Department of Plant Pathology
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- ItemAgricultural practices and their potential role in mycotoxin contamination of maize and groundnut subsistence farming(Academy of Science of South Africa, 2019-09-26) Phokane, Sylvia; Flett, Bradley C.; Ncube, Edson; Rheeder, John P.; Rose, Lindy J.Mycotoxigenic fungi are common pathogens of maize and groundnuts; they produce mycotoxins which reduce the yield and quality of these grain crops. Numerous agricultural practices including crop rotation and storage methods have been shown to impact mycotoxin accumulation. Therefore, the farming and storage practices in maize and groundnut subsistence farming systems in Pongola, Vryheid, Jozini, Manguzi and Mbazwana Districts of northern KwaZulu-Natal (South Africa) were surveyed to determine their potential role in promoting or mitigating mycotoxin contamination. A questionnaire about agricultural farming practices and storage facilities was presented to 65 subsistence maize and/or groundnut farmers. At least 90% of the farmers surveyed were not aware of mycotoxins and their consequences to animal and human health. The majority of the farmers did not practise crop rotation. However, they practised intercropping and sorted damaged and mouldy grain (maize and groundnuts) before storage. The damaged or mouldy grain was largely used as animal feed, thereby exposing animals to an increased risk of mycotoxicoses. Metal tanks and inqolobane (a type of wooden structure) were identified as the most common storage structures. Harvested homegrown maize was mostly used for the farmers’ own consumption but also sometimes sold to the local community. The implementation of mycotoxin awareness campaigns is necessary, particularly in these districts. The storage facilities used by the subsistence farmers allowed increased moisture and insect invasion. The need for the surveillance of mycotoxins in subsistence-farmed food crops is vital.
- ItemApple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) : detection and isolate characterization in South African pome fruit(Stellenbosch : Stellenbosch University, 2015-03) Gagiano, Magdalena Christiena; Bellstedt, D. U.; Mostert, Lizel; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) are known to infect pome fruit in all pome fruit producing regions of the world. In this study, a comparison between double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) for ASGV detection in pome fruit was performed. A total of 15 ASGV positive orchard leaf samples were detected using RT-PCR whilst DAS-ELISA could only detect 13. RT-PCR was found to be at least 126 fold more sensitive than DAS-ELISA. In an assessment of the genetic variation of ASGV in South Africa, the coat protein (CP) gene of isolates was sequenced, aligned and phylogenetically analysed with ASGV CP gene sequences from GenBank. Parsimony analysis identified groups of isolates, but could not resolve the relationships between them. In order to obtain better resolution, whole genome sequences of international ASGV and Citrus tatter leaf virus (CTLV) isolates were aligned with ASGV and CTLV CP gene sequences and phylogenetically analysed with parsimony. South African ASGV isolates grouped into three clades and showed multiple origins and no geographical trend. In an assessment of the genetic variation of ASPV in South Africa, the CP gene sequences of infected samples were aligned with international CP gene sequences obtained from GenBank and phylogenetically analysed using parsimony. Results from the analysis using parsimony revealed low CI and RI values indicating homoplasy in the CP gene data. To address the homoplasy, two additional analyses were performed in which the gene sequences were converted to amino acid sequences and in which the third position of the codon was excluded from the alignment. Both of these approaches resulted in a reduction in homoplasy. In an attempt to further increase the resolution of the phylogeny, the phylogenetic analysis was repeated using maximum likelihood. In the first codon unpartitioned analysis a tree with low support was retrieved followed by, as with the parsimony analysis, an analysis performed on the data translated to amino acid sequences, which showed better resolution and higher clade support. The tree with the highest resolution and clade support was retrieved by codon partitioning into first, second and third positions. South African ASGV isolates grouped into five clades and showed multiple origins and no geographical trend. This study is the first in which ASGV and ASPV have been detected using RT-PCR in South Africa. Dual infections of ASGV and ASPV were recorded in 24.7% of samples analysed. This is the first report of South African pear trees exhibiting symptoms of pear stony pit and fruit deformation associated with ASPV infection.
