Masters Degrees (Plant Pathology)
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- 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.
- 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
- 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.
- 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.
- ItemColletotrichum diseases of Proteaceae(Stellenbosch : Stellenbosch University, 2004-03) Lubbe, Carolien M.; Denman, S.; Lamprecht, Sandra, C. ; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: This thesis consists of four chapters that present research findings on Colletotrichum diseases associated with Proteaceae worldwide. The first chapter is a review of literature regarding the taxonomy and histology of Colletotrichum species associated with Proteaceae. The literature is not restricted to Proteaceae hosts, as information regarding Colletotrichum on Proteaceae is very limited. In chapter two, Colletotrichum spp. associated with proteaceous hosts growing in various parts of the world were identified based on morphology, sequence data of the internal transcribed spacer region (ITS-I, ITS-2), the 5.8S gene, and partial sequences of the B-tubulin gene. Four species of Colletotrichum were associated with Proteaceae. Colletotrichum gloeosporioides was isolated from Protea cynaroides cultivated in South Africa and Zimbabwe and from a Leucospermum sp. in Portugal, but is known to occur worldwide on numerous hosts. A recently described species, C. boninense, was associated with Zimbabwean and Australian Proteaceae, but also occurred on a Eucalyptus sp. in South Africa. This represents a major geographical and host extension for the species, and a description of the African strains is provided. Colletotrichum crassipes was represented by a single isolate obtained from a Dryandra plant in Madeira. Colletotrichum acutatum was isolated from Protea and Leucadendron in South Africa as well as from other proteaceous hosts occurring elsewhere. Colletotrichum acutatum f. sp. hakea was isolated from Hakea in South Africa. In chapter three, pathogenicity of these Colletotrichum species to certain proteas was established, relative aggressiveness of the different species tested and host response to them were compared as well as the effect that wounding had on host response. From the results obtained it is concluded that C. acutatum and C. gloeosporioides are the primary pathogens associated with Colletotrichum leaf necrosis, and C. acutatum is the main cause of anthracnose and stem necrosis of Proteaceae in South Africa. A histological study was performed in chapter four in response to the findings from the previous chapter. The behaviour of two C. acutatum isolates (one originating from Protea and the other from Hakea, C. acuataum f.sp. hakea) was studied on inoculated Protea leaf surfaces using light and scanning electron microscopy. Colletotrichum acutatum from Protea formed melanised appressoria on the leaf surface, whereas C. acutatum from Hakea formed very low numbers of both melanised and unmelanised appressoria. Most of the appressoria formed by C. acutatum from Protea were formed on the cell junctions and on the periclinal walls of the epidermal cells. From this study it is clear that C. acutatum f. sp. hakea is not a pathogen of Protea. Consequently the current use of this isolate as a biological control agent of Hakea in South Africa poses no threat to indigenous Protea species. Colletotrichum acutatum from Protea (although closely related to C. acutatum f. sp. hakea), is a pathogen of Protea, which was confirmed by histological observations. In conclusion, the present study has shown that several species of Colletotrichum are associated with diseased Proteaceae. These species differed in their pathogenicity and aggressiveness when inoculated onto certain protea cultivars. These differences could be partially explained by examining the behaviour of C. acutatum on the leaf surface. It is clear, however, that the distribution of the different species, their aggressrveness on different Proteaceae and their modes of infection needs to be investigated further. This work provides a basis for future research on the long-term effective management of these pathogens in fynbos production.
