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Bangor University DOCTOR OF PHILOSOPHY Endophytic fungi in Elms:implications for the integrated management of Dutch Elm Disease Blumenstein, Kathrin Award date: 2016 Awarding institution: Bangor University Swedish University of Agricultural Sciences Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 Endophytic Fungi in Elms Implications for the Integrated Management of Dutch Elm Disease Kathrin Blumenstein Faculty of Forest Sciences Department of Southern Swedish Forest Research Alnarp & Bangor University, School of Environment, Natural Resources and Geography, UK Doctoral Thesis Swedish University of Agricultural Sciences Alnarp & Bangor University, UK 2015 Acta Universitatis agriculturae Sueciae 2015:106 Cover: Endophytic fungi isolated from Ulmus spp. and other microorganisms; below: endophyte Monographella nivalis var. neglecta (left side) inhibiting growth of Dutch elm disease pathogen Ophiostoma novo-ulmi (right side). (Photos: K. Blumenstein) ISSN 1652-6880 ISBN (print version) 978-91-576-8410-3 ISBN (electronic version) 978-91-576-8411-0 © 2015 Kathrin Blumenstein, Alnarp Print: Repro, Alnarp 2015 Endophytic fungi in elms: Implications for the Integrated Management of Dutch Elm Disease Abstract Integrated pest management calls for new biocontrol solutions in management of forest diseases. Endophytic fungi that are commonly found in tree tissue may have potential in biocontrol. However, the links between endophyte status and disease tolerance are still unclear, and we know little about the mechanisms by which the endophytes can influence tree pathogens. The first goal of the thesis was to compare the endophyte status in elm (Ulmus spp.) trees with low vs. high susceptibility to Dutch elm disease (DED), caused by Ophiostoma fungi, and to find correlations between endophytes and the susceptibility pattern of the trees. The second goal was to investigate the potential mechanisms of antagonism by the endophytes towards the pathogen. Thus, endophytes were isolated from leaves, bark and xylem of elms that differed in DED susceptibility. The isolates were screened for their potential to counteract the pathogen in dual cultures. Selected strains were investigated using Phenotype MicroArrays to obtain the substrate utilization profiles that reflect the endophytes’ ability to compete with the pathogen for a nutritional niche. To test for a protective effect against the disease, promising isolates were injected into young elms. Preliminary analyses were done to identify the extracellular chemicals that some of the endophytes released into the growth medium. The results showed that the frequency and diversity of endophytes was higher in xylem of elms with high susceptibility to DED. Some endophytes deadlocked the pathogen with extracellular chemicals in vitro, while others had a faster growth rate. Several endophytes were able to utilize substrates more effectively than the pathogen. A preventive treatment with endophytes protected elms against DED, but the effect was unstable across years. Bioactive fungal extracts had a complex chemical profile, and the individual compounds in the extracts remain to be identified. Because endophytes antagonized the pathogen through different mechanisms, I suggest that an endophyte- based biocontrol of DED could be best achieved through a synergistic effect of several endophyte strains. Keywords: Biocontrol, Phenotype MicroArray, Nutrient utilization, Fungus-fungus interactions, Antagonism, Niche differentiation, Ophiostoma spp., Ulmus spp. Author’s address: Kathrin Blumenstein, SLU, Department of Southern Swedish Forest Research, P.O. Box 49, 230 53 Alnarp, Sweden and Bangor University, School of Environment, Natural Resources and Geography (SENRGY), Deniol Road, Gwynedd, LL57 2UW, UK E-mail: [email protected] Dedication To my husband Michael who supported me in every possible way and to our son Kristian, my little sunshine. Contents List of Publications 7 Abbreviations 9 1 Introduction 11 1.1 Endophytes 12 1.2 Overview of biological control systems 15 1.2.1 Specifics on the role endophytes could play in priming host defence responses 17 1.3 Dutch elm disease 17 1.3.1 Strategies to control DED 21 1.3.2 Breeding programs 24 2 Objectives of thesis 27 3 Material and Methods 29 3.1 Sample collection sites 29 3.2 Isolation of endophytic