Thesis (MSc.(Microbiology and Plant Pathology)-University of Pretoria, 2006. / Summary in English and Afrikaans. Includes bibliographical references.

Memoria para optar al Título Profesional de Ingeniero Agrónomo / La frecuencia de síntomas debido al ataque de hongos de la madera de la vid, ha aumentado significativamente en todo el mundo durante la última década. Las especies fungosas de la familia Botryosphaeriaceae provocan la muerte parcial de la planta, disminuyendo la productividad y la rentabilidad del cultivo. Una de las especies presentes en Chile es Diplodia mutila, asociada principalmente a la enfermedad del brazo muerto de la vid. El objetivo de esta investigación fue evaluar la susceptibilidad de sarmientos de cultivares de vid vinífera (Cabernet Franc, Malbec, Merlot, Sauvignon Blanc y Syrah) y de mesa (Crimson Seedless, Flame Seedless, Red Globe y Thompson Seedless) a D. mutila. Para ello, se realizó un ensayo “in vivo” usando sarmientos de un año, los cuales fueron inoculados con discos de agar con micelio de D. mutila, en el sector medio del entrenudo después de producir una herida. Los sarmientos fueron mantenidos en una cámara de crecimiento durante cuatro a seis semanas, en oscuridad, a una humedad relativa del 95% y a una temperatura de 25°C. Se consideraron 10 repeticiones por cultivar y al término del período de incubación, se evaluó la longitud de la lesión necrótica en forma independiente, en los cultivares viníferos de los de mesa. No se encontraron diferencias estadísticamente significativas al comparar la susceptibilidad de los cultivares viníferos y de mesa investigados, los que en su totalidad fueron susceptibles al patógeno. / The frequency of symptoms associated to fungal attack on grapevine trunk, increased significantly worldwide during the last decade. Fungal species belonging to the Botryosphaeriaceae family are responsible for the death of part of the plant, reducing productivity and profitability of this crop. Diplodia mutila is one of the species associated to the dead arm disease of grapevine in Chile. The objective of this research was to evaluate the susceptibility of vine shoots of wine grape cultivars (Cabernet Franc, Malbec, Merlot, Sauvignon Blanc and Syrah) and table grapes (Crimson Seedless, Flame Seedless, Red Globe and Thompson Seedless) to D. mutila. In vivo trials were performed using one year old vine shoots, inoculated with mycelia disks of D. mutila between the internode after performing a wound. The shoots were kept during four to six weeks in a growth chamber, in the dark, at 95% relative humidity and at 25 ° C. Ten replicates per cultivar were considered, where necrotic lesion length was evaluated. No significant differences were observed on susceptibility among wine and table grape cultivars, being all susceptible to the pathogen.

The fungal genus Botryosphaeria including its anamorphs has a cosmopolitan distribution and occurs on a wide range of monocotyledonous, dicotyledonous and gymnospermous hosts, including woody twigs and branches, herbaceous leaves, stems of grasses, and even lichen thalli. These fungi give rise to a wide variety of symptoms such as shoot blights, stem cankers, fruit rots, die-back and gummosis. They are also known as saprophytes on dead or dying stems, branches or leaves of plants. In Venezuela, the following species have been reported: Lasiodiplodia theobromae (the anamorph of Botryosphaeria rhodina), Diplodia pinea. D. mutila and Dothiorella spp. However, their characterization has been based only on morphological descriptions. The most common and well characterized species. L. theobromae, is associated with pines and other hosts. The focus of the studies presented in this thesis as to survey Eucalyptus and Acacia plantations in Venezuela for the presence and influence of Botryosphaeria spp., and to characterize these fungi using morphological characteristics and DNA sequence data. I also evaluated the pathogenicity and population biology of Botryosphaeria species present in the plantations. It was anticipated that the various studies would characterize a relatively large number of Botryosphaeria species, potentially recognize new species and provide some perspective of their relative