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 dieback pathogens of apple trees to be removed and the detection of diplodia seriata on chipped apple wood(Stellenbosch : Stellenbosch University, 2022-03) Jacobs, Vernon Guy; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: The use of chipped fruit trees as mulch in apple orchards is a common practice in the deciduous fruit industry of South Africa. Apple trees destined to be removed are usually old, do not produce optimally or need to make way for new varieties. These apple trees often have visible dieback symptoms, caused by canker and wood rot pathogens. The use of mulch produced from infected trees holds a risk to young or newly established apple orchards that could get infected with the spread of diseased wood chips as mulch. However, the presence and ability of canker and wood rot pathogens to survive on mulch made from apple trees need to be determined. Therefore, the aim of this study was to assess the diversity of canker and wood rot pathogens present on apple trees destined to be removed and chipped, and to determine the presence and viability of the canker pathogen, Diplodia seriata, on chipped apple tree wood used for mulches. The diversity of canker and wood rot pathogens present in apple trees to be removed was determined. Branches or trunks showing dieback symptoms, cankers or wood rot were sampled from 14 orchards in the Grabouw, Vyeboom and Kouebokkeveld apple producing areas in the Western Cape of South Africa. Isolations were made from a diseased branch or trunk sample taken from ten trees of every orchard. Canker and wood rot pathogens were isolated from 118 of the 144 trees sampled. Known canker pathogens identified included Cytospora parasitica, Diaporthe eres, Diplodia seriata, Eutypa lata, Phaeoacremonium fraxinopennsylvanicum, Pm. minimum and Pm. viticola. Of the canker pathogens, E. lata was predominantly isolated, followed by D. seriata and species of Phaeoacremonium. Species not frequently reported were Cy. parasitica and Dia. eres. Lesser-known fungi reported included Coniochaeta ligniaria, C. velutina and Pleurostoma richardsiae. The most diverse taxonomic group identified was the wood rotting fungus Agaricomycetes. Trametes versicolor was predominantly isolated. Chondrostereum purpureum and Schizophyllum commune were reported less. Lesser-known wood rot species identified included Bjerkandera adusta, Coprinellus micaceus, Fomitiporia capensis, Fomitiporella americana, Fo. viticola, Inocutis spp. (Taxon 3), Oxyporus latemarginatus, Phanerochaeta chrysosporium, and Phlebia sp. Several fungal species were reported for the first time from apple trees: C. ligniaria, Pl. richardsiae, F. capensis and two species of Fomitiporella, Fo. americana and Fo. viticola. Diplodia seriata is an important canker pathogen of apple trees often associated with dieback symptoms of young and mature trees. The presence of D. seriata on chipped wood pieces was investigated. Chipped apple wood pieces were sampled from heaps and when spread onto the orchard floor of three orchards in the Grabouw and Vyeboom production regions. Wood chips were sampled four times from October 2020 (heaps) to April 2021 (orchard floor). Visual inspections were done on samples collected, whereas quantitative PCR (qPCR) analyses of DNA isolated from water washes of wood pieces were done on samples from the first and last sampling. Two apple wood chip heaps were generated in the winter of 2020 (F1-2020, F2-2020), and one heap from 2019 (F1-2019). The presence of D. seriata pycnidia and viable conidia was investigated on a selection of samples. Pycnidia and/ or conidia of D. seriata were present on all wood chips assessed for F1-2019, F1-2020 and F2- 2020 sampled in October 2020 and April 2021. Diplodia seriata cultures obtained from 52 of 60 wood chips were able to survive for 20-months (F1-2019). A qPCR assay was developed to detect D. seriata from mulch wood pieces. The species-specific primers developed was specific for D. seriata and the limit of detection and limit of quantification were 571 fg and 2859 fg, respectively. Diplodia seriata was detected from DNA extracted from water washes of wood chips sampled in October 2020 and April 2021 from all three orchards. From 120 samples, 84% tested positive for D. seriata. This study showed that apple trees, chipped and used for mulch, harbour important canker and wood rot pathogens. Many of these pathogens can form fruiting structures on the wood. Visual field observations during this study confirmed the presence of basidiocarps for wood rot pathogens such T. versicolor and S. commune and pycnidia of canker pathogen, D. seriata, from cankered areas on trees in orchards sampled from. Spores could easily be distributed from wood chips made from infected trees as inoculum could be present. The presence of viable D. seriata inoculum on apple tree wood chips used for mulch in younger orchards illustrates a risk of using wood chips made from old orchards. A similar risk could be expected for other canker and wood rot pathogens associated with dieback of apple trees. However, further investigation should be done to assess the risk of transmission of other known disease-causing pathogens. The level of dieback of older orchards further contributes to the risk of the mulch. Decision-making should, therefore, include assessing the health status of orchards to be removed and if it should be used as mulch. Alternatively cankered wood or dead wood can be removed before the removal of orchards and chipping. If apple tree wood chips are used as mulch other options can be explored such as composting and the application of heat treatments to ensure that mulch material does not contribute to inoculum of canker and wood rot pathogens.
