The Rootstock-Scion Combination Drives Microorganisms’ Selection and Recruitment in Grapevine Rhizosphere Thesis by Hend Alturkey In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia July 2020 2 EXAMINATION COMMITTEE PAGE The thesis of Hend Alturkey is approved by the examination committee. Committee Chairperson: Daniele Daffonchio Committee Members: Peiying Hong, Arnab Pain 3 © July, 2020 Hend Alturkey All Rights Reserved 4 ABSTRACT The Rootstock-Scion Combination Drives Microorganisms’ Selection and Recruitment in Grapevine Rhizosphere Hend Alturkey In the last decade, several studies demonstrated that plants have developed a tight partnership with the edaphic microbial communities, mainly bacteria, fungi, and archaea. Such microbiome accomplishes essential functions and ecological services complementary to the functions encoded by the host plant, conferring adaptive advantages to the plant, particularly during stressful conditions. The interaction between microbial communities and their host plants in the natural ecosystem are complex and the mechanisms regulating these mutualistic associations are not fully elucidated. Several biotic and abiotic factors have been shown to be important during this process, including the plant properties (species, age, stage, etc.), the soil type and agronomic practices, the geo-climate conditions, and the biotic interaction. In this context, the vineyard ecosystems represent a unique biogeography model to study and disentangle microbial biodiversity patterns (compositional diversity and potential functionality) across plants cultivated in different geographical regions. Here, I used the rhizosphere and bulk soil of seven different rootstock-scion combinations (Vitis spp.) to dissect the main factors driving the microbial communities’ recruitment in ten different vineyards in Tuscany (Italy), distributed in the Pomino and Nipozzano estates of Frescobaldi company. Among the factors investigated, I focused my attention on the geographical area, soil type and rootstock-scion combination. By using high-throughput sequencing of bacterial 16S rRNA gene and fungal ITS region, I show how both bacterial and fungal communities associated with grapevine rhizosphere and bulk soils are mainly affected by the geographical area and the soil. Nonetheless, I also revealed that the rootstock-scion combination is an important driver in shaping the microbial community, explaining a higher percentage of variability in comparison with the factors rootstock and scion taken alone. Overall, the results obtained in my thesis offer a new perspective of 5 research that aim to develop a deep understanding about the contribution of scion- rootstock combinations in the microbial community ecology of the plant holobiont. Keywords: Plant-microbe interactions, edaphic microorganisms, Microbial ecology, Plant Growth Promoting Bacteria, Rootstock-scion, Grapevine. 6 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor professor Daniele Daffonchio for giving me the opportunity to work in his lab and for his guidance, immense knowledge, encouragement, and support throughout the course of this research. Besides my advisor, I would like to thank my thesis committee: prof. Peiying Hong and prof. Arnab Pain for their insightful comments, and support. Thanks to Michele Brandi, Andrea Bellucci and the Frescobaldi s.p.a. (Nipozzano and Pomino Frescobaldi estates) for their collaboration in this project and their assistance during sampling procedures. I would like to express my deepest thanks to my mentor Dr. Ramona Marasco who personally supervised, supported, and motivated me all through this project. She has trained me on how to be a scientist and showing me that it can be done and how to do it. I am truly thankful for her efforts, patience, and immense expertise that were a key motivations throughout my master research journey. Thank you for always keeping me on track, I honestly cannot think of a better mentor to have. Thanks to Kholoud Seferji for her support whenever I ran into a problem or had question about laboratory work and for being very friendly and always helpful. Thanks to my family and friends for supporting me in many ways along the way, I am forever grateful. This thesis stands as a testament to your unconditional love and encouragement. Special thanks to my mom for her kind words, encouragement, baked goods, and love, which without I would be lost. Finally, I must express my thanks to my fellow labmates for providing me with continuous support and encouragement throughout my research project. Thanks to all of you whom provided a great cheerful environment that makes it easy to work and share the laboratory with you. Thanks to the lab manager Taskeen Begum for everything she does in the lab. It has been an honor to be a member of Extreme Systems Microbiology Lab, a group whose passion for science is truly inspirational. 