importance to the Eucalyptus growing industry. The thesis has been divided into chapters that reflect discrete units prepared for future publication. The first chapter presents a review of the relevant literature. Two chapters treat the taxonomy of the Botryosphaeria spp. collected in this study. An additional two chapters consider the population biology of the most commonly encountered species and the remaining chapter deals with the pathogenicity of these fungi to Eucalyptus. In the literature review I treat the taxonomy of Botyosphaeria, which has been the subject of much uncertainty for many years. I also briefly review recent findings related to their molecular characterization. A focus is also placed on the pathogenicity of Botyosphaeria species on various host plants and their known importance in Venezuela. The focus concerns mainly Eucalyptus spp. Surveys that formed part of this study logically gave rise to a large collection of Botryosphaeria isolates. An important component of this thesis was to characterize these fungi. This was done based on morphological characteristics and also comparisons or DNA sequence data for various gene regions known to be informative for these fungi. Lasiodiplodia theobromae (anamorph of B.rhodina), B. ribis and B. parva were studied from a population biology perspective. These fungi were chosen for study because they were the most common species encountered on Eucalyptus in Venezuela. To study populations, I made use of simple sequence repeat markers (SS R). Three populations of each of L. theobromae (Venezuela, Mexico and South Africa) and B.ribis-B. parva complex (Venezuela, Colombia and Hawaii) were analysed and the data considered in terms of population differentiation, gene now, mode of reproduction, gene and genotype diversity. In the final chapter of this thesis, I evaluated the pathogenicity or the seven Botryosphaeria species identified from Venezuela. These seven species were thus inoculated on Eucalyptus urophylla x E. grandis hybrid clones. A second series of inoculations was then carried out with the two most pathogenic fungi to determine the relative tolerance or the most important clones to infection. This thesis was conducted over a period of four years. Work was undertaken both in Venezuela and South Africa and this necessitated long periods of time away from my home University and family. Surveys were conducted in many parts of Venezuela to collect the Botryospaeria spp. of interest for latter study in South Africa. The research chapters have been completed systematically and they evolved over time. Each represents a discrete unit implying that there is some overlap, at least in the references, between them. I would like to believe that the thesis will provide a firm foundation for further studies of Botryospaeria spp. and prove valuable to the small but important Eucalyptus growing industry in Venezuela. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-22Bitstream added on 2014-06-13T20:06:04Z : No. of bitstreams: 1 colturato_ab_dr_botfca.pdf: 347881 bytes, checksum: 15d06deb19c4ba13f8196a81c5a97eb1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A seca de ponteiro, causada pelo fungo Botryosphaeria ribis (Dothyorella sp.), que ataca principalmente a região apical da planta, causa lesões que podem chegar a anelar e provocar a quebra do ponteiro ou o secamento da planta. Este trabalho teve por objetivo avaliar métodos de controle químico e por extratos vegetais; estudar a dinâmica espacial de foco; a veiculação do patógeno pelas sementes e realizar a análise de perdas e danos. O teste “in vitro” dos fungicidas constou de oito tratamentos, com 4 doses cada, havendo cinco repetições para cada tratamento. A avaliação foi feita através de medição diária do crescimento radial do micélio em centímetros. O controle químico apresentou cinco tratamentos e três métodos de aplicação, num esquema fatorial 5x3, com quatro repetições. Foram feitas quatro aplicações, com intervalo de 15 dias. Simultaneamente foram feitas avaliações seguindo uma escala de notas de 1 a 6. O teste “in vitro” dos extratos vegetais constou de cinco tratamentos e quatro concentrações: 5, 10, 15 e 20%, em esquema fatorial 5x4 com três