- ItemAssessment of hot water treatment for control of grapevine trunk diseases in nurseries(2020-12) Webber, Matthew; Halleen, Francois; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Grapevine trunk diseases (GTDs) have been identified as a major factor contributing towards the decline of grapevines. The main GTDs in South Africa, and the pathogen species commonly associated with them, include Petri disease (Phaeomoniella chlamydospora, Phaeoacremonium minimum, Phaeoacremonium parasiticum, Cadophora luteo-olivacea and Pleurostoma richardsiae), Black foot disease (Campylocarpon fasciculare, Campylocarpon pseudofasciculare, Dactylonectria macrodidyma and Ilyonectria. liriodendri), Botryosphaeria canker and dieback (Neofusicoccum australe and Neofusicoccum parvum) and Phomopsis dieback (Diaporthe ampelina). Although GTDs are commonly associated with mature vines in established vineyards, it is of particular concern that planting material supplied by grapevine nurseries may already contain infections. Unfortunately, once infected, there are limited management strategies available to control GTD infections, with chemical and biological controls largely focusing on protection of pruning wounds. Hot water treatment (HWT) has shown to be effective in controlling a wide range of fungal pathogens from grapevines. Until recently, the HWT protocol recommended to South African nurseries was 50°C for 30 min. Although this protocol is already well studied and has shown to be effective against a wide range of GTD pathogens, it is also known that it does not completely eradicate all infections. A new HWT protocol of 50°C for 45 min has, however, been recommended to South African nurseries for the control of Aster Yellows. The effect of this HWT protocol on controlling fungal GTD pathogens has, however, yet to be determined under South African conditions. The aim of this study was, therefore, to determine the effect of HWT (50°C for 45 min) on fungal pathogens associated with GTDS found in South African grapevine nurseries, firstly in vitro, followed by in artificially inoculated rootstock cuttings of Ramsey, Richter 110, US 8- 7, Paulsen 1103 and 143B Mgt. Pathogen species evaluated in this study include Pa. chlamydospora, Pm. minimum, Pm. parasiticum, Ca. luteo-olivacea, Pl. richardsiae (Petri disease), Camp. fasciculare, Camp. pseudofasciculare, I. liriodendri, D. macrodidyma (Black foot disease), N. australe, N. parvum (Botryosphaeria canker and dieback) and D. ampelina (Phomopsis dieback). In vitro results concluded HWT (50°C for 45 min) was able to cause complete inhibition of conidial germination and mycelial growth of all pathogen species associated with Black foot disease, Botryosphaeria canker and dieback and Phomopsis dieback. Pathogens associated with Petri disease were, however, more tolerant of HWT (50°C for 45 min), with Pl. richardsiae identified as the most tolerant species within the disease complex. Of the Petri disease pathogens, Pa. chlamydospora was seen to be the most sensitive to HWT, followed by Ca. luteo-olivacea. A general trend observed for all pathogen species was that conidial germination was more sensitive to HWT than mycelial growth. Additionally, the effect of HWT using water temperatures greater than 50°C were also investigated using pathogen species able to tolerate 50°C. Pleurostoma richardsiae once again showed the highest tolerance to HWT with temperatures of up to 60°C not able to achieve complete control, suggesting HWT is not an effective means for controlling this pathogen. Results from the in vivo experiments concluded, with the exception to Pl. richardsiae, HWT (50°C for 45 min) was highly effective in reducing the presence of the inoculated pathogens, completely inhibiting the recovery of Pa. chlamydospora and Ca. luteo-olivacea from HWTed material. Although HWT did not completely remove the presence of Pm. minimum and Pm. parasiticum, the incidence and severity of which these species were able to be recovered from HWTed cuttings was significantly reduced. The effect of HWT on recovery of Pl. richardsiae was less consistent. The treatment was not able to significantly reduce the incidence of cuttings from which Pl. richardsiae was recovered from, however, was able to significantly reduce the severity of the infections, although inconsistently. Even though HWT (50°C for 45 min) may not eradicate all internal infections, it still provides nurseries a convenient and effective means of controlling a wide range of GTD pathogens in a single application. HWT of grapevine nursery material remains highly recommended and should be used in an integrated approach combined with all other available management strategies for optimal control of GTDs.
