Plantation eucalypts are a recent and rapidly growing industry in Australia, and will eventually replace the logging of old-growth forests. Over 40% of these plantations have been established in south-western Australia, where more than 160 000 ha of Eucalyptus globulus plantations now occur. In the early 1900’s, this species was widely planted as an exotic in South Africa, but succumbed to severe pest (Gonipterus sp.) and disease (Mycosphaerella sp.) problems. Similarly, in south-western Australia E. globulus is an exotic species, but with the additional threat that it is planted adjacent to indigenous eucalypts, which increases the possibility of pests and pathogens switching between closely related eucalypt hosts. Over the past ten years, there have been anecdotal reports of increasing levels of Mycosphaerella leaf disease (MLD) in E. globulus plantations in south-western Australia. This increase in disease level is of concern to the industry. To date there have been no comprehensive studies into the taxonomy, biogeography and population genetics of MLD in south-western Australia. This thesis investigated the impact of MLD in south-western Australia with a focus on its impact, taxonomy, biogeography and population genetics. It is the first study worldwide to quantify the relative impact of different Mycosphaerella species in a regional plantation estate. A survey of pest, disease and nutritional disorders (Chapter 2) found that MLD was the most severe and frequently occurring, single taxonomic health threat to 1 and 2-year-old E. globulus plantations in south-western Australia. For the first time, this survey identified and quantified the impact of pest and disease damage to E. globulus plantations in the region. There were differences in the disease levels between plantations and this was due to initial Mycosphaerella species composition and inoculum level, and local climatic conditions favourable for disease, rather than to the provenance planted or the nutritional status of the individual plantations. The survey for Mycosphaerella pathogens of eucalypts (Chapter 3) identified two new species of Mycosphaerella (M. ambiphylla and M. aurantia) and extended the known geographic range of eight other species (M. cryptica, M. gregaria, M. lateralis, M. marksii, M. mexicana , M. nubilosa, M. parva and M. suberosa). Of these: M. lateralis and M. mexicana were new records for Australia; and M. gregaria, M. nubilosa and M. parva were new records for Western Australia. A new anamorph, Phaeophloeospora ambiphylla was described and linked to M. ambiphylla. The occurrence of these new species and disease records in south-western Australia is significant for the plantation-eucalypt industry worldwide. The finding of two new species highlighted the need to quantify the disease impact of these on eucalypt plantations; and the extension of the range the remaining species raised important quarantine issues, concerned with the movement of plant material between regions and countries. The biogeographical investigation of Mycosphaerella (Chapter 4) identified that the most widespread and serious cause of MLD in south-western Australia is M. cryptica. In addition to occurring on the exotic E. globulus, it also occurs on two of the three important indigenous forestry eucalyptus species in this region. That is, on E. diversicolor, and E. marginata, but not on Corymbia calophylla. In terms of the plantation estate of E. globulus, however, M. nubilosa is the most widespread pathogen. The current study found that MLD on E. globulus is a complex of several different species, whereas, on E. diversicolor and E. marginata it is caused by only M. cryptica. Two species, M. cryptica and M. marksii were found commonly on adult E. globulus leaves. Although M. cryptica was the most frequent and serious cause of disease on adult leaves, M. marksii levels appear to be increasing and the future epidemiology of this pathogen should be closely monitored. There is some concern that these two MLD species could become an economically important problem on adult leaves of E. globulus. At present severe levels of MLD is significantly more common on juvenile than on adult foliage. The phylogenetic analysis (Chapter 5), based on ITS rDNA sequences from the present study and those obtained from GenBank accessions, found that Mycosphaerella is an assemblage of largely polyphyletic anamorph genera. Ten distinct clades emerged from the analysis. With the exception of the Dissoconium and the M. recutita clade, which comprised of two and one species respectively, none were comprised entirely of one anamorph genus alone. The anamorph genera represented were often dispersed across more than one clade, indicating that these anamorphs have arisen separately in different phylogenetic lineages. Cercospora, Stenella and Uwebraunia anamorphs each occurred in more than one separate clade. Although on the basis of rDNA sequence data Mycosphaerella appeared mostly monophyletic there was some evidence that the Mycosphaerella genus may be polyphyletic. This was particularly evident from the Dissoconium clade which grouped as closely to the outgroup Botryosphaeria taxon as it did with the remaining Mycosphaerella species. It was argued that a multi-gene phylogeny, which includes sequencing many species in other genera aligned with Mycosphaerella, is required in order to satisfactorily answer the question of whether Mycosphaerella is truly monophyletic. The phylogenetic analysis also showed that the taxonomy of Mycosphaerella based on ITS sequence data needs further clarification. Some species that are morphologically distinct, such as M. vespa and M. molleriana, shared identical ITS sequences. Other morphologically distinct