repetições. A avaliação foi feita através de medição diária do crescimento radial do micélio em centímetros. No teste com mudas foram utilizadas duas procedências de sementes, dois modos de aplicação (preventivo e convencional) e quatro tratamentos, sendo dois extratos, álcool e água, com oito repetições. A inoculação do patógeno foi feita no caule das mudas. Foram realizadas aplicações dos produtos e avaliações semanais. A avaliação foi feita por contagem de plantas doentes. Para a análise da dinâmica e estrutura de focos foram gerados 19 mapas com a distribuição espacial de plantas com sintomas de seca de ponteiro, provenientes de áreas de Botucatu e de Pratânia/SP. A partir dos mapas foram calculados número de focos, número e percentagem de focos unitários, índice... / Tip drought is caused by Botryosphaeria ribis (Dothyorella sp.) Which attacks mainly the apical region of the plant and causes damage that can ring and cause the breakdown of pointer or a drying plant. This study aimed to evaluate methods of disease control and plant extracts, the spatial dynamics of focus, pathogen association with seeds and analysis of damages. The test in vitro fungicide consisted of eight treatments, with four dose and five replications. The evaluation was done by measuring daily radial growth of mycelium in centimeters. The chemical control had five treatments and three methods of application: brushed, pruned and powdered, in a 5x3 factorial design with four replications. Four applications were made with an interval of 15 days. At the same time were evaluated following a scale from 1 to 6. The test in vitro of plant extracts consisted of five treatments: extracts of a thousand leaves, melon sao caetano, eucalyptus, alcohol and control and four concentrations: 5, 10, 15 and 20%, factorial 5x4 com three replications. The extracts and the alcohol were mixed to the culture medium previously autoclaved, they were placed in Petri dishes and after hardening of the medium was placed a disc of the fungus and the plates were kept in BOD at ± 26 ° C for four days. The evaluation was done by measuring daily radial growth of mycelium in centimeters. In the test plants were used with two origins, two modes of aplication (preventive and conventional) and four treatments: extract of melon sao caetano, Corymbia citriodora extract, alcohol and water, a fatorial design 2 x 2 x 4 and eight repetitions. The inoculation was made in the stem of seedlings. Products applications and evaluations were made weekly. The evaluation was done by counting the diseased plants. For the analysis of the dynamics and structure of foci were generated 19 maps with the spatial distribution... (Complete abstract click electronic access below)

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Galvão.pdf: 1560472 bytes, checksum: 5434a76f0b516e97d5f0698b73fa8050 (MD5) Previous issue date: 2008-02-22 / In this work three Brazilian B. rhodina, and also two strains from Cuba, selected for their ability to produce jasmonate compounds, were submitted to molecular analysis of internal transcribed spacers- ITS regions of rDNA to characterize their genetic variability. rDNA extraction procedures were conducted according classic protocols followed by amplification, via the PCR technique, of conserved ITS regions with the primers ITS1 and ITS4. The PCR products were purified using the GFX purification kit (GE-Healthcare), resolved in “MegaBace” 1000 – DNA Analysis System (GE-Healthcar) and further identified by comparative analysis with ITS regions of rDNA sequences from the databases available at the National Center for Biotechnology Information (NCBI). Analyses were performed with the BLAST tool and the multiple alignments of sequences with Clustal W program from the computational package BioEdit. Moreover, analysis using the TCS program resulted in the identification of nine haplotypes separated by ten polymorphic sites. AFLP analyses were also implemented using four combinations of oligonucleotide primers which resulted in the generation of 300 polymorphic markers. The mean number of amplified fragments by primer pair was 79 with up to 95% of polymorphic bands. The genetic distance between B. rhodina strains was estimated in 