SZENT ISTVÁN UNIVERSITY IDENTIFICATION AND IMPACT OF ARBUSCULAR MYCORRHIZAL FUNGI ON ECLIPTA PROSTRATA L. AND CAPSICUM FRUTESCENS L. PhD dissertation VO TRUNG AU Gödöllő 2020 i | P a g e PhD school name: Biological Sciences PhD School Discipline: Biological Sciences Head: Prof. Dr. Zoltán Nagy Professor, DSc. SZIE Faculty of Agricultural and Environmental Sciences, Institute of Biological Sciences Supervisor: Prof. Dr. Posta Katalin Professor, DSc. SZIE Faculty of Agricultural and Environmental Sciences, Institute of Biological Sciences Department of Genetics, Microbiology and Biotechnology ..………………………………. ……………………………….. Prof. Dr. Zoltán Nagy Prof. Dr. Posta Katalin Head of the PhD school Supervisor ii | P a g e CONTENTS ABBREVIATIONS ...................................................................................................................... vi 1. INTRODUCTION ............................................................................................................... 7 2. LITERATURE REVIEW ......................................................................................................... 9 2.1. Arbuscular mycorrhizal fungi ............................................................................................... 9 2.1.1. Taxonomy of Arbuscular mycorrhizal fungi (AMF) ...................................................... 9 2.1.2. Identification of arbuscular mycorrhizal fungi ............................................................... 10 2.1.3. Life cycle of arbuscular mycorrhizal fungi ..................................................................... 10 2.1.4. Benefits of arbuscular mycorrhizal fungi ........................................................................ 11 2.1.5. The role of arbuscular mycorrhizal fungi in uptake of phosphorus and nitrogen by target plants ................................................................................................................................. 13 2.1.6. Effects of arbuscular mycorrhizal fungi on plant tolerance against abiotic stresses .. 16 2.1.7. Effects of mycorrhizal fungi on secondary metabolism in medicinal plants ............... 18 2.2. Eclipta prostrata L. ............................................................................................................. 19 2.2.1. Taxonomy, and distribution of Eclipta prostrata ............................................................ 20 2.2.2. Molecular characteristics of Eclipta prostrata ................................................................. 21 2.2.3. Phytochemical constituents of Eclipta prostrata .............................................................. 21 2.2.4. Pharmacological activities of compounds originated from Eclipta prostrata ............... 25 3. MATERIALS AND METHODS ............................................................................................ 28 3.1. Target plants ......................................................................................................................... 28 3.2. Arbuscular mycorrhizal fungi inocula ............................................................................... 28 3.3. Plant growth and experiment design .................................................................................. 29 3.3.1. Isolation and characterization of arbuscular mycorrhizal fungi potential for a field application in a low input agriculture on chilli (Capsicum frutescens) plant. ........................ 29 3.3.2. Impact of arbuscular mycorrhizal inoculation and growth substrate on growth, biomass, and polyphenols content in Eclipta prostrata ............................................................. 29 3.3.3. Impact of arbuscular mycorrhizal fungi on growth, biomass, and polyphenols content in Eclipta prostrata under salt stress .......................................................................................... 30 3.3.4. Impact of different arbuscular mycorrhizal inoculation on growth, biomass and total phenolic content in Eclipta prostrata .......................................................................................... 31 3.4. Assessment of mycorrhizal colonization .......................................................................... 31 3.5. Spore counting in soil ........................................................................................................ 32 3.6. Assessment of plant growth rate and biomass ................................................................ 32 iii | P a g e 3.6.1. Treatment of media and arbuscular mycorrhizal fungi effect on the growth, biomass, and polyphenolic content in Eclipta prostrata ........................................................... 32 3.6.2. Treatment of salt stress and effect of different AMF species on the growth and secondary metabolism, and enzymatic activity of Eclipta prostrata ....................................... 32 3.7. Measurement of leaf area .................................................................................................. 32 3.8. Chlorophyll a fluorescence determination ...................................................................... 32 3.9. Proline determination ........................................................................................................ 33 3.10. Determination of total phenolic content .......................................................................... 33 3.11. Determination of polyphenols by using high performance liquid chromatographic analysis ......................................................................................................................................... 33 3.12. Determination of antioxidant enzymatic activities ......................................................... 34 3.13. Root DNA extraction ......................................................................................................... 35 3.14. PCR and cloning ................................................................................................................ 35 3.15. Sequence editing and phylogenetic analysis .................................................................... 36 3.16. Restriction fragment length polymorphism analysis ...................................................... 36 3.17. Phylogenetic analysis ......................................................................................................... 36 3.18. Statistical analysis .............................................................................................................. 37 4. RESULTS AND DISCUSSIONS ........................................................................................... 38 4.1. Isolation and characterization of promising arbuscular mycorrhizal fungal strains for field application in chilli plants in low input agriculture. ....................................................... 38 4.1.1. Assessment of root colonization by arbuscular mycorrhizal fungi............................... 38 4.1.2. Amount of AMF spores in the soils .................................................................................. 38 4.1.3. Plant growth parameters and shoot, root biomass ......................................................... 39 4.1.4. Molecular analyses for identification of arbuscular mycorrhizal fungi in the roots .. 40 4.1.5. Phylogenetic analyses ........................................................................................................ 41 4.1.6. Discussion ........................................................................................................................... 42 4.2. Impact of arbuscular mycorrhizal inoculation and growth substrate on biomass and polyphenols content in Eclipta prostrata .................................................................................... 45 4.2.1. Mycorrhizal inoculation rate ............................................................................................ 45 4.2.2. Effect of AMF inoculation and different nutrient supplies on measured plant parameters ................................................................................................................................... 45 4.2.3. Proline concentration and chlorophyll fluorescence parameters of E. prostrata ........ 48 4.2.4. Total phenolic concentration from aerial part of E. prostrata ...................................... 48 4.2.5. HPLC analysis of polyphenols from the aerial part of E. prostrata .............................. 49 4.2.6. Discussion ........................................................................................................................... 51 4.3. Impact of salt stress and arbuscular mycorrhizal fungi on plant performance of Eclipta prostrata ........................................................................................................................................ 56 iv | P a g e 4.3.1. Root colonization rate ....................................................................................................... 56 4.3.2. Growth parameters ........................................................................................................... 57 4.3.3. Proline concentration ........................................................................................................ 58 4.3.4. Chlorophyll fluorescence .................................................................................................. 59 4.3.5. Polyphenol compounds ..................................................................................................... 60 4.3.6. Antioxidant