- ItemComparative studies on genetic variability and fungicide resistance in Tapesia yallundae(Stellenbosch : Stellenbosch University, 1998-12) Ntushelo, Khayalethu; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Eyespot is an important disease of spring wheat (Triticum aestivum L.). Four species of Ramulispora are associated with this disease, of which Tapesia yallundae and T. acuformis. are common. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance in Tapesia yallundae. Each of the chapters treats specific but related topics. T. yallundae, which is the only species thus far reported from South Africa, has been associated with yield losses of up to 50%. To enable the implementation of more accurate and effective control measures, understanding the dynamics of reproduction and the genetics of the pathogen is of utmost importance. Of the many plant disease control measures such as cultural practices, sanitation, biological control, etc., fungicide application is the most commonly resorted to measure in eyespot control. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance of Tapesia yallzll7dae. Fungicide application, however, is not without problems. The pathogen can build up resistance to fungicides. The most commonly used fungicides in eyespot control include the benzimidazole carbendazim, triazoles such as flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol, fenbuconazole, triademinol, and the imidazole, prochloraz. Cases of resistance to the groups listed above have been reported. Frequent monitoring for resistance is thus crucial to prevent wastage of fungicide and unnecessary impregnantation of the environment with potentially ineffective chemicals. In chapter 2 of this thesis 300 isolates of T. yallundae from 15 fields were evaluated for resistance against carbendazim, flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol and fenbuconazole. These results indicated that to some triazoles, such as fenbuconazole, a high level of resistance was already present in field populations. In a sexually reproducing fungus such as T. yallundae, knowledge pertaining to its ability to pass resistance factors to offspring is equally important. Mating studies were, therefore, also conducted with parental strains that showed signs of triazole resistance. Three generations were subsequently tested for resistance to five triazoles, namely flusilazole, tebuconazole, propiconazole, bromuconazole and flutriafol. Results of this study showed variable sensitivity in progeny, which indicated quantitative inheritance of resistance to triazoles. Although the sexual stage has not yet been observed in the field in South Africa, this knowledge lays the foundation for the long-term understanding of the population dynamics of the fungus. The ability of a heterothallic ascomycete population to reproduce sexually is dependent on the availability of its two mating types, MATI-I and MATI-2, their distribution, and female fertility amongst other factors. In the UK. the teleomorph is commonly observed in the field, which is in contrast to the situation in South Africa, where it has only been induced in the laboratory. A comparative study between the South African and the UK. populations was therefore undertaken. Isolates representative of the two populations were mated with tester strains as both sperm recipients and as sperm donors. This allowed the percentage of hermaphrodites to be determined. No difference in terms of female fertility was observed between the South African and the UK. populations, with both populations showing low effective population numbers. These data suggested, therefore, that the teleomorph would also occur more frequently in South Africa if the climate was more indusive to its development. The overall results of this study indicated that eyes pot could still be controlled by means of fungicide application in South Africa. Although a shift in sensitivity was observed towards fenbuconazole and flusilazole, no resistance was detected towards carbendazim. The latter might be due to the absen<.:eof the sexual stage in the field, coupled by the monocyclic nature of the pathogen and sensible fungicide regimes. The absence of T. acujormis makes the disease situation less complicated in terms of fungicide application and management. Continuous surveys will have to be conducted, however, to monitor this situation in future.
- ItemDectection and quantification of soilborne pathogens in grapevine nurseries(Stellenbosch : Stellenbosch University, 2017-12) Langenhoven, Shaun; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Black-foot disease (BFD) and crown and root rot (CRR) are important decline diseases of grapevines, worldwide. These diseases occur in both nurseries and vineyards. Black-foot disease is caused by species in the genera: Dactylonectria, Ilyonectria, Campylocarpon, Cylindrocladiella and Thelonectria, whereas CRR is caused by Phytophthora, Pythium and Phytopythium species. Plant stress, caused by improper planting holes, waterlogging, drought, nutrient deficiencies and high temperatures, is a major predisposing factor for BFD and CRR. The symptoms of these two diseases are similar and include necrotic root lesions, reduced root biomass, leaf chlorosis and ultimately, plant death. The first aim