- ItemApplication of fungicides against postharvest botrytis bunch rot of table grapes in the Western Cape(South African Society for Enology and Viticulture, 1994) De Kock, P. J.; Holz, G.Fungicide programmes for the control of postharvest Botrytis bunch rot on table grapes were evaluated in six trials from 1984/85 to 1991/92 in the Western Cape. The study demonstrated the ineffectiveness of dicarboximide applications during bloom to early pea size in well managed vineyards. Dicarboximides were most effective when applied from bunch closure to ripening. lprodioue/sulphur treatments at veraison and before harvest reduced Botrytis bunch rot, but they were ineffective in inhibiting infection during storage. Control was only achieved when grapes were exposed to S02 during storage. Although bunch dip treatments reduced infection in the vineyard, this control was not commercially acceptable. Therefore no real advantage was found when bunches were dipped in fungicide at veraison to ensure better coverage. The fact that berries became infected primarily during harvest, package operations and storage, emphasised the necessity for reducing B. cinerea inoculum on harvested grapes. It is suggested that the results of this investigation may lay the foundation for incorporating biological control in Botrytis bunch rot control.
- ItemAssessing genotype-by-environment interactions in aspergillus ear rot and pre-harvest aflatoxin accumulation in maize inbred lines(MDPI, 2017) Okoth, Sheila; Rose, Lindy J.; Ouko, Abigael; Netshifhefhe, Nakisani E. I.; Sila, Henry; Viljoen, AltusAspergillus flavus, causal agent of the Aspergillus ear rot (AER) of maize, also produces aflatoxins that cause aflatoxicosis in humans and livestock. Ten maize inbred lines were evaluated in replicated trials in two aflatoxicosis outbreak hot spots in Kenya and in three maize-growing areas in South Africa for resistance to AER, A. flavus colonization, and pre-harvest aflatoxin accumulation during the 2012/13 growing season. AER severity was measured by visual assessment, while A. flavus colonization and aflatoxin content were quantified by real-time polymerase chain reaction (PCR) and liquid chromatography tandem mass spectrometry, respectively. Genotype by environment interaction (GEI) was determined using analysis of variance (ANOVA), additive main effects and multiplicative models (AMMI), and genotype plus by environment (GGE) biplot analyses. Stability of genotypes was evaluated using AMMI analysis. AER severity and fungal colonization significantly (p < 0.001) varied between genotypes. GEI influenced the severity of AER symptoms and aflatoxin accumulation significantly (p < 0.001), while fungal colonization was not affected. The inbred lines response was consistent for this trait in the test environments and was thus considered a desirable measure to indicate maize lines with a high risk of aflatoxin accumulation. CML495, CKL05019, LaPosta, and MIRTC5 were the least diseased lines, with the lowest aflatoxin contamination and a stable phenotypic response across the environments. Kiboko was determined as the ideal representative test environment, with discriminative ability of the genotypes for selection of the desired stable responses of the three traits.
- ItemAssessing Phytophthora cinnamomi seasonal root colonisation patterns and pathogen response to management practices (phosphonates and rootstock tolerance) in South African avocado orchards(Stellenbosch : Stellenbosch University, 2019-04) Jolliffe, Jenna Bryanne; McLeod, Adele; Dann, Elizabeth; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Phytophthora root rot (PRR), caused by Phytophthora cinnamomi (Pc), is a destructive soilborne disease that can cause major economic losses in commercial avocado orchards. Despite this, there is limited information on the pathogen’s seasonal colonisation patterns, as well as which sampling strategy and quantification method would be best for assessing it. Current limitations in Pc quantification methods can lead to inaccurate assessments of PRR management strategies including phosphonate fungicides and PRR-tolerant rootstocks. The current study was able to identify a peak in seasonal Pc root colonisation in late autumn (May) in mature avocado orchards situated in two main production regions (Mooketsi and Letaba) in the Limpopo province of South Africa. During two investigated growing seasons (2017 and 2018), Pc root quantities were significantly higher in May 2018 than in March (early autumn), August (late winter) and October/November (late spring) of the same season (2018). In 2017, colonisation trends were less evident, which is likely due to the less conducive PRR conditions that prevailed, especially in the Mooketsi region. In Letaba (2017), August and May yielded the highest Pc root quantities in most orchards, but these did not differ significantly from the other months (March and October/November). In May, Pc root quantities were furthermore significantly positively correlated with the number of hours at soil temperatures of 15-19°C, but negatively with 20-24°C. Soil moisture fluctuations were not associated with Pc root quantities. Evaluation of two sampling strategies consisting of four tree groups (each containing five trees) and one tree group (20 trees), showed that both approaches are suitable for investigating Pc colonisation patterns. A traditional root baiting method, where leaf baits were plated onto selective media, was as effective in identifying colonisation trends as a molecular approach using small-scale root DNA extractions and quantitative real-time PCR (qPCR) analysis. A large-scale root DNA extraction and qPCR analysis method was deemed less effective. A molecular quantification (qPCR) approach was shown to be ineffective for evaluating two management strategies (phosphonate treatments and rootstock tolerance) in avocado orchards showing no obvious aboveground symptoms of PRR decline. Although root phosphite (breakdown product of phosphonates) concentrations of a 2x trunk injection treatment applied at the