29.19%. Dendogram constructed with Jaccard’s coefficient of similarity showed that analyzed strains were assembled in four groups with high genetic variability found in all strains. / Os microrganismos produzem substâncias químicas como resultado do metabolismo primário e secundário. No metabolismo secundário estão envolvidas vias metabólicas para a síntese de produtos naturais que não são essenciais para o crescimento do organismo produtor. O fungo Botryosphaeria rhodina, tem despertado um interesse crescente devido à produção de uma serie de ácidos graxos ciclopentanos, do tipo ácido jasmônico (AJ), com propriedades reguladoras do crescimento de plantas superiores. Usualmente o AJ é um regulador de crescimento vegetal endógeno, sintetizado de maneira natural por uma grande variedade de plantas, pertence aos reguladores de crescimento vegetal (RCV) denominados jasmonatos, sendo os mais representativos o ácido (-)-jasmônico (-)-AJ e o ácido(+)-7-isojasmônico [(+)-7-isoAJ] os quais se encontram amplamente distribuídos nas plantas. AJ tem inúmeras aplicações conhecidas tais como a utilização do AJ na fabricação de perfumes; na produção do chá preto; na redução do consumo de estimulador de crescimento; na produção de malte em indústrias de cerveja; no aumento da produção de taxol; na produção de flavorizantes alimentícios, entre outros. Os microrganismos produzem substâncias químicas como resultado do metabolismo primário e secundário. No metabolismo secundário estão envolvidas vias metabólicas para a síntese de produtos naturais que não são essenciais para o crescimento do organismo produtor. O fungo B. rhodina, tem despertado um interesse crescente devido à produção de uma serie de ácidos graxos ciclopentanos, do tipo AJ, com propriedades reguladoras do crescimento de plantas superiores. A importância crescente do uso de microrganismos na biotecnologia nos levou a propor a prospecção dos metabólitos produzidos pelos microrganismos existentes em espécies vegetais da Amazônia. Desse modo, foram obtidos isolados de B. rhodina capazes de produzir o ácido jasmônico e seus derivados os quais foram caracterizados geneticamente por meio de seqüenciamento do DNA ribossomal e pela técnica de AFLP (Amplified Fragment Length Polymorphism ou Polimorfismo de Comprimento de Fragmentos Amplificados). Trinta e quatro linhagens de B. rhodina isoladas como patógenos de plantas tropicais, tais como: citros, manga, mogno africano, mamão, cupuaçu e madeira cortada foram analisadas para verificar a capacidade de produção de AJ utilizando-se a metodologia de Miersch et al., 1989. Os ensaios para produção de AJ e seus derivados foram realizados em meio Miersch (M1) e Miersch Modificado (M2) e, avaliados em placas de Cromatografia em camada delgada comparativa (CCDC) e quantificados por meio de Cromatografia líquida de alta eficiência (CLAE). Após a avaliação da produção de jasmonatos as linhagens foram submetidas à análise molecular de regiões ITS do rDNA para avaliação da diversidade genética. A extração do DNA foi realizada por métodos clássicos, seguido da amplificação das regiões conservadas ITS do rDNA por meio da técnica de PCR, onde foram utilizados os oligonucleotídeos ITS1 e ITS4. A distância genética entre as linhagens variou de 0 a 1,6%. A taxa de transição/transversão foi de 1,2. Os alinhamentos foram submetidos ainda ao Programa TCS, resultando na identificação de nove haplótipos e 10 sítios polimórficos. A matriz de distância genética assim como a árvore filogenética resultante mostrou pouca divergência genética nas regiões de DNA analisadas. Também foi feita a analise das linhagens de B. rhodina por meio de marcadores moleculares AFLP. Para a obtenção dos resultados foram utilizadas quatro combinações de iniciadores nas reações AFLP, possibilitando a geração de 300 marcadores polimórficos. O número médio de bandas obtidas por iniciador foi de 79% e a porcentagem de bandas polimóficas foi de 95%. A distância genética entre as linhagens de B. rhodina foi de 29,19%, e a porcentagem de loci polimórfico foi de 0,95%. Todas as linhagens investigadas foram agrupadas em quatro grupos e foram obtidos indicadores de alta variabilidade genética entre todos os isolados.

No description available.

