Department of Plant Pathology

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A cost-effective protocol for molecular detection of fungal pathogens in soil
(Academy of Science for South Africa, 2005) Damm, Ulrike; Fourie, Paul
DNA EXTRACTION FROM FUNGI IN SOIL often fails because of humic substances that are co-extracted with the DNA and subsequently inhibit PCR analyses. Moreover, it is difficult to release the fungal DNA because of the diverse fungal structures residing in soil. Since available DNA extraction protocols and commercial kits are expensive or time-consuming, we have devised a superior method by testing different components of these procedures on grapevine nursery soils. The best DNA yield and sensitivity were obtained by a short and easy extraction method with sodium dodecyl sulphate buffer using the FastPrep homogenizer. An easy-to-prepare spin column with polyvinylpoly-pyrrolidone was developed to remove PCR inhibitors. In the presence of bovine serum albumin, PCR reactions were possible without further dilutions of the DNA. Our method was more sensitive for detecting Phaeomoniella chlamydospora, the organism responsible for Petri grapevine decline, and Cylindrocarpon black-foot pathogens in grapevine nursery soils than the FastDNA SPIN Kit for Soil and enabled us to perform 25 extractions for the price of one with the kit. This is the first report of molecular detection of Cylindrocarpon macrodidymum from soil and the first account of Pa. chlamydospora from soils in South Africa.
Agricultural practices and their potential role in mycotoxin contamination of maize and groundnut subsistence farming
(Academy of Science of South Africa, 2019-09-26) Phokane, Sylvia; Flett, Bradley C.; Ncube, Edson; Rheeder, John P.; Rose, Lindy J.
Mycotoxigenic fungi are common pathogens of maize and groundnuts; they produce mycotoxins which reduce the yield and quality of these grain crops. Numerous agricultural practices including crop rotation and storage methods have been shown to impact mycotoxin accumulation. Therefore, the farming and storage practices in maize and groundnut subsistence farming systems in Pongola, Vryheid, Jozini, Manguzi and Mbazwana Districts of northern KwaZulu-Natal (South Africa) were surveyed to determine their potential role in promoting or mitigating mycotoxin contamination. A questionnaire about agricultural farming practices and storage facilities was presented to 65 subsistence maize and/or groundnut farmers. At least 90% of the farmers surveyed were not aware of mycotoxins and their consequences to animal and human health. The majority of the farmers did not practise crop rotation. However, they practised intercropping and sorted damaged and mouldy grain (maize and groundnuts) before storage. The damaged or mouldy grain was largely used as animal feed, thereby exposing animals to an increased risk of mycotoxicoses. Metal tanks and inqolobane (a type of wooden structure) were identified as the most common storage structures. Harvested homegrown maize was mostly used for the farmers’ own consumption but also sometimes sold to the local community. The implementation of mycotoxin awareness campaigns is necessary, particularly in these districts. The storage facilities used by the subsistence farmers allowed increased moisture and insect invasion. The need for the surveillance of mycotoxins in subsistence-farmed food crops is vital.
Apple 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.
Application of fungicides against postharvest botrytis bunch rot of table grapes in the Western Cape
(South African Society for Enology and Viticulture, 1994) De Kock, P. J.; Holz, G.
Fungicide programmes for the control of postharvest Botrytis bunch rot on table grapes were evaluated in six trials from 1984/85 to 1991/92 in the Western Cape. The study demonstrated the ineffectiveness of dicarboximide applications during bloom to early pea size in well managed vineyards. Dicarboximides were most effective when applied from bunch closure to ripening. lprodioue/sulphur treatments at veraison and before harvest reduced Botrytis bunch rot, but they were ineffective in inhibiting infection during storage. Control was only achieved when grapes were exposed to S02 during storage. Although bunch dip treatments reduced infection in the vineyard, this control was not commercially acceptable. Therefore no real advantage was found when bunches were dipped in fungicide at veraison to ensure better coverage. The fact that berries became infected primarily during harvest, package operations and storage, emphasised the necessity for reducing B. cinerea inoculum on harvested grapes. It is suggested that the results of this investigation may lay the foundation for incorporating biological control in Botrytis bunch rot control.