- 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
- ItemBiological control of the grapevine trunk disease pathogens : pruning wound protection(Stellenbosch : Stellenbosch University, 2008-12) Kotze, Charl; Fourie, P. H.; Van Niekerk, J. M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.In recent years, several studies have conclusively shown that numerous pathogens, including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and dieback of grapevines. These pathogens have the ability to infect grapevines through pruning wounds, which leads to a wide range of symptoms developing that includes stunted growth, cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16 weeks after pruning and sustained levels of pruning wound protection is therefore required. The aims of this study were to (i) evaluate the ability of several biological agents to protect pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of action and (iii) determine the optimal time of season for biological agent application. Several biological agents were initially evaluated in a laboratory for their antagonism against trunk disease pathogens. The best performing control agents were tested in a field trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were pruned to three buds and the fresh pruning wounds were treated with benomyl as a control treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp. (Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae), Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8 months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased products and isolates in most cases showed equal or better efficacy than benomyl, especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to colonise the wound tissue. The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were shown to be highly antagonistic toward the grapevine trunk disease pathogens, were identified by means of DNA comparison, and their ability to inhibit the mycelium growth of the trunk disease pathogens by means of volatile and non-volatile metabolite production studied. The two gene areas that were used include the internal transcribed spacers (ITS 1 and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage similarity determined and the two Trichoderma isolates were identified as Trichoderma atroviride. The volatile production of T. atroviride isolates was determined by placing an inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L. theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial inhibition than USPP-T2. The timing of pruning wound treatment and subsequent penetration and colonisation of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc cultivars were grown in a hydroponic system, pruned and spray treated with a spore suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected longitudinally. From the one half, isolations were made at various distances from the pruning surface, while the other half was observed under ultra-violet light to determine the depth of fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound, Trichoderma was isolated only from the wound surface and shallow depths into the wound (2 to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2 weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6 mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after pruning. One week after treatment, the distal nodes were removed and dissected longitudinally. Each half was observed under UV light and the pigment penetration measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper penetration of the pigment than pruning wounds treated in September. Moreover, pruning wounds made in September showed pigment particles in longitudinal sections up to 1 day after pruning, whereas wounds made in July showed pigment particles up to 3 days in the xylem vessels. These findings suggest that the best time for application of a biological control agent should be within the first 24 hours after pruning.