preventative dosage (0.3 g a.i./m2) were significantly higher than the untreated control, the Pc root and soil DNA concentrations were not significantly reduced by the phosphite, relative to the untreated control. This was for quantifications conducted in either May or October 2018 and using the best of three evaluated Pc-specific qPCR assays. The potentially more PRR-tolerant R0.06 rootstock yielded higher Pc root DNA concentrations than the Dusa® rootstock in November 2017, but not in the other two sampling months (March and May 2018). The identification of effective sampling strategies, Pc root quantification methods and the Pc root colonisation patterns in avocado orchards in the current study is important. Since May had the highest root colonisation levels, PRR management practices should be put in place to achieve optimal root protection during, or just prior to, this period (late autumn). The effective sampling and quantification methods that were identified for studying seasonal root colonisation patterns in avocado, will be useful for other studies that are conducted on the over 5000 host plant species of Pc. Alternative quantification methods to qPCR for assessing management strategies must be investigated. However, it is possible that qPCR analysis may be successful for evaluating management strategies if improvements are made to the trial design, and if analyses are conducted in diseased rather than asymptomatic orchards.
- ItemAssessment of inoculation techniques to evalute apple resistance to Phytophthora cactorum(Stellenbosch : Stellenbosch University, 2001-03) Zondo, Patience Thembelihle; Denman, Sandra; Labuschagne, Iwan; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Phytophthora cactorum (Lebert & Cohn) Schrot. is the primary cause of crown, collar and root rot diseases of apple (Malus domestica Borkh.) trees worldwide. This pathogen is most destructive in commercial apple orchards under waterlogged soil conditions and has recently been identified as causing serious disease in some South African apple orchards. Crown, collar and root diseases are difficult to control because of their unpredictability and catastrophic nature. The use of resistant cultivars and rootstocks is economical and environmentally considerate. Therefore the need to develop screening techniques that will enable the selection of desirable disease resistant traits as part of an apple-breeding program in South Africa was identified. The work undertaken in this study was aimed at optimizing different techniques to test resistance. Using two direct inoculation techniques (excised stem and intact stem) the aggressiveness of lO isolates of P. cactorum on apple rootstocks was determined. The susceptibilities of five apple rootstocks were also compared. Results have shown isolate by rootstock interaction which means isolate aggressiveness was influenced by rootstocks tested. The selectivity of isolates suggests that there may be several strains of the pathogen. Population studies of the pathogen might contribute valuable information that could lead to better interpretation of results. Rootstock susceptibility was monitored in vitro throughout the season by inoculating at monthly intervals for 26-months. It was observed that during winter, rootstock susceptibility was low compared to high susceptibility during summer. These results have revealed new information regarding changes in the relative resistance of the different rootstocks over the growing season, e.g. the susceptibility pattern of rootstock MMl06 occurred 1 to -2 months later than that of other rootstocks. This finding has important implications on the way in which resistance test results are interpreted, and emphasizes the importance of not relying on point sampling. Furthermore, useful information has been acquired regarding the epidemiology of the disease with regard to "windows of susceptibility". The phenomenon of a phase shift in susceptibility of different rootstocks needs to be tested on a broader scale to assess whether it has any practical application on resistance testing. Although different inoculation techniques are applied in breeding programs, up to now there is no consensus on which technique works best for seedling selections. Since large numbers of individuals must be tested to improve the chances of detecting resistant genotypes, mass inoculations of young seedlings is a rapid way of identifying resistant individuals. Two different screening methods were tested during this study. Using the sand-bran technique, seedlings were transplanted onto inoculated soil and the root mass was used as a measure of resistance. In a second method zoospore inoculum was applied to seedlings growing in a sand:bark mixture at different concentrations and the seedlings were subjected either to water drenching or not. In both trials the aggressiveness of isolates differed significantly from each other and only higher inoculum concentrations were effective in causing disease. The age of seedlings used in tests emerged as an important factor. Seedlings under five-months-old should not be used. Drenching inoculated seedlings enhanced disease development but the production of sufficiently high numbers of zoospores was a laborious task. Thus, it is recommended that the sand-bran inoculum technique be tested with the drenching treatment for mass selection. In conclusion this study confirms the importance of both choice of isolate and choice of inoculation intervals in determining susceptibility of rootstocks to infection. In spite of the fact that stem inoculation bioassays have limited resemblance to natural disease situations, these bioassays are useful for obtaining an indication as to whether genotypes have a degree of resistance and merit further testing. For this reason refinement of the stem inoculation bioassay is worthwhile pursuing. With regard to seedling trials, both the sand-bran and the zoospore technique appear promising but refinement of these techniques is necessary in order to present a more practical way of testing large volumes of seedlings.
