Masters Degrees (Plant Pathology)

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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.
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.
Characterization 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.
The 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.
Colletotrichum 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.
Comparative 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.
Epidemiology and control of Pseudocercospora angolensis fruit and leaf spot disease on citrus in Zimbabwe
(Stellenbosch : Stellenbosch University, 2005-12) Pretorius, Mathys Cornelius; Holz, G.; Crous, P. W.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Fruit and Leaf Spot Disease (FLSD) of citrus, caused by Phaeoramularia angolensis, is found only in 18 countries in Africa, the Comores Islands in the Indian Ocean and Yemen in the Arabian peninsula. The major citrus export countries in Africa are Morocco, South Africa, Swaziland, and Zimbabwe. Zimbabwe is the only country affected by FLSD. FLSD is a disease of major phytosanitary and economic importance and its devastating effect on citrus is highlighted by the fact that the damage is cosmetic, which renders the fruit unmarketable. Total crop losses are not uncommon in Kenya. The aims of the present study, therefore, was was to determine the occurrence of P. angolensis in Zimbabwe and neighbouring Mozambique, to compare these isolates with the Cercospora Fresen. isolates from Swaziland and South Africa, to determine the epidemiology of the pathogen and to implement an effective control strategy to prevent the spread of FLSD. Leaf samples with citrus canker-like lesions collected in the early 1990’s in Zimbabwe were found to be infected by the fungus, Phaeoramularia angolensis. Surveys were undertaken to determine the spread and intensity of FLSD in Zimbabwe and Mozambique. In Zimbabwe, P. angolensis was limited to an area above the 19° south latitude, predominantly the moist areas and not the low-lying drier parts of the country. In Mozambique, no P. angolensis symptoms were found. Observations during the survey indicated that no proper management systems were implemented by Zimbabwean growers. A cercosporoid fungus causing a new Fruit and Leaf Spot Disease on Citrus in South Africa was identified. From morphological and rDNA sequence data (ITS 1, 5.8S and ITS 2), it was concluded that the new disease was caused by Cercospora penzigii, belonging to the Cercospora apii species complex. The genera Pseudophaeoramularia and Phaeoramularia are regarded as synonyms of Pseudocercospora, and subsequently a new combination was proposed in Pseudocercospora as P. angolensis. Cercospora gigantea was shown to not represent a species of Cercospora, while Mycosphaerella citri was found to be morphologically variable, suggesting that it could represent more than one taxon. A control strategy for the control of FLSD was evaluated in the study. The data showed that P. angolensis in Zimbabwe can be managed successfully by the removal of all old and neglected orchards, and on timely fungicide applications. Trifloxystrobin + mancozeb + mineral spray oil (20 g + 200 g + 500 ml/100 l water) applied in November, January and March was the most effective treatment. Three applications of benomyl + mancozeb + mineral spray oil (25 g + 200 g + 500 ml/100 l water) applied during the same period, was the second most effective treatment, and two applications (November and January) of trifloxystrobin + mineral spray oil (20g + 500 ml/100 l water) and difenoconazole (40 g) per 100 l/water applied twice in November and January, the third most effective treatment. The spore trap and weather data showed that P. angolensis needs high moisture and temperatures in excess of 25°C for disease development. It is concluded that P. angolensis in Zimbabwe can be managed successfully by implementing a holistic approach, which should be supported by the authorities, organised agriculture and all technical personnel involved in citrus production.