species differed by as little as one or two nucleotides. Yet in other cases, the sequence variation amongst isolates from the same species differed substantially. Much of this variation in M. cryptica and other species was attributed to poorly edited sequences that had been lodged with GenBank. It was postulated that although a part of the remaining variation reflected the existence of cryptic species, some was likely to be genuine intra-species differences. It was concluded that further genes need to be sequenced, and more standardised cultural studies conducted in order to define species boundaries within Mycosphaerella. Based on the ITS rDNA sequence data, two different molecular methods for the identification of Mycosphaerella species from eucalypts were developed (Chapter 6). The first of these was a PCR-RFLP method that enabled the identification of Mycosphaerella species present on eucalypts in south-western Australia. A key is provided, which enabled the identification of species on a combination of PCR-RFLP DNA fragment migration patterns and a small number of morphological features. This key enables the identification of Mycosphaerella species more easily than keys that rely on morphological features alone. Therefore, this has made it easier for non-Mycosphaerella specialists to identify species from this genus. The second molecular method developed for the identification of Mycosphaerella species was that of primers that selectively amplify the DNA of M. cryptica and M. nubilosa, the two most important causes of MLD (Chapter 6). This will allow the rapid identification of these two species by non-specialists in Mycosphaerella taxonomy. The primers from the current study will also enable early diagnosis of the possible causal organism of MLD in a plantation. Once the use of these primers for amplifying DNA from leaf tissue has been optimised, they will also facilitate studies into the early infection process of M cryptica and M. nubilosa. For example, the presence of the pathogen may be detected prior to the appearance of symptoms. Studies may be conducted to determine the length of a hemi-biotrophic phase, and the extent of tissue colonisation both spatially and temporally, beyond the necrotic lesion in these two Mycosphaerella species. Previously, such studies have been hampered by the slow growth rate of these fungi in culture and the lack of media that would allow their selective isolation and detection by directly plating diseased and non-diseased host tissue. This study has clearly indicated that Mycosphaerella species are the major disease threat to E. globulus plantations in Western Australia. It has also shown that over the relatively short period of time of less than ten years the number of species recorded has increased from three to ten, and that disease severity has increased in plantations. It is therefore critical to continue the research on this genus in order to understand the biology, epidemiology and population genetics of this pathogen. This is necessary in order to inform tree selection and silvicultural practise that will minimise the future impact of MLD. This is particularly important if the industry moves towards clonal and hybrid forestry as has occurred elsewhere in the world. This study has laid the foundations for future research on this disease through the elucidation of the taxonomy of Mycosphaerella in south-western Australia and by providing some important molecular tools for its diagnosis and further study.

Made available in DSpace on 2014-06-11T19:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-25Bitstream added on 2014-06-13T20:25:43Z : No. of bitstreams: 1 passador_mm_dr_botfca.pdf: 2195581 bytes, checksum: b49ee0b8f7df05ffdf472a2785c1e683 (MD5) / Muitas espécies de eucalipto são cultivadas no mundo todo e muitos patógenos evoluíram ou se adaptaram á cultura, principalmente fungos, desde a fase de viveiro até os plantios adultos. A mancha de micosferela é uma das principais doenças e o Eucalyptus globulus uma das espécies mais suscetíveis. O presente estudo foi iniciado a partir de isolamentos monospóricos de espécies fúngicas associadas à mancha de micosferela a partir de folhas e ramos de E. globulus provenientes de Bagé-RS, Pedras Altas-RS, Botucatu-SP, Jacareí-SP e Itapeva-SP, que possibilitou a obtenção de 46 isolados, que foram observados quanto à forma de germinação e crescimento micelial. A determinação das espécies deu-se por meio de PCR com primers da região genômica ITS1 e ITS4 e sequenciamento (vide capítulo 1). Como resultado, várias espécies foram encontradas e descritas. Mudas de E. globulus foram inoculadas pelo método da exposição de mudas sadias à folhas com sintomas de Teratosphaeria nubilosa, por ejeção de ascósporos, em casa de vegetação. Estas mudas foram avaliadas e folhas com os sintomas do fungo foram coletadas a partir da terceira semana até a décima terceira semana da inoculação. Para realização de cortes e estudos histológicos, que permitiram observar a formação dos pseudotécios desde o seu início três semanas após a inoculação, as hifas ascógenas dentro do pseudotécio após nove semanas e a formação das ascas e ascósporos após 11 e 13 semanas, respectivamente (vide capítulo 2). Através de estudos anatômicos, verificou-se que as folhas adultas apresentaram menor intensidade dos sintomas, o que pode estar relacionado com a compactação das células do parênquima, enquanto que as folhas jovens apresentam espaços intercelulares no parênquima lacunoso, no parênquima paliçádico e na câmara subestomática... / Many Eucalyptus species are grown worldwide, and many pathogens have evolved and adapted to the culture. The cultivation of eucalyptus is affected by a number of diseases. Many