changes in potassium calcium and magnesium concentration of potato tubers during cold storage were influenced by treatments. Sprouting of progeny tubers (physiological quality) of treated plants was influenced by varying cation ratios, boron foliar spray and cultivars Sub-apical necrosis was observed in treatments with the lowest concentration of calcium Sprout mineral concentration was influenced by treatments. especially the calcium and magnesium contents. Susceptibility of potato tubers to Fusarlum so/ani can be influenced by fertilizing with varying cation ratios. The influence of treatments on susceptibility changed with cold storage. Pectin bound calcium and magnesium concentrations were determined in the medulla of potato tubers The pectin bound calcium concentration at harvest and after cold storage, was influenced most and the pectin bound magnesium was less by the treatments. The sugar concentration in the medulla of cold stored potato tubers was mainly influenced by cultivars, the effect of the varying cation ratios being less marked The fructose and glucose concentration increased and the sucrose concentration decreased with cold storage The magnitude of this change in sugar concentration was influenced by cultivar and to a lesser extent by the varying cation ratios The cortex fructose and glucose concentration was higher, and the sucrose concentration lower than that of the medulla The difference in sugar concentration between the cortex and medulla was mainly influenced by different cultivars. This study therefore shows that varying ratios of potassium to calcium to magnesium, a boron foliar spray and different cultivars have a marked effect on potato tuber quality at harvest and after cold storage.

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