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The taxonomy, phylogeny and impact of Mycosphaerella species on eucalypts in South-Western Australia

Maxwell, Aaron (2004) The taxonomy, phylogeny and impact of Mycosphaerella species on eucalypts in South-Western Australia. PhD thesis, Murdoch University.

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      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 1900s, 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.

      Publication Type: Thesis (PhD)
      Murdoch Affiliation: School of Biological Sciences and Biotechnology
      Supervisor: Dell, Bernard
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