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Charles University in Prague Faculty of Science Ensyeh Sarikhani

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Charles University in Prague Faculty of Science Ensyeh Sarikhani Charles University in Prague Faculty of Science Ensyeh Sarikhani Soil microbial communities in agroecosystems and natural habitats contributing to resistance and resilience of the soil environment Půdní mikrobiální společenstva přispívající k rezistenci a resilienci půdního prostředí v agroekosystémech a na přírodních stanovištích Ph.D. Thesis Supervisor: Ing. Jan Kopecký, Ph.D. Assistant Supervisor: RNDr. Markéta Marečková, Ph.D. Study program: Microbiology Prague, 2019 Obsah Acknowledgements ........................................................................................................ 8 Summary ........................................................................................................................ 9 Souhrn .......................................................................................................................... 11 List of Abbreviations .................................................................................................... 13 1. Aims of the thesis ..................................................................................................... 14 2. Introduction .............................................................................................................. 15 2.1. Preface to potato common scab ........................................................................ 15 2.2. Management strategies in use and the necessity of new studies ....................... 16 2.3. Description and taxonomy of causal agents of CS ........................................... 17 2.4. Thaxtomin and other pathogenicity determinants ............................................ 19 2.5. Horizontal transfer of mobile genetic elements ................................................ 21 2.5.1. The first region on PAI ...................................................................... 22 2.5.2. The second region on PAI ................................................................. 22 2.6. Research methodology to study CS .................................................................. 24 2.6.1. Phenotypical methods ........................................................................ 24 2.6.2. Molecular methods ............................................................................ 25 2.7. Strategies for control of common scab ............................................................. 29 2.7.1. Chemical methods and environmental factors ................................... 29 2.7.2. Biological methods and the role of nutrients ..................................... 31 2.7.3. Macro and micro nutrients ................................................................. 40 2.8. Future research requirements ............................................................................ 48 2.8.1. Biological and chemical factors associated with natural soil suppressivity ................................................................................................ 48 2.8.2. Increasing the availability of iron ...................................................... 52 2.8.3. Microbial communities associated with disease-suppressive or conducive soil and a cultivar resistant or susceptible to common scab ....... 54 2.8.4. Interaction between bakteria in potato tuberosphere ......................... 56 3. Materials and methods .............................................................................................. 59 3.1. Experimental sites ............................................................................................. 59 3.2. Pot experiment .................................................................................................. 59 3.3. Field experiment ............................................................................................... 60 3.4. Sampling ........................................................................................................... 60 3.5. Soil and potato skin analyses ............................................................................ 61 5 3.6. Cultivation of actinobacterial strains ................................................................ 62 3.6.1. Submerged culture experiment .......................................................... 62 3.6.2. Cultivation media: .............................................................................. 63 3.6.3. Sensitivity test: ................................................................................... 63 3.6.4. Vermiculite medium experiment: ...................................................... 64 3.6.5. Vermiculite media .............................................................................. 64 3.7. DNA extraction ................................................................................................. 65 3.8. Quantitative real-time PCR ............................................................................... 65 3.9. Illumina MiSeq sequencing and analysis. ......................................................... 66 3.10. 16S rRNA gene-based taxonomic microarray. ............................................... 67 3.11. Statistical analysis ........................................................................................... 69 3. Results ...................................................................................................................... 70 3.1. Determination of biological and chemical factors associated with natural soil suppressivity to potato common scab at contrasting sites. ....................................... 70 3.2. The effect of peat and iron supplements on the severity of potato common scab and bacterial community in tuberosphere soil .......................................................... 95 3.3. Bacterial, archaeal and micro-eukaryotic communities characterize a disease- suppressive or conducive soil and a cultivar resistant or susceptible to common scab. ................................................................................................................................ 100 3.3.1. Common scab severity and quantities of thaxtomin biosynthetic genes. ......................................................................................................... 100 3.3.2. Chemical composition of tuberosphere soil and periderm. ............. 100 3.3.3. Quantities of total bacteria and actinobacteria. ................................ 101 3.3.4. Bacterial community composition in bulk soil and tuberosphere by microarray analysis. ................................................................................... 101 3.3.4.1. Discriminant 16S microarray probes according to soil and potato cultivar. ...................................................................................................... 102 3.3.5. Bacterial community composition in bulk soil and tuberosphere by Illumina sequencing. .................................................................................. 103 3.3.6. Archaeal community composition in bulk soil and tuberosphere by Illumina sequencing, and discriminant OTUs. .......................................... 105 3.3.7. Micro-eukaryotic community composition in bulk soil and tuberosphere by Illumina sequencing. ....................................................... 106 3.4. Interaction between isolated actinobacteria from suppressive soil and Streptomyces scabiei in vitro. ................................................................................. 113 3.4.1. Liquid culture experiments .............................................................. 113 3.4.2. Vermiculite experiment. .................................................................. 113 6 4. Discussion .............................................................................................................. 123 4.1. Biological and chemical factors associated with natural soil suppressivity to potato common scab. .............................................................................................. 123 4.2. The effect of iron availability on potato common scab and tuberosphere bacterial community ............................................................................................... 129 4.3. Bacterial, archaeal and micro-eukaryotic communities in potato tuberosphere ................................................................................................................................ 133 4.4. Strains antagonistic to the CS pathogen ......................................................... 136 4.5. Conclusions ..................................................................................................... 140 4.6. The main outcomes ......................................................................................... 143 5. References .............................................................................................................. 144 Appendix .................................................................................................................... 174 Author’s publication activity ...................................................................................... 175 Supplementary material .............................................................................................. 176 7 Acknowledgements I am heartily thankful to my supervisor Ing. Jan Kopecký, Ph.D. for his guidance and support during the years of cooperation and for his incredible patience with finishing the thesis. I also would like to show my gratitude to RNDr. Markéta Marečková, Ph.D. who made many suggestions and helpful comments attached to the
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