pathogens occur in several eucalypt species, especially fungi, from the nursery to the planting adults. The Mycosphaerella leaf disease is a major disease and Eucalyptus globulus one of the most susceptible species. This study consisted of spore isolates of fungal species associated with Mycosphaerella leaf disease from leaves and stems of E. globulus from Bage-RS, Pedras Altas-RS, Botucatu-SP, Jacareí-SP, Itapeva-SP, which allowed the collection of 46 isolatesm sequenced (see Chapter 1). As a result, several species were found and described. Seedlings of E.globulus were inoculated by exposure to leaves of healthy seedlings with symptoms of Teratosphaeria nubilosa, for ejection of ascospores in the greenhouse. Seedlings were evaluated, and leaves with the symptoms of the fungus were collected from the third week up to thirteenth week of inoculation. To carry out studies and histological sections, which allowed the formation of the pseudothecia from its beginning three weeks after inoculation, the formation of the ascogenous hyphae within pseudothecia after nine weeks, and the formation of asci and ascospores after 11 and 13 weeks, respectively (see Chapter 2). Through anatomical studies, it was found that if the adult leaves had a lower intensity of symptoms, which may be related to compression of the parenchyma cells, while the young leaves have intercellular spaces in the spongy parenchyma, palisade parenchyma and in substomatal chambers facilitating the formation of stroma (see Chapter 3). Essential oils of leaves and young adults, healthy and stain micosferela were extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The data of chemical composition and the respective percentages... (Complete abstract click electronic access below)

Orientador: Edson Luiz Furtado / Banca: Christiane Ceriani Aparecido / Banca: Celso Garcia Auer / Banca: Roberto Antonio Rodella / Banca: Marli Teixeira de Almeida Minhoni / Resumo: Muitas espécies de eucalipto são cultivadas no mundo todo e muitos patógenos evoluíram ou se adaptaram á cultura, principalmente fungos, desde a fase de viveiro até os plantios adultos. A mancha de micosferela é uma das principais doenças e o Eucalyptus globulus uma das espécies mais suscetíveis. O presente estudo foi iniciado a partir de isolamentos monospóricos de espécies fúngicas associadas à mancha de micosferela a partir de folhas e ramos de E. globulus provenientes de Bagé-RS, Pedras Altas-RS, Botucatu-SP, Jacareí-SP e Itapeva-SP, que possibilitou a obtenção de 46 isolados, que foram observados quanto à forma de germinação e crescimento micelial. A determinação das espécies deu-se por meio de PCR com primers da região genômica ITS1 e ITS4 e sequenciamento (vide capítulo 1). Como resultado, várias espécies foram encontradas e descritas. Mudas de E. globulus foram inoculadas pelo método da exposição de mudas sadias à folhas com sintomas de Teratosphaeria nubilosa, por ejeção de ascósporos, em casa de vegetação. Estas mudas foram avaliadas e folhas com os sintomas do fungo foram coletadas a partir da terceira semana até a décima terceira semana da inoculação. Para realização de cortes e estudos histológicos, que permitiram observar a formação dos pseudotécios desde o seu início três semanas após a inoculação, as hifas ascógenas dentro do pseudotécio após nove semanas e a formação das ascas e ascósporos após 11 e 13 semanas, respectivamente (vide capítulo 2). Através de estudos anatômicos, verificou-se que as folhas adultas apresentaram menor intensidade dos sintomas, o que pode estar relacionado com a compactação das células do parênquima, enquanto que as folhas jovens apresentam espaços intercelulares no parênquima lacunoso, no parênquima paliçádico e na câmara subestomática... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Many Eucalyptus species are grown worldwide, and many pathogens have evolved and adapted to the culture. The cultivation of eucalyptus is affected by a number of diseases. Many pathogens occur in several eucalypt species, especially fungi, from the nursery to the planting adults. The Mycosphaerella leaf disease is a major disease and Eucalyptus globulus one of the most susceptible species. This study consisted of spore isolates of fungal species associated with Mycosphaerella leaf disease from leaves and stems of E. globulus from Bage-RS, Pedras Altas-RS, Botucatu-SP, Jacareí-SP, Itapeva-SP, which allowed the collection of 46 isolatesm sequenced (see Chapter 1). As a result, several species were found and described. Seedlings of E.globulus were inoculated by exposure to leaves of healthy seedlings with symptoms of Teratosphaeria nubilosa, for ejection of ascospores in the greenhouse. Seedlings were evaluated, and leaves with the symptoms of the fungus were collected from the third week up to thirteenth week of inoculation. To carry out studies and histological sections, which allowed the formation of the pseudothecia from its beginning three weeks after inoculation, the formation of the ascogenous hyphae within pseudothecia after nine weeks, and the formation of asci and ascospores after 11 and 13 weeks, respectively (see Chapter 2). Through anatomical studies, it was found that if the adult leaves had a lower intensity of symptoms, which may be related to compression of the parenchyma cells, while the young leaves have intercellular spaces in the spongy parenchyma, palisade parenchyma and in substomatal chambers facilitating the formation of stroma (see Chapter 3). Essential oils of leaves and young adults, healthy and stain micosferela were extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The data of chemical composition and the respective percentages... (Complete abstract click electronic access below) / Doutor