- ItemBars van tafeldruiwe met spesiale verwysing na Queen of the Vineyard(Stellenbosch : Stellenbosch University, 1956-12) Meynhardt, J. T. (Johann Theron); Theron, C. J.; Le Roux, M. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: no abstract available
- ItemBeheer van vrotpootjie en oogvlek van koring in Wes-Kaapland(Stellenbosch : Stellenbosch University, 1990) Bester, Frederick Christoffel Johannes; Knox-Davies, P. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Wheat (Triticum aestivum) is grown in monoculture in much of the winter rainfall region of the Western Cape Province of South Africa. This has led to a dramatic increase in the incidence of take-all (caused by Gaeumannomyces graminis var. tritici). In a tillage experiment on the Langgewens research farm of the Department of Agricultural Development, a close correlation was found between disease incidence following cultivation with a chisel plough, a mouldboard plough or no tillage. Cultivation with a mouldboard plough, or no tillage resulted in a low disease incidence and higher yields.
- ItemBiochemiese veranderinge in druiwemos veroorsaak deur Botrytis cinerea en Rhizopus nigricans(Stellenbosch : Stellenbosch University, 1964-12) Hofmann, Gerhard; Van Zijl, J. A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: no abstract available
- ItemThe 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.
- ItemBotryosphaeria 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.
- ItemThe 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.
- ItemCharacterisation 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 19992000 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.
- ItemCharacterisation of mites and peniciccium species associated with apple core rot diseases(Stellenbosch : University of Stellenbosch, 2009-03) Van der Walt, Lene; McLeod, Adele; Spotts, R. A.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Dry core rot (DCR) and wet core rot (WCR) are among some of the most important postharvest diseases of apples in South Africa. Mouldy core (MC) is also a symptom associated with the core region of apples, but it is not of economical importance since apple tissue surrounding the core region is not affected as is the case with DCR and WCR. The incidence of core rots in harvested fruits can be as high as 12%, but in general ranges from 3 to 8%. Infections and losses can also occur during fruit handling in pack houses and during storage. Additionally, yield losses also occur prior to harvest within orchards due to premature fruit drop of core rot affected fruits. The incidence of core rot diseases in apples differ among apple cultivars, with most Red Delicious varieties being susceptible to the development of core rots, whereas core rots have rarely been reported in other cultivars such as Granny Smith. The etiology and epidemiology of WCR and DCR are poorly understood. Although many fungal genera have been associated with the diseases, small-spored Alternaria species are mainly associated with DCR, whereas Penicillium species including P. roquefortii, P. expansum and P. funiculosum have mainly been associated with WCR. Dry core rot infections have long been known to occur pre-harvest, whereas WCR is primarily known as a post-harvest disease where infections take place during fruit handling in pack houses. Recently, Tarsonemus mites have also been indicated as being a potential role player in the etiology of core rot diseases. The mites have been hypothesised to carry pathogen spores into the core region of apples, and they may also possibly cause small wounds that facilitate pathogen entry. In South Africa, apple growers have recently reported WCR as being present prior to harvest, which has not been reported previously. Therefore, the first aim of the study was to investigate the incidence, as well as the causal agent/s of pre-harvest WCR. The incidence of WCR ranged from 0% to 1.7% in eleven orchards, and was in general lower than that of DCR (0.4% to 6%). Isolation studies from eight internal positions in WCR apples showed that Penicillium was the predominant fungal genus in most of the positions, including the lesion area. Morphological and molecular characterisation of Penicillium isolates from WCR showed that P. 2 ramulosum prov. nom. was the main species isolated from lesions, as well as other isolation positions. However, this species was also the main species isolated from DCR, MC and asymptomatic apples. Penicillium expansum was only isolated at low frequencies from WCR and DCR apples. Other Pencillium species that were occasionally isolated included P. glabrum, P. chloroloma, P. chermisinum and a putative new species with closest affinity to P. dendriticum (P. species aff. dendriticum) on a DNA nucleotide sequence basis. Pathogenicity and virulence studies using three different inoculation methods showed that P. expansum was