Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Doctoral Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Coutinho, T. A."
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Item Open Access Biology of botryosphaeria dothidea and sphaeropsis sapinea as endophytes of eucalypts and pines in South Africa(University of the Free State, 2001-12) Smith, Hendrik; Wingfield, M. J.; Coutinho, T. A.; Crous, P.W.English: Botryosphaeria dothidea and Sphaeropsis sapinea both very important pathogens in the South African forestry context. These fungi are well established in the country and contribute substantially to annual losses incurred. Currently very little can be done to control the fungi and the damage they cause. The understanding of their respective disease etiologies is thus of great importance to develop relevant counter measures. The overall aim of this dissertation was to investigate various poorly understood aspects of these two fungi and to try and relate the results to practical contributions towards controlling the impact the two pathogens have. Studies have been conducted during the course of five years and each study represents an independent research investigation. The introductory chapter presents a review of the literature pertaining to all aspects of biology, history and taxonomy of B. dothidea and S. sapinea. The two fungi are clearly very similar in all these aspects and perhaps the only clear difference is that S. sapinea is restricted to pines in South Africa. Many other similarities and some differences between these two important pathogens are highlighted and many of these have provided the background for further investigations. In chapter two the presence of B. dothidea and S. sapinea lS demonstrated as symptomless endophytes in healthy, pine and eucalypt tissue. Botryosphaeria dothidea was found to be common in all the Eucalyptus spp. tested, occurring at high percentages in symptomless leaves of Eucalptus smithii, E. camaldulensis, E. grandis and E. nitens. Sphaeropsis sapinea was, in contrast, only present in young, green Pinus patuIa and P. radiata cones, but virtually absent from the cones of P. elliottii and P. taeda. Botryosphaeria dothidea is associated with die-back and canker diseases of eucalypts in South Africa. Despite this fact, little is known about the infection process. The fungus is known to occur endophytically in leaves of various Eucalyptus species in South Africa. In chapter three I consider the ability of B. dothidea to infect apparently healthy Eucalyptus leaves and the subsequent location and structure of these infections once inside leaf tissue. Scanning electron microscopy revealed that conidia of B. dothidea can infect healthy leaves through stomata. These infections ultimately reside amongst mesophyll cells and constitute a number of individual infections per leaf. Two morphologically similar fungi are associated with die-back and canker of eucalypts in South Africa. The one was identified as part of the Botryosphaeria dothidea-complex. In chapter four, the identity of the second fungus was determined by comparing morphology, pathogenicity and DNA sequence analysis of isolates of both taxa. Based on results obtained, Botryosphaeria eucalyptorum, and its anamorph Fusicoccum eucalyptorum, are described as a new species. I found that the teleomorph is morphologically similar to other taxa in the B. dothidea-complex, but conidial characteristics of the anamorph are distinct, as well as the sequences of the nrDNA internal transcribed spaeers ITS 1 and ITS2. As is the case with B. doth idea , the fungus is pathogenic to Eucalyptus, there do not, however, appear to be differences in pathogenicity between the two. Sphaeropsis sapinea is the most important pathogen of pines in South Africa. The fungus, which reproduces only asexually, occurs only on exotic pines. In chapter five, I investigated the diversity of the S. sapinea population in South Africa and compared it with a population from Northern Sumatra. Both populations were obtained from exotic P. patuIa plantations. The phenotypic diversity of these populations was assessed using vegetative compatibility tests. The percentage maximum genotypic diversity, based on Stoddard and Taylor's index, for the South African population was much higher than the Northern Sumatran population, thus indicating that the South African S. sapinea population was more diverse than the Northern Sumatran population. These results support the hypothesis that the population of S. sapinea in South Africa has been introduced from various parts of the world, during the last century. In chapter six, I investigated the role that latent S. sapinea infections in seed cones of P. patuIa, play in post-hail associated die-back. Pinus patuIa seed cones were found to be infected during the second year of development, with extensive colonization only occurring m the third year when cones mature, prior to seed discharge. Vegetative compatibility tests revealed that the presence of S. sapinea in individual third year seed cones is confined to a single genetic entity. Sphaeropsis sapinea colonisation of third year seed cones thus, apparently results from a single successful infection per cone. The probable role of latent infections by S. sapinea indicated that tree age and by implication, increased numbers of attached seed cones, contributes to more severe die-back after hail damage. The control of damage caused by S. sapinea is highly dependant on a dynamic hybridisation programme. Alternative species of pines is thus constantly evaluated for potential. In chapter seven, 65 families representing both the northern and southern populations of P. greggii were evaluated for their tolerance to infection and subsequent die-back caused by S. sapinea. Families were evaluated following natural infection after hail damage, as well as by artificial inoculation. Variation in tolerance occurred and was highly significant between the two provenances, with the northern provenance proving to be very tolerant. Pinus greggii trees of the southern provenances were comparable with P. patula. The potential of the families from northern origins has to be investigated further. Cultures of Cytospora isolated from Eucalyptus trees in South Africa, Congo, Thailand, Venezuela, Mexico, Uganda and Australia, as well as Cytospora-like isolates from Indonesia were compared in chapter eight. Comparisons were based on the homology of the internal transcribed spaeer regions and the 5.8S ribosomal DNA of the nuclear ribosomal DNA repeat unit. Isolates clustered into at least three unrelated groupings, with a fourth grouping that included isolates that morphologically resembled Cytospora. Results from this chapter indicated that the current description of Valsa ceratosperma encompasses several