- ItemCharacterisation and detection of mefenoxam sensitivity in phytophthora nicotianae and phytophthora citrophthora from citrus in South Africa(Stellenbosch : Stellenbosch University, 2024-03) Moller, Heike; Rose, Lindy J. ; Van Niekerk, Jan; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: In South Africa, citrus is of high agricultural and economic importance, representing one of the country's major fruit crops. This sector plays a pivotal role in the nation's economy by substantially contributing to export earnings and employment opportunities. Citrus production is, however, threatened by oomycete pathogens, particularly Phytophthora, that can cause citrus diseases resulting in significant economic losses. Phytophthora nicotianae and P. citrophthora have been reported in every citrus-producing province in South Africa including citrus nurseries. These soil-borne pathogens primarily target the roots and the lower parts of citrus trees, causing root rot, lesions, gummosis, and brown rot of citrus fruit. Infected trees experience a decline in vigour, leading to stunted growth, wilting, and death in severe cases. These diseases also compromise the tree's ability to translocate water and nutrients, resulting in reduced fruit production and poor fruit quality. Mefenoxam is routinely used in citrus nurseries and orchards to treat Phytophthora infections. This chemical inhibits RNA polymerase I, responsible for rRNA synthesis. Its action prevents mycelial growth, sporangia formation, and germ tube growth, but due to its site-specificity, there is a high risk of resistance development. Continuous use of mefenoxam by citrus growers has led to the detection of mefenoxam-resistant Phytophthora isolates globally, including in South African nurseries and orchards. The monitoring of resistance to mefenoxam is important to ensure the lasting efficacy of this highly effective chemical and is reliant on the rapid and accurate detection of mefenoxam sensitivity. In this study, mefenoxam-insensitive and -sensitive P. nicotianae and P. citrophthora isolates were identified by in vitro fungicide sensitivity testing using Ridomil Gold 480 SL. These isolates were subjected to whole genome sequencing (WGS) using an optimised DNA isolation protocol to obtain high-quality, intact DNA from Phytophthora mycelia. A complete genome assembly of P. citrophthora was generated, for the first time, using PacBio HiFi long-read sequencing and used as the reference genome for WGS obtained by Illumina sequencing. Single nucleotide polymorphisms (SNPs) were detected in ABC transporter and cytochrome P450 genes as well as in RNA polymerase III subunits for P. nicotianae isolates and in RNA polymerase II and III subunits for P. citrophthora isolates. A quantitative polymerase chain reaction (qPCR) assay was developed to differentiate between mefenoxam-sensitive and homozygous-resistant P. citrophthora isolates. The specificity of this assay for P. citrophthora was validated against various other citrus soil-borne pathogens. The low number of insensitive isolates significantly limited the design of qPCR assays for P. nicotianae. Additionally, we evaluated a multiplex assay to detect P. citrophthora and assess mefenoxam sensitivity, simultaneously, although the amplification products could not be differentiated from each other, necessitating further optimisation. Overall, this study offers important genetic insights into mefenoxam sensitivity in Phytophthora, setting a foundation for the development of diagnostic tools to monitor fungicide resistance and manage citrus diseases caused by oomycetes more effectively.
- ItemCharacterisation and management of trunk disease-causing pathogens on table grapevines(Stellenbosch : Stellenbosch University, 2006-04) Bester, Wilma; Fourie, P. H.; Crous, P. W.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens.
- ItemCharacterisation and pathogenicity of soilborne fungi from Western Cape olive orchards(Stellenbosch : Stellenbosch University, 2022-03) Bishop, Robyn; Spies, Chris, F. J.; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Soilborne diseases are of concern to the olive industry worldwide, causing significant financial losses. The most devastating of these diseases is Verticillium wilt caused by Verticillium dahliae, followed by Phytophthora root rot. Species of Cylindrocarpon-like fungi, Fusarium, Macrophomina, Pythium and Rhizoctonia have also been associated with these diseases on olive, resulting in symptoms of dieback, defoliation, wilting, root rot, stunted growth, and tree