Assessing genotype-by-environment interactions in aspergillus ear rot and pre-harvest aflatoxin accumulation in maize inbred lines
(MDPI, 2017) Okoth, Sheila; Rose, Lindy J.; Ouko, Abigael; Netshifhefhe, Nakisani E. I.; Sila, Henry; Viljoen, Altus
Aspergillus flavus, causal agent of the Aspergillus ear rot (AER) of maize, also produces aflatoxins that cause aflatoxicosis in humans and livestock. Ten maize inbred lines were evaluated in replicated trials in two aflatoxicosis outbreak hot spots in Kenya and in three maize-growing areas in South Africa for resistance to AER, A. flavus colonization, and pre-harvest aflatoxin accumulation during the 2012/13 growing season. AER severity was measured by visual assessment, while A. flavus colonization and aflatoxin content were quantified by real-time polymerase chain reaction (PCR) and liquid chromatography tandem mass spectrometry, respectively. Genotype by environment interaction (GEI) was determined using analysis of variance (ANOVA), additive main effects and multiplicative models (AMMI), and genotype plus by environment (GGE) biplot analyses. Stability of genotypes was evaluated using AMMI analysis. AER severity and fungal colonization significantly (p < 0.001) varied between genotypes. GEI influenced the severity of AER symptoms and aflatoxin accumulation significantly (p < 0.001), while fungal colonization was not affected. The inbred lines response was consistent for this trait in the test environments and was thus considered a desirable measure to indicate maize lines with a high risk of aflatoxin accumulation. CML495, CKL05019, LaPosta, and MIRTC5 were the least diseased lines, with the lowest aflatoxin contamination and a stable phenotypic response across the environments. Kiboko was determined as the ideal representative test environment, with discriminative ability of the genotypes for selection of the desired stable responses of the three traits.
Assessing 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.
Assessment 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.
Bars 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
Beheer 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.
Biochemiese 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
Biological 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.
The biology of Endophyllum osteospermi, and its use for the biological control of Chrysanthemoides monilifera ssp. monilifera
(Stellenbosch : Stellenbosch University, 2004-12) Wood, A. R. (Alan Robert); Crous, P. W.; Lennox, C. L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Chrysanthemoides monilifera ssp. monilifera is a shrub indigenous to South Africa, which has become a serious weed of native vegetation in Australia. Endophyllum osteospermi is a microcyclic, autoecious, rust fungus that induces witches' brooms on C. monilifera ssp. monilifera. This rust is considered as a candidate biocontrol agent for use against C. monilifera ssp. monilifera in Australia. The vegetative growth and reproductive output of healthy branches on bushes with different levels of E. osteospermi infections were measured at three sites. The growth of healthy branches on infected bushes was 26- 81% less than that of healthy branches on uninfected bushes. The number of buds, flowering capitulae, fruiting capitulae, and cypselas on healthy branches of infected bushes was 35-75%, 45-90%, 15-99%, and 15-90% less, respectively, than those on uninfected bushes. At five sites, the infection levels and number of witches' brooms were determined every two months. The increase in number of witches' brooms per bush ranged between o and 282 within one year, with an average increase per bush of28 (SE ± 4.8) and 39 (SE ± 9.2) during two years. The average simple interest rate (rs) increase of infection levels for all bushes was 0.015 month-I (s.e. ± 0.0041, n = 72) and 0.0098 month" (s.e. ± 0.0073, n = 43) during two years. Aecidioid teliospores germinated between 10 and 20oe, with 15°e as optimum. Light, and particularly near-uv light, stimulated germination. A period of 6 to 8 hours of light was needed to obtain optimum germination levels. The temperature requirements for basidiospore development differed from that of aecidioid teliospore germination. Optimum was at 15°e, but a rapid decrease in basidiospore production occurred at higher temperatures, few developed at 19°e. Two nuclear divisions occurred within 12 hours of germination to produce a metabasidium with three or four nuclei. A third nuclear division occurred in the basidiospores between 24 and 48 hours. Plants inoculated under controlled conditions took 5 to 24 months before witches' brooms began to develop. A Geographic Information System (GIS) approach was used to model the potential distribution of E. osteospermi in South Africa, based on monthly average climate surfaces with parameters derived from the above experiments. The same model was applied to Australia to suggest a potential distribution of the rust if released in Australia. This potential distribution was similar to one generated using the climate matching computer programme CLIMEX©, but gave greater spatial accuracy. Both approaches indicate that E. osteospermi should establish in temperate Australia. Chrysanthemoides species, as well as other South African asteraceaus plants, were monitored for E. osteospermi between 1992 and 2003. Endophyllum osteospermi was recorded on C. monilifera ssp. monilifera, C. monilifera ssp. pisifera, C. monilifera ssp. rotundata, C. monilifera ssp. canescens, C. monilifera ssp. subcanescens, C. incana, an undescribed taxon of Chrysanthemoides, Osteospermum ciliatum, 0. polygaloides and 0. potbergense. Endophyllum dimorphothecae sp. nov. is described on Dimorphotheca cuneata. Aecidium elytropappi, which was recorded on Elytropappus rhinocerostis and Stoebe plumose, is transferred to Endophyllum as E. elytropappi comb. nov. Germination of aecidioid teliospores and penetration by basidiospores were observed on the surface of excised leaves of 32 plant species at 4 days after inoculation. Germinating aecidioid teliospores aborted on 14 plant species, whilst no penetration was attempted on a further 12. Penetration only occurred on 9. Therefore only these 9 plant species need to undergo traditional host specificity testing. Pending these results, E. osteospermi could be safely released in Australia for the biological control of C. monilifera ssp. monilifera.
