| dc.description.abstract | European ash, Fraxinus excelsior, has huge economic and environmental value on the island of Ireland for forestry and ecosystem services. Given the rapid spread of Hymenoscyphus fraxineus, ash dieback disease, since its first Irish finding in 2012, and the extensive and severe damage caused by the pathogen in mainland Europe, methods are required to minimise the impact of this fungus. The pathogen is now past the point of eradication in Ireland, being present in all 32 counties. There is, therefore, a need to develop methods to manage the disease and minimise its impact. Manipulation of its microbiome, and its fungal endophytes in particular, is one such solution. Endophytes of ash include other pathogens that can worsen the symptoms of a dieback infected tree but also beneficial symbionts that can improve the health of the trees. This thesis explores the fungal microbiome of ash. It takes a culture based endophyte isolation approach to characterise endophytes from a European provenance trial of ash in Roosky, Co. Rosscommon, a sample from a dieback infected population in the French Alps, and a collection of different Fraxinus species and taxa from the National Botanic Gardens, Glasnevin. Methods were tested to optimise endophyte isolation from different tissues with contrasting health (diseased vs non-diseased) and tissue types (position on leaf, rachis, roots). The diseased material from Roosky could have been infected with Hymenosycphus fraxineus but we did not detect it even though it has subsequently been recorded at the site. A total of 628 endophyte isolates were cultured and these represented 214 morphotypes. Different media (MEA and MEA+Fraxinus leaf extract) recovered different endophyte communities.
A comprehensive sample of these isolates were further identified using DNA barcoding with the nrITS region and 119 fungal endophyte species discovered from 2 phyla and 10 classes. Fungal endophyte communities were shown to differ markedly between root and leaf samples and some evidence was found for variation between source populations. Identification of four Hymenoscyphus culture strains from the island of Ireland was confirmed with nrITS DNA barcoding and three distinct haplotypes discovered. These match other European strains from differing countries but phylogenetic analysis using TCS network analysis could not resolve the invasion route into Ireland. One haplotype matched UK samples but the other two matched with the samples from Latvia, Lithuania, Slovakia and Switzerland. A culture independent approach was also undertaken in parallel to directly characterise the fungal endophytes of leaves and seeds. In this case, the nrITS region was sequenced using Illumina HiSeq (high throughput sequencing; HTS) of DNA amplicons directly from plant DNA extracted from ash (without the isolation and culture of the endophyte). The analysis also revealed a huge alpha diversity of fungi including a greater taxonomic depth of endophytes than the culture dependent method (5 phyla, 16 classes). The seed endophyte community was markedly different from the leaf communities and communities could be separated by NMDS and PCoA according to geographical location (France, Glasnevin, Roosky). There is also evidence for differing fungal communities in other ash species as the diverse sample of Fraxinus taxa differed from the communities found in F. excelsior alone. Despite the variation detected, core endophytes found in all samples of leaf, root and seed could be identified. However, there was little overlap between the community composition estimated by the culture dependent and culture independent approaches. This suggests that only a small proportion of endophytes were cultured and also that primers used to in the nrITS HTS did not amplify all the fungal taxa.
Methods are required to test the interactions of the ash dieback pathogen with endophytic fungi to assess how they influence the growth of each other. In particular, we wanted to know if any endophytes were antagonistic to H. fraxineus. Therefore, we undertook dual culture, in vitro, antagonism testing of endophytes against two strains of the pathogen. Pyronema domesticum, Meyerozyma guilliermondi and Lecanicillium attenuatum were found to significantly reduce the growth of the pathogen. Methods are also required to test the interaction of endophyte, pathogen and Fraxinus plants. This is a challenge because field-based inoculation experiments release the pathogen into the environment. Furthermore, it is also harder to control other environmental variables in field or glasshouse experiments. We therefore developed an in vitro tube screening system to grow ash in tissue culture, to break dormancy and to co-culture the plants with the endophytes and pathogen. We compared tissue culture of embryos with seed culture methods and found the seed culture methods to be most appropriate for establishing clonal lines of ash despite higher germination success with embryos. Survival rate of ash, post germination, was higher (100%) for seeds than embryos (43%) presumably because of their endosperm reserves. We showed that the in vitro method can be used for screening of dieback resistance and for endophyte interaction studies but more work is required to optimise the system. We found that the tissue culture methods can also be used to remove endophytes from ash.
The ultimate aim of the research was to provide evidence-based guidance for foresters and woodland managers to minimise the impact of the disease and to maximise the recovery of timber or minimise the damage to ash woodland ecosystems. This thesis has, therefore, made the most comprehensive assessment and characterisation of Irish ash endophytes to date and laid the foundation for further endophyte manipulation studies aiming to manage and control the disease. | en |