An investigation of soilborne fungi associated with roots and crowns of nursery grapevines
(Stellenbosch : Stellenbosch University, 2004-03) Van Coller, Gerhardus J. (Gerhardus Johannes); Denman, S.; Lamprecht, Sandra, C. ; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Soilborne diseases of grapevines represent a complex problem with limited information available, both locally and internationally. Previous research in South Africa indicated that Phytophthora and Pythium spp. were the most widespread and devastating pathogens in grapevine nurseries and vineyards in the Western Cape province. The local grapevine industry is currently expanding; new cultivars, methods and agricultural chemicals are being used which can affect soilborne pathogens. It has therefore become necessary to reassess the status of soilborne pathogens in nurseries, since information in this regard is crucial for the development of disease management practices for the expanding local grapevine industry. Soilborne fungal genera associated with roots and crowns of declining nursery grapevines were assessed in surveys conducted at three different grapevine nurseries in the Western Cape province. Cylindrocarpon, Fusarium, Pythium, and Rhizoctonia spp. were consistently isolated from roots and crowns of declining nursery grapevines. Cylindrocladiella spp. and Phytophthora cinnamomi were infrequently isolated from diseased roots, crowns and soil whereas Pythium spp. were abundant in most of the soils. Results suggest that the status of soilborne fungal pathogens in grapevine nurseries in the Western Cape province has changed over the last 30 years. The DNA phylogeny and pathogenicity of the isolates of Cylindrocladiella were determined. Four species of Cylindrocladiella occur on grapevines in South Africa, namely C. lageniformis, C. parva, C. peruviana, as well as a new species, described in this study as C. viticola, which forms part of the C. infestans species complex. Pathogenicity trials were inconclusive. Ten Fusarium spp. were isolated from roots and crowns of declining nursery grapevines, namely F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum and F. solani. The dominant species was F. oxysporum, followed by F. proliferatum and F. solani. In pathogenicity trials F. oxysporum and F. solani significantly reduced root volume, root dry mass, length of new shoots, stem diameter and number of leaves, but increased the percentage of chlorotic leaves and root rot severity. Fusarium proliferatum also caused a significant reduction in new shoot growth, number of leaves and increased root rot severity compared to the controls. Fusarium so/ani seems to be more virulent than F. oxysporum, followed by F. pro/iferatum. This is the first report of F. oxysporum, F. pro/iferatum and F. so/ani as pathogens of grapevines in South Africa, and the first report of F. proliferatum as a pathogen of grapevines in the world. Phytophthora cinnamomi was isolated at low frequencies from declined grapevines, although present in the rhizosphere soil. It is possible that the extensive use of downy mildew chemicals in grapevine nurseries may protect grapevines from infection by P. cinnamomi. The effect of chemicals used to combat downy mildew on Phytophthora root rot of nursery grapevines was evaluated in a glasshouse. There was very little discernable effect of the chemicals tested relative to the control plants for the parameters measured and it was concluded that the inoculation technique needed refinement. However, plants treated with phosphorous acid tended to be taller and have more leaves, greater stem diameter and root volume than controls or plants treated with the other chemicals. The data obtained in this study are not conclusive, but indicated certain trends that more glasshouse trials and field trials would resolve. Results presented in this thesis indicate that a major shift has occurred in the status of soilborne fungi associated with roots and crowns of grapevines in nurseries in the Western Cape since the 1970s when Phytophthora and Pythium were predominant. The prevalence and role of soilborne fungi need to be determined so that new appropriate disease management strategies can be developed to limit losses in grapevine nurseries and ensure the sustainable production of healthy plants for the grapevine industry.
Pathogens associated with diseases of Protea, Leucospermum and Leucadendron spp.