of this study was to conduct a survey of BFD and CRR pathogens in nursery grafted plants over a 3 year period (2013-2015) as well as from weeds and rotation crops in the alternate season (2014). Furthermore, pathogen detection using quantitative realtime polymerase chain reaction (qPCR) was conducted on soil samples which were collected in close proximity to the sampled grapevine plants, weeds and rotation crops. Soil was collected up to a depth of 30 cm and 60 cm. The decline pathogens that were quantified in the soil with qPCR were Dactylonectria and Ilyonectria genera, Phytophthora species and Pythium irregulare. The predominant BFD pathogens isolated from grapevines were Dactylonectria macrodidyma, Campylocarpon pseudofaciculare and Ca. fasciculare. The predominant CRR pathogens were Pythium irregulare followed by Phytopythium vexans. Furthermore, Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, Py. irregulare, Py. ultimum and Py. heterothallicum were isolated from Triticale roots. Dactylonectria was also isolated from the weed corn spurry, while Py. irregulare and Py. ultimum were isolated from numerous weeds and rotation crops. Dactylonectria, Ilyonectria, Phytophthora and Py. irregulare DNA was detected in all nurseries across all years at varying concentrations with Nursery E having the lowest concentrations. Ilyonectria and Dactylonectria mean soil DNA concentrations ranged from 0.04 pg.µL-1 to 37.14 pg.µL-1, while the Py. irregulare mean soil DNA concentrations ranged from 0.01 pg.µL-1 to 3.77 pg.µL-1. The Phytophthora mean soil DNA concentrations ranged from 0.01 pg.µL-1 to 33.48 pg.µL-1. This study successfully used existing SYBR green I qPCR assays to quantify BFD and CRR pathogens in grapevine nursery soil. During this study nursery soils were confirmed as a sources of infection for BFD and CRR pathogens, and that weeds may serve as alternative hosts to these pathogens. This is also the first report of D. alcacerensis and D. pauciseptata in South African nurseries. The second aim was to describe a putative new Phytopythium species which was isolated from nursery grapevine roots and to determine if it is a pathogenic species. Six isolates of an unknown Phytopythium species were isolated from the roots of an asymptomatic plant. Morphological observations and phylogenetic analyses, based on the ITS rDNA and cytochrome oxidase subunit 1 gene regions, proved that it was indeed a novel species. Together with an undescribed Japanese isolate, the six South African isolates were named Phytopythium paucipapillatum and displayed occasionally papillate, direct sporangial germination as well as sporangia which proliferates internally. The sporangia were globose, subglobose, ovoid, obovoid, limoniform to ellipsoid or distorted. The oogonia were small, globose, with some oogonia also displaying short papillae protruding from the surface. This putative new species was also insensitive to the isoxazole fungicide, hymexazol. A pathogenicity trial, on the rootstock 110-Richter, revealed that Pp. paucipapillatum was not pathogenic towards grapevines. In accordance with other studies regarding Phytopythium species, Pp. paucipapillatum may be a soil inhabitant.
- ItemThe detection of cherry leaf-roll nepovirus and the use of molecular markers for germplasm identification in walnuts (Juglans regia L.)(Stellenbosch : Stellenbosch University, 2003-12) Mkhize, Thokozani M; Botha, F. C.; Mansvelt, E. L.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed.
- ItemDetermining the resistance or susceptibility of grapevine rootstocks used in South Africa towards fungal trunk disease pathogens(Stellenbosch : Stellenbosch University, 2017-03) Sieberhagen, Madeleine; Halleen, Francois; Mostert, Lizel; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Grapevine fungal trunk diseases are responsible for reduced grapevine production world-wide. Trunk diseases are caused by endophytic, xylem-inhabiting pathogens associated with a wide range of symptoms. Currently the largest concern is the presence of pathogens in rootstock mother vines and propagation material. Very little information is available on the susceptibility of grapevine rootstocks used in the South African industry. The susceptibility of the most popular grapevine rootstocks in South Africa to Petri disease (Phaeomoniella chlamydospora, Phaeoacremonium minimum, Pm. parasiticum, Cadophora luteo-olivacea and Pleurostoma richardsiae), black-foot disease (Ilyonectria liriodendri, Dactylonectria macrodidyma, Campylocarpon fasciculare and C. pseudofasciculare) and Botryosphaeria dieback (Neofusicoccum australe and N. parvum) pathogens were evaluated. Firstly, virulence screenings were conducted by plug inoculating dormant Paulsen 1103 cuttings with 10 isolates of each pathogen. The two most virulent isolates were identified according to lesion lengths that developed after six weeks of incubation at 22 °C. There was significant variability between the isolates of all the pathogens, except Pleurostoma richardsiae. Dormant rootstock cuttings of Ramsey, Ruggeri 140, Paulsen 1103, Richter 99, Richter 110, US 8-7, 101-14 Mgt and SO4 were vacuum inoculated with conidial suspensions (1 x 106 conidia mL) of the two most virulent isolates of each pathogen. After eight months in nursery