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Ph.D Thesis Diversity and Antagonistic Activity of Endophytic Ph.D Thesis Diversity and Antagonistic Activity of Endophytic Fungi from Sweet Cherry and Pepper Neda Haddad Derafshi Supervisors: Dr. Krisztián Halász Prof. Dr. Noémi Lukács Department of Plant Physiology and Plant Biochemistry Budapest Corvinus University 2015 1 Ph.D School Name: Doctoral School of Horticultural Sciences Field: Crop Sciences and Horticulture Head of Ph.D School: Dr. Magdolna Tóth Academic Professor, DSc Department of Fruit Sciences, Faculty of Horticultural Science, Corvinus University of Budapest Supervisors: Dr. Noémi Lukács Academic Professor, DSc Department of Plant Physiology and Plant Biochemistry, Faculty of Horticultural Science, Corvinus University of Budapest Dr. Krisztián Halász Assistant Professor, Ph.D Department of Plant Physiology and Plant Biochemistry, Faculty of Horticultural Science, Corvinus University of Budapest The applicant met the requirement of the Ph.D regulations of Corvinus University of Budapest and the thesis is accepted for the defence process. …………………………….... …..………..……………… …..………..……………… Prof. Dr. Tóth Magdolna Prof. Dr. Noémi Lukács Dr. Krisztián Halász Head of the Ph.D School Supervisor Supervisor 2 According to the Doctoral Council of Life Sciences of Corvinus University of Budapest on 9th June, 2015, the following committee was designated for the public discussion: COMMITTEE: Chair: Dr. Mária Hőhn, CSc, Corvinus University of Budapest Members: Dr. Zoltán Illyés, Ph.D Zalaegerszeg-Botfa Dr. Zoltán Bratek, Ph.D Eötvös Loránd University Dr. Marietta Horváthné Petróczy, Ph.D Corvinus University of Budapest Opponents: Dr. István Parádi, Ph.D Eötvös Loránd University Dr. Erzsébet Koósné Szathmáry, Ph.D Corvinus University of Budapest Secretary: Dr. Erzsébet Kissné Bába, Ph.D Corvinus University of Budapes 3 Table of Contents 1. Introduction ................................................................................................................................. 6 2. Literature Review...................................................................................................................... 10 2.1. Definition of endophytes .................................................................................................... 10 2.2. Ecology............................................................................................................................... 11 2.3. Biology ............................................................................................................................... 12 2.3.1. Reproduction and transmission mode of endophytic fungi ......................................... 12 2.3.2. Partner fidelity and evolution of virulence .................................................................. 14 2.4. Impacts of plant-endophyte interactions on endophyte diversity....................................... 15 2.5. Pathophysiological aspects of plant-endophyte symbiosis ................................................ 21 2.5.1. Host protection against herbivores .............................................................................. 22 2.5.2. Effects to stress tolerance of the host plant ................................................................. 22 2.5.3. Induction of plant resistance to pathogens .................................................................. 23 2.6. Agricultural use of endophytic fungi ................................................................................. 24 2.7. Occurrence and biodiversity of endophytic fungi .............................................................. 25 2.8. Current status of the issue in the area of the present study ................................................ 28 2.9. Endophytic fungi in Capsicum ........................................................................................... 30 3. Materials and Methods .............................................................................................................. 31 3.1. Locality and sampling strategy of the study....................................................................... 31 3.2. Tissue preparation .............................................................................................................. 32 3.3. Primary isolation of endophytic fungi ................................................................................ 33 3.4. Single spore isolation of endophytic fungi ......................................................................... 33 3.5. Morphological study .......................................................................................................... 34 3.6. PCR amplification of ribosomal internal transcribed spacer regions ................................. 34 3.7. Analysis of endophyte – pathogen antagonism by dual culture method ............................ 37 3.8. Statistical analysis .............................................................................................................. 38 3.9. Sampling, cultivation and identification of endophytic fungi in Capsicum annuum L. .... 39 4. Results ....................................................................................................................................... 40 4.1. Identified endophytic fungi associated with Prunus sp., rootstocks .................................. 41 4.2. Diversity of identified endophytic fungi in different sampling periods ............................. 41 4 4.3. Pattern of colonization and distribution of endophytic fungi on examined cherry rootstocks .................................................................................................................................. 52 4.4: Relative frequency of identified endophytic fungi on cherry rootstocks ........................... 58 4.5. Endophytic fungi associated with Capsicum annuum L. ................................................... 67 4.6. The evolutionary relationship of identified fungal endophyte taxa ................................... 70 4.7. Dual-culture of identified endophytic fungi with selected pathogens and potential antagonistic activities ................................................................................................................ 74 4.8. Novel scientific achievements by the present study .......................................................... 92 5. Discussion ................................................................................................................................. 93 5.1. Characterization of endophytic fungi isolated from cherry rootstocks .............................. 94 5.2. Impact of host species, colonized tissue, and temporal changes on biodiversity of endophytic fungi ........................................................................................................................ 97 5.2.1. Sweet cherry ................................................................................................................ 97 5.2.2. Capsicum annuum L. ................................................................................................. 101 5.3. Pathogen growth inhibition by endophytic fungi ............................................................. 102 Summary ..................................................................................................................................... 105 Acknowledgement ...................................................................................................................... 108 Appendix ..................................................................................................................................... 109 A1. Biodiversity of endophytic fungi associated with Prunus sp., rootstocks ........................ 109 A.1.1. Biodiversity of endophytic fungi in Prunus mahaleb L., rootstocks ........................ 109 A.1.2. Biodiversity of endophytic fungi associated with other Prunus sp., rootstocks ....... 125 A.2. Radial growth measurements for antagonistic activity of selected fungal endophytes ... 132 References ................................................................................................................................... 143 5 1. Introduction Synthetic agents, widely used for medicinal and industrial purposes, are also essential to reduce economical losses caused by pathogens in agriculture. Due to their effect on nature and to the health risks of their application reduction of their use would be highly advisible. Recently, there has been an emerging attempt to explore nature-friendly compounds which could substitute for chemically synthesized products. A growing body of research have perceived plants as „bio- factories‟ of potentially valuable bioactive compounds, but slow growing rate and harvesting of rare, endangered species pose a risk in themselves, therefore this approach is not generally applicable. This is why alternative sources are outmost essential, since organic synthesis of natural products is not yet economically feasible and cost-effective (Pereira & Castro, 2014). By searching for new potential sources of novel bioactive molecules special attention has been given to symbiotic microorganisms that are associated with a wide-range of plant species and are termed as „endophytes‟(Guo et al., 2008). Endophytic symbionts including bacteria and fungi live within plant tissues without causing any obvious negative effects and have been found in every plant species examined to date. It became evident that endophytes are rich sources of bioactive natural products, and many different agents have been isolated from
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