distinctly different species and needs to be further refined. Botryosphaeria dothidea and S. sapinea are two of the most important pathogens of eucalypts and pines in South Africa. The fact that they exist as symptom less endophytes in trees has added a fascinating aspect to our understanding of their role in tree diseases. In the past, they have generally been considered to be wound infecting opportunistic fungi. Results of these studies have shown that this is not so and that they are clearly able to infect healthy trees. They are unlikely to be able to infect dead or moribund tissue. The investigations presented in this dissertation have added considerable knowledge to our understanding of B. dothidea and S. sapinea and will also promote efforts to reduce disease caused by them. However, there are many questions that remain to be answered pertaining to them and it is my hope that this study will provide a foundation and stimulus for further work.Item Open Access Factors associated with coniothyrium canker of Eucalyptus in South Africa(University of the Free State, 1999-05) Van Zyl, Leonel Merwe; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.English: English: In chapter one of this thesis, the literature pertaining to the genus Coniothyrium and its importance in plant pathology, is reviewed. Special attention is given to Coniothyrium species associated with Eucalyptus but the focus is on Eucalyptus stem canker pathogen, C. zuluense. Coniothyrium zuluense is an important pathogen in South Africa and has, since its discovery, become widespread throughout plantation areas of KwaZulu-Natal. The current means for reducing the impact of this disease is to plant disease resistant species and clones of Eucalyptus. It is evident from this review that very little information is available pertaining to the biology, reproductive system, or the population structure of C. zuluense. Such information is essential for managing the disease successfully in the future. The strategy currently used to reduce the impact of Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Results of the study represented in chapter two showed that there is considerable variation in colony colour and pathogenicity of a large collection (344) of C. zuluense isolates. Conidial morphology and growth requirements are, however, similar for all isolates tested. The considerable variation in pathogenicity indicates that C. zuluense has been present in South Africa for an extended period of time, or that virulence is changing rapidly due to strong directional selection pressure. Results of the taxonomic and pathogenicity studies in chapter two, suggest that the C. zuluense population is well established. In chapter three, the population diversity of 108 C. zuluense isolates, differing in their pathogenicity to a susceptible Eucalyptus clone, was investigated using Amplified Fragment Length Polymorphism (AFLP) technology. Results indicated that the level of genetic diversity is relatively low, but higher than expected for an asexually reproducing pathogen. Genetic similarity values also indicated a significant population differentiation between different plantation regions (subpopulations), suggesting that gene flow, together with selection, might be responsible for most of the gene diversity. New epidemics would, therefore, not be as a result of the emergence of new aggressive strains, but would rather be due to the introduction of susceptible Eucalyptus species, together with environmental conditions favouring disease development. A Coniothyrium species associated with similar symptoms to those associated with C. zuluense in South Africa was observed on E. camaldulensis in Thailand in 1996. It was previously thought that C. zuluense was restricted to South Africa. In chapter four, I show using morphological and molecular comparisons, as well as pathogenicity studies, that C. zuluense and the Coniothyrium sp. from Thailand are the same organism. This is, thus, the first record of this important Eucalyptus stem canker pathogen, C. zuluense, outside South Africa. Bacteria commonly exude from necrotic cankers on severely infected Eucalyptus clones in plantations. In chapter five, it was shown that bacteria associated with Coniothyrium canker in the field are species of the genus Pantoea. These species were identified based on 16S rDNA sequence data as P. ananatis pv. ananatis and a species closely related to P. stewartii subsp. stewartii. It was also shown that a synergistic interaction between C. zuluense and both Pantoea species exists. Inoculation studies, using both Pantoea species together with C. zuluense isolates, resulted in a significant increase in pathogenicity as opposed to inoculations where the bacterial and fungal isolates were used alone. Future studies should consider the presence or absence of both bacteria species in disease development in Thailand. During plant-pathogen interactions, pathogens are known to produce cell wall degrading enzymes, in particular pectin degrading enzymes. Polygalacturonase (PG) is the first enzyme produced during such interactions and is known to be a determining factor in pathogenicity. Chapter six showed that C. zuluense isolates and both Pantoea species, P. ananatis pv. ananatis and an unknown Pantoea sp., produce PG. Experimental assays show that levels of PG activity for both Pantoea spp. are significantly higher than those obtained for C. zuluense isolates. As PG is the first enzyme produced during disease development it is hypothesised that the two Pantoea species might play a significant role in the development of Coniothyrium canker. Production of PG could also be used as an assay to evaluate pathogenicity in different isolates of C. zuluense. Pathogen-produced cell wall-degrading enzymes play a key role in activating plant defence responses. Most inducible defence responses are the result of transcriptional activation of genes. Various plant resistance (R) genes, as well as pathogenesis-related proteins, such as polygalacturonase inhibiting proteins (PGIPs), have been linked with resistance to various fungal and bacterial pathogens. In chapter seven, a partially sequenced resistance gene from disease resistant E. grandis clone, TAG 5, was shown to be similar to a gene associated with a disease resistance gene in Arabidopsis thaliana. The most exciting aspect of this study was, however, the discovery of a shift in reading frame of this gene for the susceptible Eucalyptus clone, ZG 14. The complete sequence of this gene should provide a more complete view of its importance in disease resistance. Screening for similar interruptions in the open reading frame of various commercially available Eucalyptus clones could significantly speed up breeding programmes aimed at producing improved disease resistant clones.