fields, the dormant plants were uprooted and assessed for un-dried shoot and root weight. The percentage disease severity was determined by cutting the plant 10 cm from the base and assessing the internal vascular discolouration. All of the rootstocks inoculated with Petri disease, black-foot disease and Botryosphaeria dieback pathogens showed a significant reduction in root mass, shoot mass and a significant increase in percentage disease severity. None of the rootstocks were completely resistant to fungal trunk disease pathogens, but differential levels of tolerance did exist. The reduction in shoot and root mass did not show consistent results between the rootstock cultivars, but rootstocks 101-14 Mgt and SO4 were the most susceptible and Ramsey the most tolerant, in terms of percentage disease severity. In an attempt to explain the variable tolerance of rootstocks to infection, suberin production and xylem morphology was investigated. Transversal cuts of Ramsey and 101-14 Mgt were made and stained using two suberin staining techniques. The important role suberin plays in the compartmentalization of a pathogen in grapevine wood was confirmed. Suberin was located around vessels filled with tyloses, tyloses itself was also suberized, ray parenchyma cells and cells located on the growth boundary ring. The suberized zones form impermeable barriers that restrict pathogen spread to uninfected and newly developed vascular tissue. The mean vessel diameters were determined for each cultivar using 40 μm thick transversal cuts stained with toluidine O. A strong correlation between mean vessel diameter and rootstock tolerance to fungal trunk pathogens was established. Ramsey had the smallest mean vessel diameter and 101-14 Mg the largest. The quality of plant material is crucial in the success and longevity of newly established vineyards. By combining existing knowledge of disease management in the propagation process with knowledge obtained from this research, can the quality of plant material be optimized to ensure the sustainability of the South African grapevine industry.
- ItemThe development of novel molecular diagnostic assays for fusarium oxysporum f. Sp. Cubense(Stellenbosch : Stellenbosch University, 2019-04) Matthews, Megan Ceris; Viljoen, Altus; Rose, Lindy J. ; Mostert, Diane; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Banana (Musa sp.) is an important crop for food security and income generation in developed and developing nations. Most bananas are grown for local consumption, with approximately only 14% exported to Europe, the US, Japan and Russia. The export industry is almost exclusively reliant on Cavendish bananas. Cavendish bananas also constitute approximately 47% of bananas grown globally. A major constraint to sustainable banana production is Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Race 1 and 2 Foc isolates cause disease of Gros Michel, other dessert varieties and some cooking banana like Bluggoe, but not Cavendish bananas. Subtropical (STR) and tropical (TR4) race 4 Foc isolates affect Cavendish and most banana cultivars susceptible to Foc races 1 and 2 in the subtropics and tropics, respectively. Fusarium wilt is difficult to manage as Foc chlamydospores can survive in the soil for decades. Phytosanitary regulation and clean planting material to exclude Foc, and the planting of resistant banana cultivars, are therefore required to manage the disease. Early detection and the geographic mapping of Foc can inform farmers and governing bodies about the distribution of the fungus and aid in containment strategies. DNA-based detection with PCR is favoured over phenotypic identification due to its speed and accuracy. PCR detection, however, is qualitative and lacks sensitivity when DNA is isolated from environmental samples. Quantitative (q)PCR has been developed to directly detect plant pathogens in environmental samples. Molecular markers are available to quantitatively detect Foc races 4 and TR4, but the Foc TR4 markers lack specificity. Molecular markers for Foc Lineage VI (race 1 and 2) and STR4 are also required. In this study, DNA markers and qPCR assays were developed to detect Foc Lineage VI, TR4 and STR4 in plant, water and soil samples. Markers were designed from the RNA polymerase III subunit, a hypothetical protein and the Foc supercontig 1.57 gene regions. Marker suitability and specificity was evaluated, and standard curves produced for Foc detection based on specificity, repeatability, reproducibility, limit of quantification (LOQ) and limit of detection (LOD). The Foc TR4 and Lineage VI markers were specific for qPCR detection, but the Foc STR4 markers amplified two non-target Fusarium members and two non-pathogenic F. oxysporum isolates. Quantitative PCR can detect Foc collected in the environment. DNA from non-viable cells, however, is then also amplified, which can lead to an overestimation of inoculum levels. In this study, propidium monoazide (PMA) and qPCR were combined to quantify Foc Lineage VI and TR4 spores that survive following sanitiser treatments. PMA applied at 20 μM, incubated in the dark for 1 min and activated with light for 5 min effectively separate viable from non-viable Foc spores. The PMA-qPCR results also correlate well with colony forming unit counts.