the most virulent species, followed by P. species aff. dendriticum. The P. ramulosum prov. nom. isolates varied in their virulence, but were all considered to have low virulence. The role of Tarsonemus mites in the etiology and epidemiology of core rot diseases is poorly understood, and therefore the second aim of the study was to investigate some of these aspects. The specific aims of the study were to (1) investigate the ecology of Tarsonemus mites in Red Delicious and Granny Smith orchards during different apple developmental stages, (2) determine if there is a significant association of Tarsonemus mites with diseased (WCR and DCR) fruits and (3) determine if potential core rot pathogenic fungi are associated with the mites. Tarsonemus mites were found in all of the investigated apple developmental stages (buds, blossoms, 4cm diameter fruit, mature fruit and mummies), having the highest incidence in mummies and mature fruits from Red Delicious and Granny Smith orchards. In Red Delicious fruits the Tarsonemus mites were found within the core and/or calyx tube, whereas in Granny Smith fruits the mites were restricted to the calyx tube. In Red Delicious fruits there was a significant association between dry core rot as well as total core rot (wet- and dry-core rot) with the presence of mites in the core, as well as total mites (mites in core and calyx tubes). Fungal isolation studies from the Tarsonemus mites showed that they carried potential core rot fungal pathogens within the genera Penicillium and Alternaria. The Penicillium species isolated from the mites included two of the most virulent WCR species, P. expansum and P. species aff. dendriticum.
- ItemCharacterisation of pathogens associated with trunk diseases of grapevines(Stellenbosch : Stellenbosch University, 2004-04) Van Niekerk, Jan Marthinus; Crous, P. W.; Fourie, P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology .ENGLISH ABSTRACT: In an attempt to combat some of the pathogens that are associated with trunk diseases and disorders of grapevines, research in this thesis focused on the taxonomy and pathological aspects of Coniellai/Pilidiella, Botryosphaeria and Phomopsis spp. Previously, conidial pigmentation was used to separate Pilidiella from Coniella. Recently, however, the two genera have been regarded as synonymous, with the older name, Coniella, having priority. The most important species in the Coniellai/Pilidiella complex of grapevines is C. diplodiella (Speg.) Petr. & Syd., the causal organism of white rot of grapevines. Previous studies found it difficult to distinguish between C. diplodiella and C. fragariae (Oudem.) B. Sutton, which is known to occur in soil and caused leaf diseases of Fragaria and Eucalyptus. Both these species have previously been reported from South Africa. None of the reports on C. diplodiella could be scientifically substantiated; therefore it is still a quarantine organism. However, this status has been questioned. Based on sequence analyses of the internal transcribed spacer region (ITS 1, ITS 2), 5.8S gene, large subunit (LSU) and elongation factor 1- α gene (EF l- α) from the type species of Pilidiella and Coniella, Coniella was separated from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by species with hyaline to pale brown conidia (avg. length: width >1.5), with Coniella having dark brown conidia (avg. length: width ≤1.5). Pilidiella diplodiella, previously C. diplodiella, causal organism of white rot of grapevines, was shown to be an older name for C. petrakii. This fungus is present in South Africa and is therefore no longer of quarantine importance. Based on analyses of the histone (H3) gene sequences of isolates in the P. diplodiella species complex, P. diplodiella was separated from a newly described species, P. diplodiopsis. A new species, P. eucalyptorum, is proposed for isolates formerly treated as C. fragariae, associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens was newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. The genus Botryosphaeria Ces. & De Not. are known to be cosmopolitan, with broad host ranges and geographical distributions. Several saprotrophic species have been reported from grapevines, while others are severe pathogens of this host. These species include B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.J.L. Phillips and B. ribis Grossenb. & Duggar. Species reported from South Africa as grapevine pathogens are B. obtusa, B. dothidea, B. ribis and B. vitis (Schulzer) Sacco. In the present study, morphological, DNA sequence data (ITS 1, 5.8S, ITS 2 and EFI-α) and pathological data were used to distinguish 11 Botryosphaeria spp. associated with grapevines from South Africa and other parts of the world. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. were confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme were described as new species. Although isolates of B. dothidea and B. stevensii were confirmed from grapevines in Portugal, neither of these species, nor B. ribis, were isolated in this study. All grapevine isolates from Portugal, formerly presumed to be B. rib is, are identified as B. parva based on EF1-α sequence data. Artificial inoculations on grapevine shoots showed that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes was identified as morphologically similar, but phylogenetically distinct from D. sarmentorum, while D. sarmentorum was confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria, and is thus regarded as a probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs. The genus Phomopsis (Sacc.) Bubak contains many species that are plant pathogenic or saprotrophic. Ten species are known from grapevines. However, only two have been confirmed as being pathogenic, namely P. viticola (Sacc.) Sacc., causal organism of Phomopsis cane and leaf spot and P. vitimegaspora Kuo & Leu (teleomorph Diaporthe kyushuensis Kajitani & Kanem.), causal organism of swelling arm disease of grapevines. P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, a known pathogen from Prunus sp., was shown to be a possible pathogen of grapevines in a previous study. D. perjuncta Niessl. causes bleaching of dormant canes only and is therefore of little importance as a grapevine pathogen. Recently a number of Phomopsis isolates were obtained from grapevines in the Western Cape province of South Africa. Isolations were made from Phomopsis-like symptoms, pruning wounds and asymptomatic nursery plants. These isolates showed great variation in morphology and cultural characteristics. Earlier taxonomic treatments of Phomopsis, based species identification on host specificity, cultural characteristics and morphology. Recent studies have indicated that these characteristics can no longer be used to distinguish species of Phomopsis due to wide host ranges and morphological plasticity of some species. The use of anamorph/teleomorph relationships in species identification is also untenable, since Diaporthe teleomorphs have only been described for approximately 20% of the known Phomopsis species. In this study morphological data, DNA sequences (ITS-I, 5.8S, ITS-2) and pathogenicity data were combined to distinguish Phomopsis spp. from grapevines. Fifteen species of Phomopsis were delineated by phylogenetic analysis of ITS sequence data. Diaporthe helianthi, a sunflower pathogen, was reported from grapevines for the first time, with a further six, unknown species also distinguished. Three different clades contained isolates previously identified as D. perjuncta. Based on type studies, it appeared that the name D. viticola was available for collections from Portugal and Germany, a new species, D. australafricana, was proposed for South African and Australian isolates, formerly treated as D. perjuncta or D. viticola. An epitype specimen and culture were designated for D. perjuncta. This species was distinguished from D. viticola and D. australafricana based on morphology and DNA phylogeny. Artificial inoculations of green grapevine shoots indicated that, of the species tested, P. amygdali, a known pathogen of peaches in the USA, and P. viticola were the most virulent.
- ItemCharacterisation of South African isolates of Fusarium oxysporum f.sp. cubense from Cavendish bananas(Academy of Science of South Africa (ASSAf), 2010-04) Visser, Marinda; Gordon, Tom; Fourie, Gerda; Viljoen, AltusFusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f.sp. cubense (Foc), is a serious vascular disease of bananas in most subtropical and tropical regions of the world. Twenty-four vegetative compatibility groups (VCGs) and three pathogenic races have been identified in Foc, reflecting a relatively high genetic diversity for an asexual fungus. To characterise a South African population of Foc, a collection of 128 isolates from diverse geographic origins were isolated from diseased Cavendish bananas and subjected to VCG analysis and sequencing of the translation elongation factor 1-α (TEF) gene region. The presence of mating type genes was also determined using MAT-1 and MAT-2 specific primers. VCG 0120 was established as the only VCG of Foc present in the South African population studied. Only the MAT-2 idiomorph was present in all the local isolates of Foc. A phylogenetic analysis of DNA sequences of the TEF gene region revealed that the South African isolates grouped closely with VCG 0120 isolates from Australia and Asia. These results suggest that the South African population of Foc was most likely introduced in a limited number of events and that it had spread with infected planting material within the country. The presence of only one mating type and the limited diversity in this pathogen render it unlikely to rapidly overcome disease management strategies involving host resistance. © 2010. The Authors.