Botryosphaeria diseases of proteaceae
(Stellenbosch : Stellenbosch University, 2002-03) Denman, Sandra; Crous, P. W.; Wingfield, M. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Fungi belonging to the genus Botryosphaeria are heterotrophic micromycetes that can be pathogens on woody plants. They cause serious, and in some cases devastating losses to crops through leaf necrosis, stem cankers and plant death. The Proteaceae cut-flower industry in South Africa accounts for 70% of the national cut-flower enterprise. Botryosphaeria diseases are a major impediment to production and trade of Proteaceae and there is an urgent need to investigate the etiology, epidemiology and control of these diseases. Losses of one of the most important proteas, P. magnifica, amount to 50% or more, locally. The main aims of this study were therefore to establish the etiology and aspects of epidemiology of Botryosphaeria stem cankers on P. magnifica and other Proteaceae, and to investigate methods of disease control. Although there is a vast body of information pertaining to this fungus, which was reviewed in Chapter 1, there is relatively little information available on Botryosphaeria on Proteaceae. The taxonomy of Botryosphaeria requires thorough review, and molecular techniques need to be employed to resolve species identities. In Chapter 2, it was found that Phyllachora proteae, a leaf pathogen of proteas, produced a Fusicoccum anamorph, which is described as F. proteae. A sphaeropsis-like synanamorph was associated with F. proteae and a new combination for P. proteae is proposed in Botryosphaeria, as B. proteae. The taxonomy of Botryosphaeria is in disarray at both the generic and the specific level. In Chapter 3 the taxonomic history of Botryosphaeria is reviewed, and the genus circumscribed and distinguished from other morphologically similar genera. Although several anamorph genera have been linked to Botryosphaeria, based on morphological observations and phylogenetic analysis of lTS rDNA sequence data, two anamorph genera are now recognised, those with pigmented conidia (Diplodia), and those with hyaline conidia (Fusicoccum). Botryosphaeria proteae should thus be excluded from Botryosphaeria. Several pathogenic Botryosphaeria spp. have an endophytic phase within their hosts. They are therefore imported unwittingly into other countries where they may pose a risk to agriculture and indigenous vegetation. The current global distribution of Botryosphaeria spp. associated with Proteaceae is clarified and a key to these taxa associated with Proteaceae is provided in Chapter 4. Five Botryosphaeria spp. are associated with cut-flower Proteaceae worldwide viz. B. lute a, B. obtusa, B. protearum, B. proteae and B. rib is. B. protearum is described as a new species. A thorough understanding of disease epidemiology is essential to effect a reduction of losses. In Chapter 5, I show that on P. magnifica, lesions caused by Botryosphaeria protearum, which lead to the formation of stem cankers, are initiated in the mid-rib vein or margin of leaves. Koch's postulates were satisfied and it was found that the number of lesions that developed from artificial inoculations correlated with starch levels present in leaves at the time of inoculation. In Chapter 6 it is shown that B. protearum exists as an endophyte in leaves of P. magnifica in naturally occurring as well as cultivated plants. In natural stands of proteas stem cankers are rare, but in cultivated plantations the incidence is high. Nutritional analyses indicate that higher levels of nitrogen occur in leaves of cultivated plants in spring, which could enhance disease development. High levels of sodium in the leaves of wild plants may restrict disease development. The severe economic losses caused by B. protearum make the search for improved methods of disease control essential. Fungicide applications form an important component of an integrated approach to disease management. In Chapter 7, in vitro tests demonstrate that tebuconazole, benomyl, prochloraz me, iprodione and fenarimol reduce the mycelial growth of B. protearum effectively. In the field there was a 25-85% reduction in the occurrence of stem cankers by applying fungicides or sanitation pruning. The best control was achieved by using benomyl, bitertanol, fenarimol, iprodione, prochloraz manganese chloride alternated with mancozeb and tebuconazole prophylactically. If sanitation pruning is combined with regular applications of fungicides, disease can be combated.
Characterisation 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.
Characterisation 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.