- ItemDiversity and detection of Kenyan and Nigerian populations of Fusarium oxysporum f. sp. strigae(Stellenbosch : Stellenbosch University, 2017-12) De Klerk, Madelein; Viljoen, Altus; Mostert, Diane; Beed, Fen; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: The Genus Striga contains some of the most destructive plant-parasitic weed species in the world. These include S. hermonthica and S. asiatica, which attack important staple food crops in sub-Saharan Africa such as maize, sorghum and millet. Of the two, S. hermonthica is more devastating and can cause yield losses on average of 50% and, ultimately, land abandonment. The parasite produces up to 200 000 seeds per plant, which causes a rapid build-up of a seed bank in the soil. Germination of seeds happens after a ripening period and when strigolactones, exuded by the roots of susceptible hosts, are detected. After a haustorium connects to the host, it parasitizes the host for water and nutrients, and in effect stunts the development of the host plant. Striga infestations are difficult to manage due to its prolific nature and late emergence. The weed affects subsistence farmers most severely, as these growers have little access to, or the financial resources available, to control the pest with chemicals. Biological control, therefore, was proposed as a means of control, as this method can be integrated with existing farming practises, as its use would be safe to both the farmer and the environment. Fusarium oxysporum f. sp. strigae (Fos), a soil-borne fungal pathogen affecting Striga plants, was identified as a possible Biological Control Agent (BCA), as Fos was host specific and did not produce harmful secondary metabolites. Its population structure in Africa, and means to survive and disseminate in farmer fields, however, was unknown. The aims of this study, therefore, were to characterize Fos populations in two African countries, Kenya and Nigeria, and to develop molecular markers to rapidly and accurately identify the fungus. The diversity of Fos in Kenya and Nigeria was investigated by means of vegetative compatibility group analysis (VCGs) and phylogenetic analysis. VCG analysis showed that the Kenyan isolates consisted of a single VCG, and that the Nigerian isolates were divided into seven VCGs and eight SMV’s. A combined maximum likelihood tree of the translocation elongation factor (TEF) 1α and mitochondrial small sub-unit (MtSSU) gene areas revealed that Fos isolates from the two countries separated into two different clades. This suggested that there was two separate events of evolution, and that the Nigerian isolate group is older than the Kenyan group due to the greater number of VCGs present in Nigeria. Mating type analysis confirmed clonality within the Kenyan group, where all isolates in VCG 04708 contained only the MAT1-1 gene. However, a larger diversity was found in the Nigerian group, where both mating type idiomorphs were present in the different VCGs. Molecular markers that distinguish between the two Fos populations in Africa have been developed in this study. Primer-set Foxy2-F/R1 was developed form a single nucleotide polymorphism (SNP) in the TEF gene area, and primer set FK3-F/R from SNPs in the secreted-in-xylem 14 gene area. Both primer sets were tested against a collection of isolates that includes non-pathogenic F. oxysporum isolated from Striga, other Fusarium species associated with Striga, and other formae speciales of F. oxysporum. Sensitivity assays revealed that the Foxy2 primer only detected target Fos DNA at a concentration of 10 ng/μl in the presence of S. asiatica DNA. Primer-set FK3 on the other hand, could detect target Fos DNA at the low concentration of 0.1 ng/μl in the presence of S. asiatica DNA. The findings in this study suggest that pathogenicity evolved at two separate events in Fos. The molecular markers designed could compliment the primer-set designed by Zimmerman et al. (2015) to aid in diagnostics and monitoring of Fos after application as a BCA.