- ItemCharacterisation, epidemiology and management of olive trunk disease pathogens in South Africa(Stellenbosch : Stellenbosch University, 2020-04) Van Dyk, Meagan; Halleen, Francois; Mostert, Lizel; Spies, Chris, F. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: The Olive Sector Development Plan of the Department of Trade and Industry identified low production and the lack of local research as weaknesses of the olive industry in South Africa. The management of trunk diseases forms an integral part of practices aimed at increasing olive production. A recent olive trunk disease survey performed in the Western Cape Province, South Africa, identified an undescribed Pseudophaeomoniella sp. as the most prevalent fungus associated with the trunk disease symptoms, with other fungal species occurring at much lower frequencies. In the current study, 40 of these isolates were selected for a pathogenicity study. The species forming lesions included several Botryosphaeriaceae, Phaeoacremonium and Phaeomoniellaceae species, as well as Biscogniauxia mediterranea, Coniochaeta velutina, Diaporthe foeniculina, Didymocyrtis banksiae, Eutypa lata, Pleurostoma richardsiae, Symbiotaphrina buchneri, isolates of the Cytospora pruinosa complex, and a Cytospora sp., Fomitiporella sp., Geosmithia sp. and Punctularia sp. The Pseudophaeomoniella sp. formed among the longest lesions, affirming its status as a potentially important trunk pathogen. Long distance dispersal of olive trunk pathogens is expected to occur via infected nursery material, similar to that found in other systems such as in grape and fruit trees. Nurseries as an inoculum source was investigated by making isolations from asymptomatic cuttings from mother blocks (Stage 1), rooted cuttings (Stage 2) and 1–2-year-old trees (Stage 3) of eight cultivars in two nurseries. Known olive trunk pathogens of the Botryosphaeriaceae, Diaporthaceae, Nectriaceae, Phaeomoniellaceae, Pleurostomataceae and Togniniaceae were recovered. Neofusicoccum australe was detected in a single Stage 1 cutting. Stage 3 material showed the highest incidence of fungi from these families, with P. richardsiae having the highest incidence in both nurseries (82.2% and 36.7% of the 1–2-year-old trees). Phaeoacremonium parasiticum was present in 28.9% of the trees from one nursery (Stage 3). The remaining pathogens occurred in 13.3% or less of the material. Pseudophaeomoniella sp. was present in the nurseries but at low frequencies. This suggests that alternative inoculum sources of this pathogen exists. A nested species-specific PCR was developed for the detection of Pseudophaeomoniella sp. from spore washes of pruning debris collected from established olive orchards. Pruning debris identified with a positive PCR was evaluated microscopically. Pycnidia of Pseudophaeomoniella sp. were observed on the pruning debris. Based on previous research, it is expected that the spore release coincides with rainfall and that the spores can be dispersed onto pruning wounds. The susceptibility of wounds from winter and spring pruning to Pseudophaeomoniella sp. was compared. Two-year-old olive branches of 16-year-old olive trees were pruned and inoculated with spore suspensions of Pseudophaeomoniella sp. at different time-points after pruning. The pruning wounds were susceptible for up to 42 days, with no difference between seasons (winter vs. spring). The wounds were the most susceptible within the first week after pruning. Eleven pruning wound protectants were evaluated and applied on pruning wounds made on 16–17-year-old trees directly after pruning. The treated wounds and positive (non-treated) controls were challenged with spore suspensions of Pseudophaeomoniella sp. at 1 or 7 days after pruning. Under low inoculum pressure (first season), Garrison, MT1, Neocil Plus and Tree Seal, reduced Pseudophaeomoniella sp. infections, while the Trichoderma-based protectant, MT1, was considered the most effective water-based protectant. Under higher inoculum pressure (during the second season), Tree Seal and Coprox Super/Bendazid consistently performed the best. In conclusion, several fungal species were identified as olive trunk pathogens, with Pseudophaeomoniella sp. being identified as one of the most important olive trunk pathogens. The propagation process was identified as a source of inoculum for some pathogens, including Pseudophaeomoniella sp. Inoculum sources of Pseudophaeomoniella sp. were also identified in established orchards. Olive pruning wounds are susceptible to Pseudophaeomoniella sp. for prolonged periods. MT1 was highly effective under lower inoculum pressure, while Tree Seal and Coprox/Bendazid were highly effective under high inoculum pressure. This study led to new knowledge with regards to olive trunk diseases, their pathogenicity, detection, epidemiology and control which can be used for the development of improved management strategies of olive trunk diseases in South Africa.
- ItemCharacterization and control of Phaeomoniella chlamydospora in grapevines(Stellenbosch : Stellenbosch University, 2000-12) Groenewald, Michelle; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry.