Les maladies tropicales négligées, telle que la leishmaniose, constituent un problème majeur de santé publique, en particulier dans les pays à faible revenu où cette maladie sévit. Le nombre limité des médicaments disponibles, leurs coûts, leurs effets indésirables et l’émergence de résistances, exige la recherche de nouveaux agents thérapeutiques. Les champignons endophytes constituent une source prometteuse de nouvelles molécules bioactives. Dans ce contexte, une étude mycochimique a été menée sur une souche endophyte de Botryosphaeria mamane, un micromycète relativement peu étudié, isolée des feuilles de Bixa orellana. Cette souche a été sélectionnée suite à un criblage préliminaire mené contre un modèle d’amastigotes axéniques de Leishmania infantum. Les études chimiques sur ce champignon ont permis d’isoler 9 molécules pures (BM1-BM9), dont 6 de structures nouvelles (BM1-BM4, BM8, BM9) et deux mélanges BM10 et BM11. Les molécules BM1-BM4 appartiennent à la famille des cyclopeptides de type thiodicétopipérazines que nous avons nommés botryosulfuranols A-D. Ils possèdent un squelette original caractérisé par deux centres spirocycliques. BM5-BM7 se sont révélés être des isocoumarines dérivés de la mélleine (cis-4-hydroxymélleine, trans-4-hydroxymélleine et 5-hydroxymélleine). BM8 et BM9 sont deux polycétides de structure nouvelle. Enfin, BM10 et BM11, sont des mélanges de cyclopentapeptides. L’évaluation de leur activité biologique sur amastigotes axéniques de Leishmania infantum a révélé que les thiodicétopipérazines avaient les meilleures activités. Le composé BM4 est le plus actif (CI50 = 0.03 µM) mais également le plus sélectif (IS = 190) de L. infantum par comparaison avec sa cytotoxicité sur des macrophages. BM1 et BM3 sont aussi actifs mais peu sélectifs (CI50 = 0.69 et 0.44 µM, IS = 3 et 15, respectivement) en raison de leur toxicité sur macrophages, et BM2 (CI50 = 3.88 µM) n’a montré aucune sélectivité. Les composés BM8 et BM9, bien que non cytotoxiques sur macrophages, ont montré une activité plus modérée contre L. infantum. Les composés BM5-BM7, et les mélanges BM10, BM11 n’ont présenté aucune activité leishmanicide. L'ensemble de ce travail souligne ainsi l'intérêt des endophytes fongiques comme sources de nouveaux composés leishmanicides. / Neglected tropical diseases, such as leishmaniasis, are a major public health problem, especially in the poorest countries of the world. The limited number of drugs available, their costs, adverse effects and the emergence of resistances, require the search for new therapeutic agents. Endophytic fungi are a promising source of new bioactive molecules. In this context, a mycochemical study was carried out on an endophytic strain of Botryosphaeria mamane, a relatively poorly studied micromycete, isolated from Bixa orellana leaves. This strain was selected from a preliminary screening for its good activity against Leishmania infantum axenic amastigotes. Chemical investigations on this fungus led to the isolation of 9 pure molecules (BM1-BM9), 6 of which are new structures (BM1-BM4, and BM8, BM9) and two mixtures BM10 and BM11. BM1-BM4 belong to the family of cyclodipeptides and are thiodiketopiperazines derivatives that we called botryosulfuranols A-D. The latter possess an unprecedent skeleton characterized by two spirocyclic centers. BM5-BM7 are isocoumarins derived from mellein (cis-4-hydroxymellein, trans-4-hydroxymellein and 5-hydroxymellein). BM8 and BM9 are two new polyketides. Finally, BM10 and BM11 are mixtures of cyclopentapeptides. Evaluation of leishmanicidal activities of these 9 compounds and the two mixtures on axenic amastigotes of Leishmania infantum showed that thiodiketopiperazines possessed the highest activities. BM4 was the most active (IC50 = 0.03 μM) and the most selective (IS = 190) when compared to its cytotoxicity on macrophages. The other thiodiketopiperazines BM1 and BM3 were active but less selective (IC50 = 0.69 and 0.44 μM, SI = 3 and 15, respectively), whereas BM2 was moderately active (IC50 = 3.88 μM) and not selective. BM8 and BM9, despite their absence of cytotoxicity against macrophages, were also moderately active against L. infantum (IC50 = 13.35 and 4.53 μM respectively). Compounds BM5-BM7, and the mixtures BM10 and BM11 did not show any leishmanicidal activities. This work highlights the interest of fungal endophytes as sources of new leishmanicidal compounds.