- ItemThe ecology of Botrytis cinerea on grape in the Western Cape Province(Stellenbosch : Stellenbosch University, 2004-03) Van Schoor, Jan Adriaan; Holz, G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Botrytis cinerea Pers.: Fr., a pathogen of grapevine (Vitis vinifera L.), moves mainly through conidia by air currents in vineyards which are deposited intermittently on the surfaces of leaves, inflorescences and bunches. Little is known about the relationship between the inoculum dosage in air and incidence of Botrytis bunch rot, and how the relationship is influenced by environmental and host factors. To better understand this relationship, information is needed on the period over which conidia have accumulated, the time they are able to survive and remain infectious, time of symptom expression in relation to conidium arrival at the infection court and host surface wetness. The aims of this study were (i) to estimate the amount of viable B. cinerea occurring in air in vineyards, and at different positions on leaves, inflorescences and bunches of grape at different phenological stages, (ii) to determine the relationships between the number of B. cinerea colonies recorded on spore traps placed in the bunch zone of vines and the incidence of B. cinerea recorded from the different tissues, and (iii) to compare the efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum with those inoculated with dry, airborne conidia. Different techniques were used to detect viable Botrytis cinerea in air currents and on plant material obtained from table (cultivars Dauphine and Waltham Cross in Paarl- and Worcester-district) and wine grape (cultivars Chardonnay, Sauvignon Blanc and Merlot in Stellenbosch- and Malmesbury district) vineyards in the Western Cape province during 2001-02 and 2002-03. For four consecutive days during prebloorn, bloom, pea-size, bunch closure, veraison and harvest, sets of Petri dishes with freshly prepared Kerssies' B. cinerea selective medium (spore traps) were left overnight in the bunch zone of vines. Plant material was collected from the vines on the fourth day. Leaves, infloresence and bunches were treated with paraquat to terminate host resistance and to promote the development of the pathogen on the tissues. The B. cinerea inoculum dosage in air, and the incidence at which the pathogen was detected at various positions on leaves and in bunches normally differed between vineyards. However, the various tests revealed that the pathogen generally occurred in a consistent pattern in air in the bunch zone of vines, on leaves and in bunches from all vineyards. The inoculum dosage in air in the bunch zone of the vine was generally highest during prebioom or during bloom, it decreased at pea size and mostly remained at a very low level at the later growth stages. The estimations of viable B. cinerea residing naturally on leaves and in bunches, showed that their amounts depicted levels occurring in air in the bunch zone of the vine. Necrotic leaves occurring early season in vineyards were identified as an important source of secondary inoculum for dispersal to the developing bunches. Latent infections at the various positions in bunches were few at véraison and harvest. However, due to the necrotrophic ability of the pathogen, extensive berry rot (due to berry-to-berry contact) and thus severe bunch rot developed from a single berry that become symptomatic at the base of the pedicel/berry attachment zone. The B. cinerea occupation pattern explains why Botrytis bunch rot develops mostly from the inner bunch and why disease management strategies should concentrate on the bloom to pre-bunch closure stage and on inhibiting B. cinerea development in the inner bunch during the early part of the season. Thus, to effectively reduce B. cinerea in grapevine, preventative applications are recommended to reduce two primary infection events: (a) between budding and pre-bloom to counteract primary leaf infection; (b) during late bloom or early pea size stage, to reduce the amount of the pathogen on leaves and infloresences and to prevent colonisation of floral debris. A third spray can be applied at bunch closure to reduce the amount of B. cinerea at various positions of the inner bunch, especially for cultivars with tight bunches. The efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum was compared with those inoculated with dry, airborne conidia. Shoots were obtained during late bloom from a vineyard (wine grape cultivar Merlot) in the Stellenbosch region. The shoots were divided into two main groups. One group of shoots was left uninoculated, the other shoots were inoculated by dusting with dry B. cinerea conidia in a settling tower. Before inoculation, equal numbers of shoots in each main group was sprayed with fenhexamid, or left unsprayed. Following inoculation and incubation, shoots of each treatment were divided in two equal groups. The one lot of shoots were rinsed in water. The other lot of shoots were immersed in paraquat solution to terminate host resistance and to promote the development of the pathogen from the tissues. For both uninoculated and inoculated shoots, irrespective of fungicide treatment, leaves remained asymptomatic at