- ItemThe characterization and control of Phomopsis cane and leaf spot on vine(Stellenbosch : Stellenbosch University, 2000-12) Mostert, Lizel; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Phomopsis cane and leaf spot disease of grapevine is an economically important disease in many of the vine-growing areas of the world. Four different Phomopsis spp. have previously been associated with this disease. The present study investigates the taxonomic significance of the different taxa found on grapevines in South Africa, as well as the endophytic growth and fungicide sensitivity of Phomopsis viticola isolates. The thesis is compiled of several different parts, which deal with specific, but related topics, and hence some duplication has been unavoidable. Understanding the epidemiology of a disease is important for the correct timing of disease control. To investigate the endophytic growth of P. viticola, asymptomatic shoots were collected at eight different growth stages. Nodes, internodes, leaf petioles, leaves, tendrils and bunch peduncles were investigated. Two Phomopsis spp., taxon 1 and 2 were identified in this study. The Phomopsis viticola-complex had a relative importance of 9% and accounted for 3% of the isolations. P. viticola (taxon 2) is mainly isolated from the nodes and internodes. Inoculations of healthy, young vine tissue confirmed taxon 2 to be a virulent pathogen, suggesting that it is a latent pathogen rather than an endophyte. In contrast, taxon 1 appeared to be a true endophyte, and did not seem to be an important pathogen on vines. The true identity of the causal organism of Phomopsis cane and leaf spot disease was investigated by collecting samples from 58 different vineyards in the grapevine growing areas of the Western Cape. P. viiicola occurred in grapevine material collected from Lutzville to Swellendam, but was not found in the Oudtshoorn and Orange River grapevine areas. Diaporthe perjuncta (taxon 1), P. vutcola (taxon 2), taxon 3 and a Phomopsis species commonly associated with shoot blight of peaches in the U.S.A., P. amygdali, were identified among the South African grapevine isolates. Examination of the Australian culture designated as taxon 4 found it to be a species of Libertella, thus excluding it from the P. viticola-complex. An Italian isolate was found to represent a species of Phomopsis not previously known from grapevines, and this was subsequently described as taxon 5. Species delimitation was based on morphological and cultural characteristics, stem inoculations and the formation of the teleomorph in vitro. The identity of each morphological taxon was confirmed by means of phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacers (ITS 1 and ITS2) and the 5' end partial sequence of the mitochondrial small subunit (mtSSU). P. amygdali, associated with peach shoot blight in the U.S.A., was isolated once only and appeared to be of lesser importance in this disease complex. Furthermore, taxa 1 (Diaporthe perjuncta) and 3 were also rarely encountered and proved to be non-pathogenic, indicating their non-functional role in Phomopsis cane and leaf spot disease. Taxon 2 (Phomopsis viticolas was common and widely distributed in diseased vineyards. This taxon was associated with the typical disease symptoms and proved to be pathogenic. Morphologically taxon 2 corresponded best with P. viticola, which was also neotypified in this study. Taxon 2 was mostly isolated from buds and nodes, indicating that these are important sites in which the fungus survives during winter. Molecular data indicated that taxon 3 and P. amygdali were not host specific to grapevine. The currently used foliar fungicides were compared to the new strobilurin fungicides. The effects of nine fungicides (azoxystrobin, flusilazole, folpet, fosetyl- Al+mancozeb, kresoxim-methyl, mancozeb, penconazole, spiroxamine and trifloxystrobin) were tested in vitro on inhibition of mycelial growth. The following EC50 (ug/ml) values were obtained: azoxystrobin (0.350), flusilazole (0.007), folpet (4.489), fosetyl-Al+mancozeb (3.925), kresoxim-methyl (1.665), mancozeb (2.891), penconazole (0.023), spiroxamine (0.321) and trifloxystrobin (0.051). Additionally, azoxystrobin, folpet, kresoxim-methyl, mancozeb, propineb and trifloxystrobin were tested for their ability to inhibit spore germination in vitro. The subsequent EC50 (ug/ml) values were obtained: azoxystrobin 0.123), folpet (0.510), kresoxim-methyl (0.0037), mancozeb (0.250), propineb (0.156) and trifloxystrobin (0.003). The results reported in part 4 showed that the strobilurin fungicides inhibited the mycelial growth and spore germination of P. viticola. However, further trials need to be conducted to verify these findings under field conditions. In the present study taxa 1, 3 and P. amygdali were infrequently isolated, suggesting that they played a less prominent role in the P. viticolacomplex.
- ItemCharacterization 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.