fungi 31 3.3 Identification and characterization of endophytes 31 3.3.1 Antagonism assays in vitro dual culture and in vivo tests 32 3.3.2 Nutritional profiling with Phenotype MicroArrays 33 3.3.3 Effect of single vs. dual culture conditions on nutritional phenotypes 33 3.3.4 Preparation of inoculum and inoculation of the microplates 34 3.3.5 Data reading and analysis 34 3.3.6 Chemical analyses of extracellular fungal products 34 4 Main results 37 4.1 Origin and isolation of the endophyte collection 37 4.2 Identification and characterization of endophytes 39 4.2.1 Antagonism assays in vitro dual culture and in vivo tests 39 4.2.2 Nutritional profiling of endophytes with Phenotype MicroArrays – carbon and nitrogen substrates 42 4.2.3 Chemical analyses of extracellular fungal products 46 5 Discussion 47 5.1 General discussion 47 5.1.1 DED complex as a system for endophyte studies 47 5.1.2 Biocontrol as an option in DED control 48 5.2 Methodological considerations 49 5.2.1 Culturable vs. “total” communities 49 5.2.2 Dual cultures 50 5.2.3 Possibilities in the application of genomics for the research of endophyte communities 51 5.2.4 Phenotype MicroArrays – possibilities and limitations 53 5.3 Qualitative and quantitative differences in fungal endophytes between elm trees that differ in their susceptibility to Dutch elm disease 56 5.4 Mechanisms of antagonism between elm endophytes and DED pathogens 57 5.4.1 Extracellular chemicals produced by endophytes 58 5.4.2 Competition between fungi – nutritional niches 58 5.5 Future research 60 5.6 Concluding remarks 61 6 Appendix 63 6.1 Recipes 63 6.1.1 Fungal culture media 63 6.2 List of fungal isolates and their origins 64 6.3 Phenotype MicroArray Plates used in this project 65 6.3.1 PM 1 MicroPlateTM Carbon sources 66 6.3.2 PM 2A MicroPlateTM Carbon sources 67 6.3.3 PM 3B MicroPlateTM Nitrogen sources 68 References 69 Acknowledgements 83 List of Publications This thesis is based on the work contained in the following papers, referred to by Roman numerals in the text: I Martín, J.A., Witzell, J., Blumenstein, K., Rozpedowska, E., Helander, M., Sieber, T., Gil, L. (2013). Resistance to Dutch elm disease reduces presence of xylem endophytic fungi in elms (Ulmus spp.). PLoS ONE 8(2), e56987. doi: 10.1371/journal.pone.0056987. II Martín, J.A., Macaya-Sanz, D., Witzell, J., Blumenstein, K., Gil, L. (2015). Strong in vitro antagonism by elm xylem endophytes is not accompanied by temporally stable in planta protection against a vascular pathogen under field conditions. European Journal of Plant Pathology, 142(1), pp. 185- 196. doi: 10.1007/s10658-015-0602-2. III Blumenstein, K., Macaya-Sanz, D., Martín, J.A., Albrectsen, B.R., Witzell, J. (2015). Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes. Frontiers in Microbiology, 6:1033. doi: 10.3389/fmicb.2015.01033. IV Blumenstein, K., Albrectsen, B.R., Martín, J.A., Hultberg, M., Sieber, T.N., Helander, M., Witzell, J. (2015). Nutritional niche overlap potentiates the use of endophytes in biocontrol of a tree disease. BioControl, 60(5), pp.655–667. doi: 10.1007/s10526-015-9668-1. V Papers I-IV are reproduced with the permission of the publishers. 7 The contribution of Kathrin Blumenstein (KB) to the papers included in this thesis was as follows: I KB was involved in developing the idea and hypothesis with Juan A. Martín and Johanna Witzell. She contributed with 10% to the planning, with 20% to the laboratory work, with 10% to the summary and analysis of results and with 15% to writing the manuscript. II KB contributed with 15% in developing the idea and hypothesis, 10% in planning of the work, 25% of the laboratory work, 10% of the analysis and summary of the results, and 15% with writing the manuscript. III The idea and hypothesis were developed by KB to 40% and 65% of the planning of the work. KB performed 60% of the laboratory work. She did 50% of the analysis and summary of the results. As one of the two first authors, KB wrote 45% of the manuscript and contributed to 30% in the correspondence with the journal. IV KB developed 70% of the idea and hypothesis and 95% of the planning of the work. All laboratory work was completed by KB. 80% of the analysis and summary of results was done by KB and 95% of writing the manuscript. KB was responsible for the entire correspondence with the journal. 8 Abbreviations AWCD Average well