both the blade and petiole position for the water rinse treatment. No symptom of B. cinerea decay developed at any of the positions on leaves from shoots sprayed with fenhexamid. Spraying of shoots with fenhexamid completely suppressed B. cinerea infection and symptom expression on both uninoculated and inoculated inflorescens. For inoculated shoots, B. cinerea developed from approximately 50% of the laterals in the water rinse treatment. However, inflorescences rinsed in water remained asymptomatic. The laboratory studies showed that fungicides, if applied properly to shoots and bunches under controlled conditions, effectively reduced the amount of B. cinerea at the various positions on leaves and inflorescence, and prevented infection and symptom expression at bloom. However, these goals are not achieved in vineyards where the fungicides are applied by conventional spraying methods. Therefore, more work is needed to evaluate fungicide application techniques by conventional spraying methods for proper fungicide coverage, and the reduction of B. cinerea in bunches.
- ItemThe effect of haloxyfop-R-methyl ester and imazamox herbicides, tine or no tillage and nine different medic cultivars on the seed and dry matter production as well as the quality of medic pastures(Stellenbosch : Stellenbosch University, 2001-12) Beyers, Hendrik Philippus; Agenbag, G. A.; Ferreira, A. V.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: The aim of this study was to determine the effect of a grass herbicide, a broadleaf herbicide with some grass control capabilities, method of tillage (tine and no-tillage) at planting of wheat as well as different medic cultivars on the regeneration, dry matter (OM) production and quality of a medic pasture. The trial was conducted at Langgewens experimental farm in the Swartland wheat producing area. Nine medic cultivars of three different species were evaluated after being sprayed with either haloxyfop-R-methyl (HAL) ester or imazamox (IMI) and subjected to either a tine tillage or a no tillage treatment at planting of wheat. Soil samples were taken during January 2000 to determine the size of the medic and weed seedbank as well as the degree of dormancy in the medic seeds, while OM samples were taken throughout the growing season to determine the OM production of the different medic cultivars and weed species. OM samples taken during October 1998 on the same pasture, were used to determine the crude protein (CP) and neutral detergent fibre (NOF) content of the pasture. The samples were subjected to in vitro digestion and the digestibility of pasture CP (OCP), NOF(ONOF) and DM (DOM)were determined. Results showed that seedling establishment differed between cultivars used, herbicide treatments applied as well as the crop stage in the rotation. The cultivars produced more seedlings where IMI was applied compared to HAL as well as where the area consisted of two year pasture compared to one year pasture (1998) and one year wheat (1999). After a year of pasture and a year of wheat, cultivars Sephi and Paraggio produced the most seedlings, while Caliph and Orion produced the least. Caliph however, showed a very high degree of seed dormancy while Orion's low seedling establishment was due to its sensitivity to the IMI herbicide used. Little difference was found between the nine cultivars early in the season (July - August) with regard to cumulative OM production, except for Orion, whose growth was severely damaged by the IMI treatment. At the end of the growing season (October), the cultivar Caliph's cumulative OM production (2010.1 kg/ha) was significantly higher than all the other cultivars, except for Parabinga (1053. 4 kg/ha). Oifferent pasture samples, of which the botanical composition was known, was analysed for CP, NOF, OOM, OCP and ONOF. There was no significant difference in pasture composition during 1998 but variation in the pasture composition did however cause the IMI treatment, compared to the HAL treatment, to have a lower ONOFand OOMcontent. A modelling procedure was used to predict the pasture quality parameters (CP, NOF, OOM,OCP and ONOF) from the pasture composition (medic hay, medic pods, grassy and broadleaf weeds). This prediction of CP, NOF, OOM, ONOF and OCP from the pasture components had a relative low accuracy (49 -74.1 %) and a further refinement of this model for possible use on farms in order to improve grazing management and animal production is advised. In conclusion it could be said that broadleaf weed control caused a definite increase in medic seed and OMproduction, but Orion should not be used with an IMI herbicide. All the cultivars, except for Orion, produced enough seedlings up to the second year to ensure sustainability of the medic pasture. All the cultivars, except for Orion, produced a sufficient amount of OM early in the growing season. Caliph however, produced by far the most OM later in the growing season. A reduction of broadleaf weeds and medic pods will increase the digestibility of NOFand OMand therefore increase the quality of the pasture. Pods however are an important part of summer forage and the aim should therefore rather be to reduce the number of broadleaf weeds in the pasture.