death. In 2019, a survey targeting Cylindrocarpon-like fungi, Phytophthora and Verticillium revealed a high incidence and wide distribution of Cylindrocarpon-like fungi (mainly the Dactylonectria macrodidyma species complex) and Pythium irregulare in Western Cape olive orchards. However, Phytophthora (three species) was recovered from only 8% of samples, and no Verticillium was found. The survey also uncovered other potential pathogenic genera such as Fusarium, Diaporthe, Macrophomina and Rhizoctonia, but these were not identified to species level. Consequently, the aims of this study were to identify the most frequently recovered fungi, refine the species identities of isolates previously identified as the Da. macrodidyma species complex, and evaluate the pathogenicity of widely distributed species and species suspected to be pathogens. Unidentified isolates form the 2019 survey were divided into morphological groups. Groups containing isolates recovered from five or more of the thirteen sampling sites were selected for identification to species level using PCR, sequencing and phylogenetic analyses. Phylogenetic analyses revealed four species of Diaporthe, fourteen species of Fusarium, one species of Macrophomina, eight species of Neocosmospora, and nine anastomosis groups (AGs) of Rhizoctonia. Morphological descriptions of two new Diaporthe species were compiled. Isolates previously identified as the Da. macrodidyma species complex were delineated into Da. macrodidyma, Da. novozelandica, Da. sp., Da. torresensis, and Da. pauciseptata. The most widely distributed species included Da. sp., Da. macrodidyma, Diaporthe sp RB-2019a., F. fabacearum, F. nirenbergiae, M. phaseolina, and Rhizoctonia AG-G. Species of Fusarium, Macrophomina, Neocosmospora, Phytophthora, Pythium and Rhizoctonia have been reported as olive pathogens in other countries, however, none of these reports identified phylogenetic species within Fusarium and Neocosmospora, and none reported AGs of Rhizoctonia. Species identified in this study that have been reported on olives previously include Da. ecuadoriensis, Da. macrodidyma, Da novozelandica, Da. sp., Da. torresensis, Da. valentina, Di. ambigua, F. oxysporum, M. phaseolina, N. solani, Pleurostoma richardsiae, and Rhizoctonia spp. In addition to the two new Diaporthe species, Cadophora constrictospora, Da. pauciseptata, F. clavus, F. udum, nine species from the Fusarium oxysporum species complex, seven species of Neocosmospora, as well as nine different AGs of Rhizoctonia are reported on olive for the first time worldwide. The pathogenicity of selected species was evaluated on three-month-old ‘Mission’ cuttings under wet, moderate and dry irrigation regimes in glasshouse trials. Twenty–nine isolates representing Da. macrodidyma, Da. novozelandica, Da. sp., Da. pauciseptata, Da. torresensis, Di. ambigua, Diaporthe. sp., F. fabacearum, F. nirenbergiae, M. phaseolina, N. solani, Ph. multivora, Ph. pseudocryptogea, Py. irregulare, Py. oligandrum, Rhizoctonia AG- G, and V. dahliae were included. Species of Da. macrodidyma, F. nirenbergiae, Ph. Multivora, Ph. pseudocryptogea, Rhizoctonia AG-G, and V. dahliae were found to be pathogenic towards olive trees. The different watering regimes had no consistent effect on pathogenicity. This study was the first to evaluate the pathogenicity of soilborne fungi on olive trees in South Africa and to report the pathogenicity of three species for the first time on olive, including F. nirenbergiae, Ph. pseudocryptogea, and Rhizoctonia AG-G. Future research should aim to address the management of these diseases on olive.
- 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 Rhizoctonia in cropping systems in the Western Cape Province(Stellenbosch : University of Stellenbosch, 2005-03) Tewoldemedhin, Yared Tesfai; Lamprecht, Sandra, C. ; Mazzola, M.; McLeod, Adele; University of Stellenbosch. Faculty of Agrisciences. Dept. of Plant Pathology.The genus Rhizoctonia includes important soilborne plant pathogens that can cause severe economic losses on a wide range of crops including cereal, canola, pasture and grain legume crops. Limited information is available on specific anastomosis groups (AGs), nuclear status, pathogenicity and cross-pathogenicity of Rhizoctonia isolates associated with each of these crops in the Western Cape province. Therefore, these aspects were investigated in this study, since information in this regard is crucial for the development of Rhizoctonia disease management practices.
- 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.