7 TABLE OF CONTENTS EXAMINATION COMMITTEE PAGE 2 COPYRIGHT PAGE 3 ABSTRACT 4 ACKNOWLEDGEMENTS 6 TABLE OF CONTENTS 7 LIST OF ABBREVIATIONS 9 LIST OF ILLUSTRATIONS 10 LIST OF TABLES 12 AIM OF THE STUDY 13 Chapter 1. Understanding the plant holobiont: the interdependence of plants and 17 their microbiome 1.1 Introduction 17 1.2 Soil microbiome: ecological services and biodiversity reservoir 19 1.3 Microbial selection and recruitment by the plant host 21 1.3.1 Two-step selection model for root-system microbial community 21 differentiation 1.3.2 Compartmentalization of plant microbiome 24 1.4 Ecological factors driving the plant-microbiome assembly and interactions 27 1.5 Functional services of plant-associated microbiomes: biopromotion and 30 bioprotection mechanisms exerted by beneficial microorganisms 1.6 Conclusion 34 Bibliography 35 Chapter 2. Grapevine associated microbiome: understanding the factors affecting the rhizosphere microbial community structure and composition in rootstock-scion 44 combinations Abstract 44 2.1 Introduction 45 8 2.2 Methods and Materials 48 2.2.1 Sample collection 48 2.2.2 Physico-chemical analysis of vineyards’ soil 48 2.2.3 Total DNA extraction 49 2.2.4 Amplification, sequencing and meta-phylogenomic analysis 49 2.2.5 Bacterial and fungal diversity, taxonomy distribution and statistical analysis 50 2.3 Results and Discussion 51 2.3.1 Geoclimatic and physico-chemical characterization of Pomino and Nipozzano 51 areas 2.3.2 Distribution and use of grape rootstocks in Tuscan vineyards 55 2.3.3 Diversity estimation of microbial communities associated to the rhizosphere 56 of different grapevine rootstocks 2.3.4 Relative abundance of bacterial taxa associated to the grape rootstocks’ 63 rhizosphere and surrounding bulk soil 2.3.5 Taxonomy of the mycobiota associated with the rootstocks’ rhizosphere and 67 the surrounding bulk soil 2.3.6 Shared microbiome and rootstock-scion specific microbial assemblages 71 2.4 Conclusion 76 Bibliography 77 Supplementary Materials 86 THESIS CONCLUSION 91 9 LIST OF ABBREVIATIONS C Carbon Ca Calcium K Potassium N Nitrogen P Phosphorus Mg Magnesium Na Sodium CEC Cation Exchange Capacity PGP Plant Growth Promoting OTU Operational Taxonomic Unit PCoA Principal Coordinate Analysis PERMANOVA Permutational Multivariate Analysis of Variance 10 LIST OF ILLUSTRATIONS Figure 1.1. Description of plant microbiome 19 Figure 1.2. The recruitment and enrichment of microbes by the plant root system 23 Figure 1.3. The relative abundance of grapevine microbial community 26 Figure 1.4. Rhizosphere Microbiome Drivers in natural and agricultural ecosystems 28 Figure 1.5. Schematic representation of the microbial activities involved in the 32 alleviation of abiotic stress in plants Figure 2.1. Satellite images of the studied vineyards in Tuscany 52 Figure 2.2. Chemical-physical analysis of vineyards’ soil 53 Figure 2.3. Microbial recruitment process in grape rhizosphere 59 Figure 2.4. Beta-diversity of microbial communities associated with grapevine 61 rootstocks’ rhizosphere and bulk soils in Tuscan vineyards Figure 2.5. Taxonomic diversity of bacterial communities associated with 64 grapevine rhizosphere and vineyard bulk soils Figure 2.6. Distribution of the main bacterial taxa across the rhizosphere of the 66 grafted grapes Figure 2.7. Taxonomic diversity of fungal communities associated with grapevine 68 rhizosphere and vineyards bulk soils Figure 2.8. Distribution of the main fungal taxa across the rhizosphere of the 70 grafted grapes Figure 2.9. Distribution of the core microbial taxa across the rhizosphere of 72 grapevine plants with different graft combinations Figure 2.10. Relative abundance of the rootstock-scion specific microbial taxa 75 across grapevine rhizospheres 11 LIST OF TABLES Table 2.1. List of vineyards with location and of the sampled rootstock-scion 49 combinations Table 2.2. Physical-chemical parameters measured in the ten selected vineyards 54 Table 2.3. Results of distance-based linear model (DistLM) marginal and sequential 55 step-wise tests Table 2.4. Rootstock name, genetic origin and main adaptation to environment are 56 reported Table 2.5. Alpha diversity estimates of the microbial communities 57 Table 2.6. Multivariate analysis of variance for each factor tested 60 Table 2.7. Bacterial and fungal reads assigned to taxonomic ranks 63 Table 2.8. Shared and specific bacterial and fungal OTUs detected across rhizosphere microbial communities associated with the seven rootstock-scion 71 combinations 12