- ItemEffect of phenolic compounds on maize ear rot pathogens and their associate mycotoxins(Stellenbosch : Stellenbosch University, 2018-12) Cassiem, Asheeqah; Rose, Lindy J. ; Viljoen, Altus; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Maize (Zea mays L.) is one of the most important staple food crops in South Africa. It is significantly affected by Fusarium ear rot (FER) pathogens such as F. verticillioides, F. proliferatum and F. subglutinans, as well as Gibberella ear rot (GER) pathogens such as F. boothii and F. graminearum which results in poor grain quality and yield. Grain contamination with these pathogens also poses a significant food safety concern, as they are known to produce mycotoxins. Mycotoxins have been associated with a number of humans and animal diseases. Mycotoxins persist during food processing as they are heat-stable molecules and thus not fully eliminated. Therefore, the most effective way to prevent mycotoxins contamination would be to limit their production and accumulation in the field. Numerous cultural methods may reduce Fusarium inoculum, however, none of these methods are totally effective under high disease pressure and ideal circumstances for mycotoxin production. Phenolic compounds, naturally produced by plants, have been shown to reduce the growth and mycotoxin production of Fusarium spp. Pre-existing phenolic compounds can act as a physical barrier to pathogens, while induced phenolic compounds have been known to accumulate in response to biotic stress such as fungal infections. Phenolic compounds are associated with plant defence mechanisms and can be seen as a potential management strategy. In this study, the phenolic compounds vanillic, ferulic, caffeic, coumaric, chlorogenic and sinapic acid were used at different concentrations (0; 0.5; 1.5 and 2.5 mM) in vitro to evaluate the effect of phenolic compounds on FER and GER causal pathogens. A significant isolate by compound by concentration interaction was observed. The growth study illustrated significant reduction in growth of all Fusarium species evaluated. The growth of all isolates was inhibited by ferulic acid (2.5 mM). Ferulic acid combined with caffeic acid did not differ significantly from ferulic acid (2.5 mM) individually, while ferulic acid combined with chlorogenic acid had a synergistic effect when compared to the compounds individually. The biomass of the FER pathogens were reduced by caffeic acid while vanillic and coumaric acid reduced the biomass of the GER pathogens. Several phenolic compounds were able to reduce the production of mycotoxins, with chlorogenic acid significantly reducing the production of all mycotoxins evaluated. Phenolic profiles in response to infection by F. verticillioides, over kernels maturation stages, of a resistant (CML 390) and susceptible (R2565y) maize inbred line was evaluated. Ferulic and caffeic acid was found at higher concentrations in the susceptible line than in the resistant line. Moreover, higher concentratrions of ferulic, sinapic and caffeic acid was observed in the water-inoculated grain of both lines compared to the fungal-inoculated grain.. No clear differences in phenolic content were established in the fungal-inoculated grain of both lines. In addition, gene expression of phenylalanine ammonia lyase (PAL) 3 was determined by reverse transcription quantitative PCR. The susceptible line displayed a delayed response to infection as PAL3 was only upregulated at 28 days after inoculation (dai). However, the resistant line illustrated a much faster defence response as PAL3 was up-regulated from 7 dai. The information obtained in this study could be used to enhance integrated disease management strategies to prevent infection by mycotoxigenic fungi and subsequent contamination with mycotoxins.