Ce travail porte sur l'étude chimique d'une souche endophyte de Botryosphaeria mamane, un micromycète relativement peu étudié, isolée des feuilles de Bixa orellana. Des travaux préliminaires portant sur le screening de 409 souches de champignons isolés à partir de plantes médicinales d'Amérique du Sud a révélé que parmi celles-ci, B. mamane E224 était l'une des souches les plus actives in vitro sur un modèle de Leishmania infantum. L'objectif de ce travail a consisté en l'induction de la production de nouveaux métabolites secondaires produits par B. mamane via l'optimisation des conditions de culture de cette souche, la mise en place de méthodes de co-cultures et l'ajout de modificateurs épigénétiques. Une analyse des métabolomes dans les différentes conditions a été réalisée à travers une approche métabolomique, utilisant un couplage UHPLC-HRMS, ainsi que grâce à différents outils statistiques. Deux grandes classes de composés ont ainsi été détectées dans les cultures axéniques de B. mamane. Premièrement, la famille des cyclopeptides, incluant les cyclodipeptides soufrés avec en particulier trois nouveaux composés, les botryosulfuranols A-C. Puis la famille des isocoumarines, avec des dérivés de la melleine (trans-4-hydroxymelleine, 4-hydroxymelleine et 5-hydroxymellein). A travers l'ajout de modificateurs épigénétiques à la culture de B. mamane, nous avons pu étudier les effets de deux inhibiteurs d'histone désacétylases (HDACis), l'acide suberoylanilidehydroxamique (SAHA) et le valproate sodique, ajoutés à deux stades différents de la culture fongique. L'ajout de HDACi dans la culture de B. mamane a entraîné des changements importants dans la production de métabolites secondaires. En effet, une induction de certains métabolites mais également une réduction et l'inactivation de la production d'autres métabolites, ont été observés, et ceci selon la nature du modificateur épigénétique ajouté. Cette étude illustre l'importance du choix des HDACis pour l'induction de la production de métabolites spécifiques. Concernant l'optimisation de la co-culture de B.[...] / This study focused on the strain of a poorly studied fungal endophyte Botryosphaeria mamane E224, isolated from Bixa orellana leaves. Our previous screening involving 409 fungal strains isolated from medicinal plants from South America revealed that among all these strains, B. mamane was shown to be the most bioactive on in vitro model against Leishmania infantum. The objectives of this work consisted in the introduction of new metabolite production by B. mamane by optimizing the fungal culture conditions, and by using co-cultivation methods and addition of epigenetic modifiers. This work was followed by an analysis of the different metabolomes via a metabolomics approach using UHPLC-HRMS and integration of informatics and statistical tools for metabolomics. Two major compound classes were detected in B. mamane. First, the cyclopeptide family including the thiodiketopiperazines (TDKPs) alkaloids with three new compounds proposed as botryosulfuranols A-C; and the isocoumarin family, with the mellein derivatives, trans-4-hydroxymellein, 4-hydroxymellein, and 5-hydroxymellein. Regarding the exploration of B. mamane metabolome cultured in the presence of epigenetic modifier, the effects of two different histone deacetylase inhibitors (HDACis), suberoylanilidehydroxamic acid (SAHA) and valproate sodium added in two different stages of fungal growth, were investigated. As expected, HDACis addition in the culture of B. mamane led to significant changes in the secondary metabolite production. Addition of modifier not only induced metabolites production but also reduced and may inactivate metabolite production in fungi, depending on the nature of the epigenetic modifier added. This study illustrates the importance in the choice of HDACis to fungal culture in order to induce specific metabolite productions. In the study of B. mamane and C. albicans co-cultivation in different culture conditions, we showed the influence of the conditions (static versus agitation) on the metabolome of the fungi. However, the co-culture with yeast did not induce any modification in the fungal metabolome. The investigation of fungal interactions between B. mamane, Fusarium solani, and Colletotrichum linicola in 6-multi well plates in time-series based analysis has been carried out. [...]