The characterisation of basidiomycetes associated with esca disease in South African grapevines
(Stellenbosch : Stellenbosch University, 2015-03) Cloete, Mia; Mostert, Lizel; Halleen, Francois; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Esca is a disease complex of grapevine that includes different foliar and vascular symptoms caused by various fungal pathogens. One of the distinguishing characteristics of the disease on mature vines is the white rot of the wood. Esca-related wood rot is caused by several lignicolous basidiomycetes from the order Hymenochaetales. The Hymenochaetales fungi associated with esca vary depending on geographic location. For example, in Europe and the Mediterranean grape-growing regions, Fomitiporia mediterranea is the prevalent species; in Argentina, Inocutis jamaicensis; in Chile “Fomitiporella vitis”, and in Australia Fomitiporia australiensis. In the United States, Fomitiporia polymorpha has been associated with esca, though not consistently. A previous study identified ten different taxa belonging to the genera Fomitiporella, Fomitiporia, Inocutis, Inonotus, and Phellinus associated with esca in South Africa. The current study was tasked with characterising these taxa and assessing their epidemiology and pathogenicity. The study has characterised three novel species, Fomitiporella viticola, Fomitiporia capensis and Phellinus resupinatus from Vitis vinifera and a first report of Inonotus setuloso-croceus occurring on Vitis vinifera and Salix spp. worldwide and in South Africa. The sporulation of F. viticola was surveyed over two seasons. The pathogenicity of all ten taxa was tested on mature field grown vines and enzymes secreted by all ten taxa were assayed. This study aimed to add in the understanding of the esca complex disease in South Africa and contributed towards the wider knowledge regarding the ecology of the Hymenochaetales. A novel Fomitiporia species, F. capensis, was described based on fruit body morphology and combined internal transcribed spacer rRNA ITS1-5.8S-ITS-2 (ITS) and large sub-unit (LSU) phylogeny, where it formed a clearly delineated and well-supported clade. Morphologically, F. capensis was similar to F. punctata in that both species essentially lack setae. Fomitiporia capensis, F. punctata and F. aethiopica produced similarly sized basidiospores, but differed in terms of host range, pore size and, possibly, fruiting body shape. Phylogenetically, F. capensis appeared to be related to F. tenuis, though morphologically the species differed significantly in that F. tenuis had smaller pores and smaller basidiospores. During all surveys conducted, Fomitiporia capensis was found to occur widely as throughout the Western Cape Province, though fruit bodies were scarce in comparison to mycelium isolated from symptomatic vines. Fruit bodies were also found in a vineyard in the Limpopo region in the north east part of the country. Phellinus resupinatus was described based on fruit body morphology, ITS and LSU phylogenies. It formed a well-supported clade closely related to Phellinus bicuspidatus, a species associated with white rot in oak trees in the United States. Morphologically, P. resupinatus was characterised by its resupinate fruit body shape, straight, ventricose hymenial setae, and broadly ellipsoid hyaline basidiospores. It was only found on diseased grapevines in the summer rainfall regions of South Africa, mainly in the Northern Cape and Limpopo provinces. Fomitiporella viticola was described from Vitis vinifera based on fruit body morphology and ITS phylogeny. It is characterised by a resupinate to effuse-reflexed fruit body with large, loosely spaced pores and fairly small yellowish-brown basidiospores. Inonotus setuloso–croceus was found occurring on Salix and Vitis vinifera and was identified based on fruit body morphology. The ITS region was sequenced from DNA isolated from cultures obtained from rotten wood or fruit bodies, and was matched to the Hymenochaetales species from Vitis previously classified as Taxon 7. The discovery of Inonotus setuloso-croceus on Salix validated the hypothesis that fruit bodies may occur on alternative hosts. Fomitiporella viticola was often isolated from white rot on vines affected by esca and fruit bodies were often found on vines in the Western Cape Province. Twelve fruit bodies of F. viticola were monitored for sporulation weekly over two seasons lasting between winter and early summer. Levels of sporulation had a weak positive correlation with rainfall and a weak negative correlation with average temperature. Sporulation was found to occur throughout the entire monitoring period. Little is known about the pathogenicity and aetiology of the Hymenochaetales taxa associated with esca in South Africa. All ten taxa were subjected to enzyme assays to determine which ligninolytic enzymes were secreted by each taxon. In addition, a field trial was undertaken to determine the pathogenicity of ten South African Hymenochaetales taxa associated with esca in grapevine. Twenty-seven fungal isolates and two negative controls were inoculated into mature grapevines and incubated for 24 months. The results of the enzyme assays indicated a difference in enzyme secretion between taxa and also among isolates of the same taxa. All isolates secreted cellulase and laccase, but there was a difference between isolates‟ ability to secrete manganese peroxidase and lignin peroxidase. The results of the pathogenicity trial showed that all of the isolates used were capable of causing the characteristic white rot symptom in the wood. There were also clear differences in susceptibility to white rot between the two cultivars tested. Cultivars also differed in which taxa proved pathogenic. On Shiraz, Taxon 6 (an Inonotus sp.), Phellinus resupinatus and Inonotus setuloso-croceus were significantly virulent. On Mourvédre, however, Taxon 3, an Inocutis sp. and Taxon 2, a Fomitiporella sp. were significantly virulent. Cultivar differences could be due to various factors, including differences in host response to colonisation and physical differences in wood structure, as well as the differences in enzyme secretion between taxa.
