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Università degli Studi del Piemonte Orientale Dipartimento di Scienze e Innovazione Tecnologica Dottorato di Ricerca in Chemistry & Biology Curriculum: Energy, Environmental and Food Sciences XXIX ciclo a.a. 2015-2016 SSD: BIO/19 Exploring the microbiota of Vitis vinifera cv. Pinot Noir in two vineyards with different soil management: metagenomic and metaproteomic analysis Giorgia Novello Supervised by Prof.ssa Elisa Gamalero and Prof.ssa Graziella Berta PhD program co-ordinator Prof. Osella Domenico Università degli Studi del Piemonte Orientale Dipartimento di Scienze e Innovazione Tecnologica Dottorato di Ricerca in Chemistry & Biology Curriculum: Energy, Environmental and Food Sciences XXIX ciclo a.a. 2015-2016 SSD: BIO/19 Exploring the microbiota of Vitis vinifera cv. Pinot Noir in two vineyards with different soil management: metagenomic and metaproteomic analysis Giorgia Novello Supervised by Prof.ssa Elisa Gamalero and Prof.ssa Graziella Berta PhD program co-ordinator Prof. Osella Domenico Contents Chapter 1 Introduction.......................................................... 1 1.1 Definition of soil ........................................................................... 2 1.2 The rhizosphere ............................................................................. 12 1.3 Microbial diversity in the rhizosphere .......................................... 19 1.4 Role of plant microbiome in plant health and productivity .......... 23 1.4.1 PGPB andv their mechanisms of action...................... 23 1.4.1.1 Improvement of plant mineral nutrition ............. 25 1.4.1.2 Phytohormone production .................................. 28 1.4.1.3 Lowering ethylene concentration ...................... 29 1.4.1.4 Soil born disease suppression ............................ 32 1.5 Methods to characterize soil microbial communities ............... 35 1.6 Grapevine (Vitis vinifera) ...........................................................42 1.6.1 Annual growth cycle of grapevine ............................. 45 1.6.2 Economical and cultural importance of grapevine ..... 48 References .................................................................................................51 Chapter 2 Outline of the thesis Outline of the thesis ....................................................................... 77 Chapter 3 Materials and methods to analysing the structure and activities of microbial communities....................................... 81 3. 1 Soil sampling .................................................................... 82 3. 2 DNA extraction from soil samples ................................... 85 3. 3 Quantification of nucleic acids ......................................... 88 3. 4 Ethanol precipitation of samples ...................................... 89 3. 5 PCR amplification of soil bacterial communities ............. 89 3. 6 Pyrosequencing ................................................................. 91 3. 7 Bioinformatic analisys........................................................ 93 3. 8 Protein extraction and digestion ........................................ 94 3. 9 MS/MS analysis of protein ............................................... 97 3. 10 Protein database search and Blast2GO data analysis ......... 98 References .................................................................................................. 99 Chapter 4 Exploring the microbiome of Vitis vinifera cv. Pinot noir in a conventional pest management vineyard....................... 101 4. 1 Biodiversity ........................................................................... 103 4. 2 Description of microbial communities in the vineyard ......... 106 4. 3 Discussion ............................................................................ 119 References ................................................................................................... 123 Chapter 5 Exploring the microbiome of Vitis vinifera CV. Pinot noir in an integrated pest management vineyard................. 129 5.1 Biodiversity ............................................................................. 131 5.2 Description of microbial communities in the vineyard ........... 134 5.3 Discussion ............................................................................... 146 References .................................................................................................. 151 Chapter 6 Functional and phylogenetic characterization in a IPM vineyard by metaproteomic approach................................. 157 6.1 Protein identification by MS/MS analysis and phylogenetic classification of acquired proteins......................................... 158 6.2 Functional and biological classification of the identified proteins................................................................................. 163 6.3 Protein expression ............................................................... 165 6.4 Discussion............................................................................. 165 References ............................................................................................. 173 Chapter 7 Conclusion......................................................... 183 Annex ................................................................................... 187 List of publications............................................................... 247 Acknowledgements .............................................................. 248 Chapter I Introduction 1 Introduction 1.1 Definition of soil As usually happens for many common words, there is not a general consensus on the definition of “soil”. Based on a traditional point of view, “soil is the natural medium for the growth of plants”. However, according to FAO, soil has also been defined as a “natural body consisting of layers (soil horizons, Figure 1.1) that are composed of weathered mineral materials, organic material, air and water. Soil is the end product of the combined influence of climate, topography, organisms (flora, fauna and human) on parent materials (original rocks and minerals) over time. As a result soil differs from its parent material in texture, structure, consistency, colour, chemical, biological and physical characteristics” (FAO, http://www.fao.org/soils-portal/about/all-definitions/en/). The current definition of soil provided by the Soil Survey Staff (Soil Taxonomy) is: “Soil is a natural body comprised of solids (mineral and organic matter), liquid, and gases that occurs on the land surface, occupies space, and is characterized by one or both of the following: horizons (Figure 1.1), or layers, that are distinguishable from the initial material as a result of additions, losses, transfers, and transformations of energy and matter or the ability to support rooted plants in a natural environment.” (1). 2 Figure 1.1 Subdivision of soil into soil horizons having typical chemical, physical and biological properties (Source:http://www.ctahr.hawaii.edu/mauisoil/a_profile.aspx) In a very recent review Hartemink (2) analysed the definitions of soil given in the past 200 years and, in addition, attempted to give a brief scientific definition of soil: “The soil is a living, four-dimensional natural entity containing solids, water (or ice) and air. Most soils are outside and are open systems, but soils also occur in shallow lakes and underneath pavement. A soil 3 can have any colour, any age, be very shallow or deep, and consists mostly of a structured mixture of sand, silt and clay (inorganics), rocks and organic material (dead and alive). The soil has one or more genetic horizons, is an intrinsic part of the landscape, and changes over time. Soil are distributed across the earth mostly in a systematic manner. Soils store and transform energy and matter. The soil often supports vegetation, carries all terrestrial life, and produces most of our food. It is an integral part of the natural world interacting with the climate, lithosphere and hydrosphere. Soils are often studied in combination with land-use, climate, geomorphology or the hydrology of an area”. Overall, soil is a heterogeneous, structured and discontinuous system, generally poor in energy sources and nutrients, resulting from the interactions among mineral, biological and chemical components present in soil (3-5). Under a physical point of view soil is a highly complex environment of aggregated particles originating an intricate 3D network of pores that are filled with water and/or air (6). The formation of aggregates plays a key role in defining soil structure. Clay particles and humus form micro- aggregates characterized by a diameter lower than 250 μm, negative charge, and resistance to mechanical action (7); macro-aggregates are larger (size higher than 250 μm), less stable and can be destroyed by soil management (8- 9). 4 Figure 1.2 Soil representation at microscale level. While soil seems to be very homogeneous when looking at macroscale (a) it appears very heterogeneous at the microscale (b and c). b) Microaggregates carrying water filled (dark blu) micropores organize in clusters and originate macroaggregate. Pores having larger size (light blue and white) are localized between aggregates and are water or air filled, depending on the soil moisture status. c) The aggregates results from the combination of primary components, plant roots, fungal hyphae, and exopolysaccharides. Bacterial cells (red and green dots) mainly live in micropores, where they found protection against predators and dehydration.

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