Dissertation (PhD (Agric))--University of Stellenbosch, 2002. / 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. / AFRIKAANSE OPSOMMING: Mikrofungi wat tot die genus Botryosphaeria behoort, is heterotrofiese organismes, wat patogenies op houtagtige plante kan wees. Hulle veroorsaak ernstige, en in sommige gevalle, verwoestende verliese, deur blaarnekrose, stamkankers en plantafsterwing. Die Proteaceae snyblom-industrie in Suid-Afrika maak 70% van die nasionale snyblomindustrie uit. Botryosphaeria siektes is 'n belangrike struikelblok in die produksie en handeldryf van Proteaceae, en daar is 'n ernstige behoefte om die etiologie, epidemiologie en beheer van siektes te ondersoek. Verliese van een van die belangrikste proteas, P. magnifica, beloop plaaslik 50% of meer. Die hoof doelstellings van hierdie studie was dus om die etiologie en epidemiologie van Botryosphaeria stamkankers op P. magnifica en ander Proteaceae vas te stel en metodes van siektebeheer te ondersoek. Hoewel daar 'n wye hoeveelheid inligting rakende die swam bestaan, wat in Hoofstuk I hersien is, is daar relatief min inligting oor Botryosphaeria op Proteaceae beskikbaar. Die taksonomie van Botryosphaeria benodig deeglike hersiening, en molekulêre tegnieke word benodig om spesie-identiteite op te klaar. In Hoofstuk 2 is gevind dat Phyllachora proteae, 'n blaarpatogeen van proteas, 'n Fusicoccum anamorf produseer, wat as F. proteae beskryf word. 'n Sphaeropsis-agtige synanamorf is met F. proteae geassosieer en 'n nuwe kombinasie vir P. proteae is as B. proteae in Botryosphaeria voorgestel. Die taksonomie van Botryosphaeria is, beide op die genus- as die spesievlak, in wanorde. In Hoofstuk 3 word die taksonomiese geskiedenis van Botryosphaeria hersien, en die genus word omskryf en van ander morfologies soortgelyke genera onderskei. Hoewel verskeie anamorf genera al met Botryosphaeria op grond van morfologiese waarnemings en filogenetiese analise van ITS rDNA volgorde data verbind is, word twee anamorf genera nou herken, dié met gepigmenteerde konidia (Diplodia), en dié met deurskynende konidia (Fusicoccum). Botryosphaeria proteae moet dus van Botryosphaeria uitgesluit word. Verskeie patogeniese Botryosphaeria spp. het 'n endofitiese fase in hul lewenssiklus. Hulle word dus onwetend in ander lande ingevoer waar hulle 'n gevaar vir landbou en inheemse plantegroei kan inhou. Die huidige wêreldverspreiding van Botryosphaeria spp. wat met Proteaceae geassosieer word is opgeklaar, en in Hoofstuk 4 word 'n sleutel tot die taksa wat met Proteaceae geassosieer word verskaf. Vyf Botryosphaeria spp. word met snyblom Proteaceae wêreldwyd geassosieer, naamlik B. lutea, B. protearum, B. proteae, B. ribis en B. obtusa. B. protearum word as 'n nuwe spesie beskryf. 'n Deeglike kennis van siekte-epidemiologie is noodsaaklik ten einde verliese te verminder. In Hoofstuk 5 dui ek aan dat letsels wat lei tot stamkankers, veroorsaak deur Botryosphaeria protearum op P. magnifica, in die hoofnerf of rant van blare ontstaan. Koch se postulate is uitgevoer en daar is vasgestel dat die aantal letsels wat vanuit kunsmatige inokulasies ontwikkel het korreleer met die styselvlakke teenwoordig in die blare ten tye van die inokulasie. In Hoofstuk 6 word getoon dat B. protearum as 'n endofiet in die blare van P. magnifica. In natuurlike standplase van proteas is stamkankers skaars, maar in verboude plantasies is die voorkoms hoog. Voedingsanalises dui aan dat hoër vlakke van stikstof in die blare van verboude plante in die lente voorkom, wat siekte-ontwikkeling moontlik kan bevorder. Hoë vlakke van natrium in die blare van natuurlike plante mag siekteontwikkeling beperk. Die ernstige ekonomiese verliese wat deur B. protearum veroorsaak word, maak die soektog na verbeterde metodes van siektebeheer noodsaaklik. Fungisiedtoedienings maak 'n belangrike deel uit van 'n geïntegreerde benadering tot siektebeheer. In Hoofstuk 7 dui in vitro toetse aan dat tebuconazole, benomyl, prochloraz me, iprodione en fenarimol die miseliumgroei van B. protearum effektief verminder. 'n Vermindering van 25-85% is aangetoon in die voorkoms van stamkankers in die veld, deur die toediening van fungisiedes en sanitasiesnoei. Die beste beheer is verkry deur die voorkomende toediening van benomyl, bitertanol, fenarimol, iprodione en prochloraz manganese chloride, afgewissel met mancozeb en tebuconazole, op plante in die veld. Indien sanitasiesnoei met gereelde toedienings van fungisiedes gekombineer word, kan die siekte bekamp word.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.