Characterisation of Cylindrocarpon spp. associated with black foot disease of grapevine
(Stellenbosch : Stellenbosch University, 2005-12) Halleen, Francois; Crous, Pedro W.; Fourie, Paul H.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: During the past few years a drastic reduction has been noted in the survival rate of grafted grapevines in nurseries, as well as in young vineyards in the Western Cape Province of South Africa. Circumstantial evidence suggested that Cylindrocarpon spp., which cause black foot disease of grapevine, were associated with this decline. Black foot disease of grapevine is a relatively new, and as yet poorly known disease affecting vines in various countries where grapevines are cultivated. Primary aims of this research have been (1) to conduct nursery surveys in order to determine which fungi are involved in the decline phenomenon, with special reference to the involvement of Cylindrocarpon spp., (2) to identify and characterise the organisms believed to be the causal organisms of black foot disease, and (3) the development of control and/or management strategies to prevent or eradicate Cylindrocarpon infections. Nursery grapevines were sampled at different stages from three commercial nurseries in the Wellington area of the Western Cape Province and were investigated during the 19992000 season by means of destructive sampling. The first samples were taken in September from callused cuttings prior to planting in nurseries. After planting, asymptomatic rooted cuttings were selected from nurseries after 3, 6 and 9 months. Isolation studies clearly demonstrated that different “Cylindrocarpon spp.” infected cuttings from nursery soils. These species rarely occurred in rootstock propagation material prior to planting. At the time of planting, the susceptible basal ends (especially the pith area) of most of the nursery cuttings are partly or even fully exposed. Callus roots also break during the planting process, resulting in small wounds susceptible to infection by soilborne pathogens. The isolation studies revealed that the first infections occurred in the roots, followed by infections of the rootstocks. These infections increased progressively during the course of the growing season. Substantial variation in cultural and morphological characters was observed among the Cylindrocarpon isolates obtained from the nursery survey, as well as from isolations that were made from diseased grapevines. Morphological and phylogenetic studies were conducted to identify these “Cylindrocarpon spp.” and to establish their association with black foot disease. Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial β-tubulin gene introns and exons were used for phylogenetic inference. Phylogenetic analyses confirmed the diversity observed among the isolates and four Cylindrocarpon-like species were identified. One of these species was initially identified as Cylindrocarpon destructans. However, further research revealed C. destructans to represent a species complex. Grapevine isolates of “C. destructans” proved to be identical to the ex-type strain of Cylindrocarpon liriodendri, which also produced a teleomorph, Neonectria liriodendri in culture. A second species was newly described in this study as Cylindrocarpon macrodidymum (Neonectria macrodidyma). The two remaining Cylindrocarpon-like species were placed in a new genus, Campylocarpon. The two species were named Campylocarpon fasciculare and Campylocarpon pseudofasciculare. Pathogenicity studies confirmed that all four species were able to reduce root and shoot mass significantly. Knowledge obtained pertaining to the disease cycle of black foot disease suggest that suitable management strategies should focus on prevention of primary infection in nurseries. However, at present, no fungicides are registered for control of this disease in South African vineyards or nurseries. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens. Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effective fungicides tested, and were subsequently included in semi-commercial field trials. Basal ends of grafted cuttings were dipped (1 min) in various chemical and biological treatments prior to planting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttings prior to planting in nurseries. Additional treatments involved soil amendments with Trichoderma formulations and hot water treatment (50°C for 30 min) of dormant nursery grapevines. Field trials were evaluated after a growing season of eight months. The incidence of black foot pathogens was not significantly and/or consistently reduced by the majority of chemical or biological treatments. However, these pathogens were not isolated from uprooted plants that were subjected to hot water treatment. It is therefore recommended that hot water treatment of dormant nursery plants be included in an integrated strategy for the proactive management of black foot disease in grapevine nurseries.
Characterisation 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.
Characterisation 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.
Characterisation 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.

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