(Stellenbosch : Stellenbosch University, 1999-12) Swart, Lizeth; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH SUMMARY: The manuscript consists of six chapters that represent research on different diseases and records of new diseases of the Proteaceae world-wide. The fungal descriptions presented in this thesis are not effectively published, and will thus be formally published elsewhere in scientific journals. Chapter one is a review that gives a detailed description of the major fungal pathogens of the genera Protea, Leucospermum and Leucadendron, as reported up to 1996. The pathogens are grouped according to the diseases they cause on roots, leaves, stems and flowers, as well as the canker causing fungi. In chapter two, several new fungi occurring on leaves of Pro tea, Leucospermum, Telopea and Brabejum collected from South Africa, Australia or New Zealand are described. The following fungi are described: Cladophialophora proteae, Coniolhyrium nitidae, Coniothyrium proteae, Coniolhyrium leucospermi,Harknessia leucospermi, Septoria prolearum and Mycosphaerella telopeae spp. nov. Furthermore, two Phylloslicla spp., telopeae and owaniana are also redecribed. The taxonomy of the Eisinoe spp. associated with scab disease of Proteaceae in Australia, California, South Africa and Zimbabwe is elucidated in chapter three. General morphology, symptomatology and phylogenetic analysis based on random amplified polymorphic DNA (RAPD) profiles and DNA sequence of the S.8S rDNA gene and its flanking ITS 1 and ITS2 regions were used. The study provides the first evidence that several distinct Eisinoe spp. are associated with scab disease of Proteaceae. The isolates from Leucospermum, Protea and Banksia represent three distinct species. The isolates from Protea in Zimbabwe represent an additional species. The isolates from Leucadendron, Leucospermum and Serruria in South Africa and Australia, and the isolates from Leucospermum in California and Zimbabwe are representative of the same species. In chapter four, fungal endophytes occurring in leaves and stems of a species of Protea, Leucospermum and Leucadendron were investigated in three localities in the Western Cape province. The aim of the study was to determine if Botryosphaeria proteae was an endophyte of Proteaceae, and is so, how the role of water stress would influence canker development. B. proteae was routinely isolated as an endophyte but was not regarded as a dominant taxon. Inoculation studies were done on non-stressed plants, as well as plants with a leaf water potential of -1.0 MPa (moderately stressed) and -2.0 MPa (severely stressed). From the results of the study it was concluded that Botryosphaeria proteae is primarily an endophyte and can cause leaf necrosis of Protea, and is not a serious stem canker pathogen. A new disease of cultivated Protea in southern Africa, Fusarium wilt, is described in chapter five. The disease is caused by Fusarium oxysporum. It occurs on various Protea cultivars in the North-Western province and in Zimbabwe. Disease symptoms first become visible as necrotic leaves on infected plants. Subsequently, a dark lesion develops from the roots along the stem, usually visible only on one side of the stem. The vascular tissue is discoloured, leading to branch die-back and plant death. Glasshouse trials were conducted to prove Koch's postulates on six Protea cultivars. Forty-five rooted plants of each of six cultivars were inoculated with isolates of F. oxysporum derived from the same cultivar. Disease symptoms similar to those occurring in the field, developed 6 weeks after inoculation. This is the first record of Fusarium wilt on Protea plants. In chapter six, Pestalotiopsis leaf spot disease of Proteaceae in Zimbabwe is described. Pestaloptiopsis Steyart causes necrotic leaf spots in Leucospermum R. Br. and Protea L. species in Zimbabwe. Inoculation studies conducted to prove pathogenicity, confirmed the Pestaloptiopsis sp. as the causal agent of the disease. A description of the fungus is given, and it is compared to other Pestalotiopsis spp. associated with Proteaceae. In conclusion, the present study has shown that several unique species of fungal pathogens are associated with the Proteaceae. Several of these have proven to be new to science, and are described in this thesis. It is clear, however, that the taxonomy of some of these pathogens, their host range and distribution needs to be further investigated. Furthermore, much more research needs to be done on the biology, epidemiology and control of the diseases of the Proteaceae.
Quarantine status of selected fungal pathogens on Malus, Prunus and Vitis species
(Stellenbosch : Stellenbosch University, 2006-04) Carstens, Elma; Crous, P. W.; Fourie, P. W.; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant and Pathology.