- ItemThe characterization of the basidiomycetes and other fungi associated with esca of grapevines in South Africa(Stellenbosch : University of Stellenbosch, 2010-12) White, Chana-Lee; Mostert, Lizel; Halleen, Francois; University of Stellenbosch. Faculty of Agrisciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: Esca is a disease affecting grapevines and is potentially devastating as there are economic losses due to a decrease in yield, wine quality and berry quality. Vineyards also need to be replaced earlier and therefore esca has a great impact on the wine, table grape and raisin industries. The disease is known to affect vineyards worldwide and has been studied extensively in Europe, but not in South Africa. Esca diseased grapevines were observed for the first time prior to 1981 in South African vineyards. The disease is primarily caused by Phaeoacremonium aleophilum, Phaeomoniella chlamydospora (both causing brown and black wood streaking) and white rot basidiomycete species such as Fomitiporia mediterranea which cause wood rot in the trunks and arms of generally older grapevines. Species of the Botryosphaeriaceae and Phomopsis (mainly Phomopsis viticola) and Eutypa lata have also been isolated from esca diseased vines, but their association with esca is unclear. Some of the symptoms associated with the disease on most grapevine cultivars include ‘tiger-stripe’ foliar symptoms, apoplexy and berry symptoms such as shriveling, discoloration and ‘black measles’. These external symptoms as well as internal symptoms are thought to be a result of toxin and enzyme production by the fungi involved. Symptom expression is erratic and varies from year to year making investigations into the causal fungi and the toxins and enzymes secreted in planta difficult. Vines with internal or external symptoms of esca were sampled in this study from table and wine grape cultivars in 37 towns in the Western Cape, Northern Cape and Limpopo provinces. The majority of sampled vines were over ten years of age, but vines as young as two to three years were also found to be infected. The external symptoms included dieback, tiger striped leaves, berry symptoms (shriveling, insufficient colouring and black spots) and apoplexy. These symptoms resembled those found on grapevines in Europe, Australia and the USA. The internal symptoms found were also similar to European symptoms and included white rot, black and brown wood streaking, brown necrosis within white rot, sectorial brown necrosis and central brown/ red/ black margin. The fungi mostly isolated from the white rot were the basidiomycetes. Black and brown wood streaking was primarily caused by Phaeomoniella chlamydospora. Brown necrosis within the white rot was caused by Phaeomoniella chlamydospora and less frequently by Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae and Pleurostomophora richardsiae. The sectorial brown necrosis and the central/ brown/ red/ black margin were dominated by Phaeomoniella chlamydospora. The fruiting bodies of the basidiomycetes were found on only a few grapevines. The fungal species associated with the internal wood symptoms were characterized on cultural growth patterns, morphology as well as phylogenetic inference. The gene areas sequenced included the internal transcribed spacers and the 5.8S rRNA gene for the basidiomycetes and Phomopsis isolates, the partial b-tubulin gene for Phaeoacremonium isolates and the partial translation elongation-1a gene for the Botryosphaeriaceae isolates. The basidiomycete isolates fell into ten taxa within the Hymenochaetales of which two could be linked to known genera, namely Fomitiporia and Phellinus. The ten basidiomycete taxa do not correspond to any published sequences. Eutypa lata, Diaporthe ambigua, Diplodia seriata, Neofusicoccum australe, Neofusicoccum parvum, Phomopsis viticola, Phomopsis sp. 1, Phaeomoniella chlamydospora and six species of Phaeoacremonium including P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae and P. sicilianum were also isolated of which the latter three are reported for the first time in South Africa. To understand the role of the basidiomycetes in the complex, toxin and enzyme analyses was determined for these fungi. Selected basidiomycete isolates were grown up in liquid broth and extractions performed to test for the presence of 4-hydroxy-benzaldehyde. All of the basidiomycete isolates were able to produce this toxin which is known to be phytotoxic. The basidiomycetes were then tested for the presence of certain wood degrading enzymes. All of the taxa were able to produce manganese peroxidase. Laccase was produced by all taxa, except Taxon 8. Lignin peroxidase was produced by Taxa 1, 2, 7, Fomitiporia sp. and the Phellinus sp. All the basidiomycete isolates were able to produce cellulose and none were able to produce xylanase. These enzyme tests showed that the basidiomycetes produce a wide variety of enzymes which are able to degrade cellulase and lignin which are both structural components of wood. Given the wide distribution of esca in the grape growing regions investigated in South Africa and the diverse amount of species found, this disease must surely be seen as a limiting factor to the productive lifespan of vineyards and quality of produce. Preventative measures such as sanitation and pruning wound protection contribute to the management of the disease, but many questions still remain about the synergy of the causal fungi, epidemiology and management of esca.
- 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.