ENGLISH ABSTRACT: Invasions of alien species into non-native environments pose one of the largest, but least addressed international threats to biodiversity, both within natural ecosystems and agricultural settings. It is without exception ranked as the greatest environmental threat of the 21st century. Their introduction and spread have been identified as one of the six major categories of change that could potentially alter the world's biodiversity. The number and variety of species introduced make it clear that it is no exaggeration to state that biological invasions are breaking down biogeographic barriers that created and maintained the major floral and faunal regions of Earth. It is, however, difficult to conceive that a single indicator could measure the impact of an invader on a country due to the difficulty in measuring their environmental as well as their financial impacts. Another contributing factor to this problem is the scarcity of data available on the impact of the thousands of invaders in other countries. For the agricultural sectors, alien invasive species are likened to a two-edged sword: on the one hand they are used in the development of new plant varieties and products and on the other hand they have the potential to threaten agricultural production and as a consequence agricultural economic development. An important challenge for countries lies in the development of improved protocols for the prevention, identification and managing of potential invasive alien species. Plant health has always influenced international trade and the backbone for nondiscriminatory, fair, predictable and transparent international trade is the set of rules that national governments have agreed to follow with the foundation of the World Trade Organisation (WTO) in 1995. With the establishment of the WTO, the "WTO - Agreement on the Application of Sanitary and Phytosanitary measures (WTO-SPS)" also came into force. This agreement lays out the provisions, rights and obligations of countries in setting measures to protect human, animal and plant life and health. It also guides quarantine policy and decision-making, with the objective to prevent the use of quarantine measures by governments as disguised or unjustified trade barriers to protect their agricultural industries from import competition. As a signatory member of the WTO-SPS, South Africa has the right to implement appropriate measures to protect our plant health and environment. To set the appropriate levels for protection, South Africa must either apply to international standards or undertake a scientific based risk analysis process, to justify quarantine measures. These measures must also be the minimum necessary to protect plant health. A key element in negotiations for market access is the provision of a list of regulated pests, as well as a list of all pests associated with the crop within the exporting country, to the trading partners. In this study lists of fungal pathogens associated with Malus spp. and Vitis spp. worldwide, including South Africa, were compiled to enable the National Plant Protection Organisation in South Africa to comply with their responsibilities as a signatory member of the international regulatory bodies and to assist them to safeguard our country against harmful invasive species. It was concluded that to have access to accurate plant health status information, all researchers are summoned to validate new pest records and to submit voucher specimen to our National Collection. Geographical distribution records of pathogens and pests are the basis for phytosanitary decision-making and therefore it is imperative for countries to have access to accurate information regarding the geographical distribution of pathogens within their boundaries. For a pathogen to be classified as an A 1-pest, the pest should be of economic importance to the endangered area and not yet present there or present, but not widely distributed and being officially controlled. Many disputes have arisen during the past years concerning the classification for Monilinia fructicola and Neonectria galligena as A 1- regulated pests for South Africa, due to official records of the presence of these pathogens dating back as early as 1917. The situation was further complicated by recent reports from some European countries that M fructicola was detected on stone fruit imported from South Africa. These issues were resolved in this study by following a molecular approach and guidelines as stipulated by the international regulatory bodies. The absence of M fructicola as well as N galligena from South African stone and pome fruit orchards was confirmed. The regulated status of M fructicola and N galligena in South Africa is therefore scientifically justified.
A study on the etiology and epidemiology of black root rot of strawberries in the Western Cape
(Stellenbosch : Stellenbosch University, 2002-12) Botha, Adri; Denman, S.; Lamprecht, Sandra, C. ; Crous, P. W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
ENGLISH ABSTRACT: Historically root diseases have been a production-limiting problem for the strawberry industry worldwide. In the Western Cape Province of South Africa the most serious root disease is black root rot, which causes losses of up to 30%, annually. The aims of this study were to investigate aspects of the etiology and epidemiology of this disease in the Western Cape, and to provide information that can be incorporated in an integrated disease management strategy. In Chapter I a summary of published information on this disease is presented. Disease symptoms include severe stunting of plants, which have black, rotted, reduced root systems. Even though this disease is of great economic importance, the etiology remains unresolved. However, soilborne fungal root pathogens, particularly Pythium and Rhizoctonia spp. have been implicated as major role players. Control of this disease, as well as the other root diseases affecting strawberries, has relied on soil fumigation with broad spectrum chemical fumigants, in particular methyl bromide. However, due to the ozone depleting action of methyl bromide it was decided at the signing of the Montreal Protocol to remove this chemical from the market. This action has caused great demand for alternative measures to control root diseases on many crops including strawberries. Development of integrated disease management strategies is dependent upon a more complete understanding of the etiology, biology and ecology of the disease complex. In Chapter 2 fungal pathogens associated with diseased plants were isolated and Koch's postulates were carried out. The most frequently isolated fungal pathogens were Pythium irregulare, Rhizoctonia spp. and Cylindrocarpon destructans. Two morphotypes of Rhizoctonia were isolated viz. a brown and a white type. Pythium irregulare was isolated more frequently in July than in September, and was not isolated at all in November. Rhizoctonia spp. were present at all sampling dates but were more frequently isolated in November than at the other times. All the fungi that were tested were pathogenic and caused root lesions. Cylindrocarpon destructans and Coniella fragariae did not have a stunting effect on the plants. These results confirm a major role for Pythium and Rhizoctonia in the black root rot complex and suggest that there is a complimentary seasonal variation in occurrence between these two pathogens. The Rhizoctonia species and anastomosis groups of isolates obtained from diseased strawberries in the Western Cape Province were determined, and their pathogenicity and relative virulence assessed. Both binucleate and multinucleate types were recovered from diseased roots and identified as R. fragariae and R. so/ani, respectively. All isolates of R. solani were members of anastomosis group (AG) 6, whereas three AG types were identified among isolates of R. fragariae, viz. AG-A, AG-G and AG-I at a relative occurrence of 69%, 25%, 6% respectively. All Rhizoctonia isolates were pathogenic to strawberry, but R. solani (AG 6) was the most virulent causing severe stunting of plants. This is the first species confirmation and AG type identification of Rhizoctonia taxa causing root rot of strawberries in South Africa. An assessment of the presence and quantity of black root rot pathogens associated with soils prior to fumigation and post fumigation with methyl bromide, was made in Chapter 4. Isolations were also made from nursery plants to determine whether any black root rot pathogens were in the plants before transplanting. Results demonstrated that after fumigation the soil was free of all pathogenic fungi associated with the disease. However, the main pathogens involved in black root rot, viz. Rhizoctonia fragariae, R. solani and Pythium spp. were isolated from nursery plants. The fact that the plants are already infected with these pathogens renders the prospects for control of this disease difficult. Further studies are urgently required to develop production practices that can be included in disease management programmes. In vitro studies were carried out to determine the ECso values of different fungicides for isolates of Pythium irregulare, Rhizoctonia fragariae AG-A, AG-G and AG-I and R. solani AG 6. Benomyl, fludioxonil and tolc1ofos-methyl were used in these tests. Field trials were also conducted using these fungicides. In Chapter 5 it is shown that in general application of fungicides improved the yield and did not affect the survival rate of strawberry plants. Fludioxonil showed potential for short-term use. Applications of fungicides that targeted specific fungal genera were not sufficient to control the disease. Seasonal fluctuation of Pythium and Rhizoctonia spp. became apparent with the occurrence of Pythium being relatively high early in the season but low late in the season. Conversely, the occurrence of Rhizoctonia was low at the beginning of the season but high late in the season. In the short-term there is potential for fungicide applications as part of an integrated disease management strategy, but the economic feasibility of this practice needs to be assessed. In this study the major pathogens causing black root rot were identified in the Western Cape Province of South Africa, and important information regarding the epidemiology of the disease was reported. These results can be incorporated in an integrated management strategy to reduce losses of strawberry production, attJibutable to black root rot.

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