Detecting AM communities in high-altitude vineyards: a next generation sequencing approach.

Since they arose from prokaryotes, eukaryotes have been coexisting in close relationship with microorganism consortia consisting of what is currently described as the microbiota. This includes symbiotic associations with surface colonizing and endophytic microorganisms that are so essential for the host life cycle that we can refer to the host-microbiota association as a single organism, also known as the holobiont. In the kingdom Plantae, the symbiotic interactions between plants and mycorrhizal fungi, due to their spread, and their ecological and agricultural value, represent an important field of study. Among these fundamental symbioses, the first one that took place was the 450-million-year-old arbuscular mycorrhizas, occurred between land plants and arbuscular mycorrhizal fungi (AMF). These obligate biotrophs are pivotal for plant life, providing nutrient uptake and pathogen protection to the plant along several other benefits. AMF interact with more than 80% of vascular plant species, including Vitis vinifera L. (grapevine), an important crop plant that has been attracting the interest of the human kind since the Neolithic thanks to wine production. Given the agricultural importance of mycorrhizal symbiosis, the work of my thesis has focused on the elucidation of the AM fungal coenosis by studying soil-dwelling and grapevine root colonising AMF communities in three high altitude vineyards in Valle d'Aosta (Italy). These communities have been investigated through a Next-Generation Sequencing (NGS) technique, i.e. Illumina MiSeq, by sequencing the ITS and the 18S rDNA regions, which are characterized by a different variability and can potentially lead to a different AMF community representation. Therefore, in addition to the ecological interest raised by the studied environments, the technical questions faced in this thesis focused on i) the understanding of how each DNA region describes the community, and ii) if the choice of a different region can affect the description of the community composition and the estimation of β-diversity. The characterization of the 18S region yielded 148 OTUs (defined by 97% sequence similarity) belonging to Glomeraceae (115), Paraglomeraceae (15), Diversisporaceae (10), Claroideoglomeraceae (5), and Gigasporaceae (3). Analyzing the ITS region, 157 OTUs were shown to belong to Glomeraceae (148), Claroideoglomeraceae (4), Diversisporaceae (4), and Ambisporaceae (1). In the three sites, the 18S and the ITS regions showed that the root communities shared 26 and 10 OTUs, respectively. Among these, the most frequent taxonomical rank was the Rhizophagus/Sclerocystis clade. According to Bray-Curtis and Jaccard distance matrix PERMANOVAs, both DNA regions made it possible to detect a significant effect of the two fixed factors (Site and Matrix) and their interaction on the community structuring. Summing up, this thesis provides an accurate and novel description of the AMF communities found in cultivated mountain ecosystems. Notwithstanding both the 18S and the ITS regions produced biases in the determination of the α-diversity, the assessment of the β-diversity proved to be repeatable, since the two OTU-based sample distance matrices were shown to be correlated (Mantel test). These results suggest the suitability of the ITS region, identified with universal fungal primers and thereby able to characterize the entire fungal community, for AMF-based β-diversity studies. However, the 18S region was demonstrated to be the best tool to describe the AMF-based α-diversity. As a future perspective, the data collected aim to contribute to the development of sustainable viticulture. In fact, the ubiquitous taxa found could represent the backbone of a grapevine-specific inoculum that could be adopted as biofertilizer under poor soil conditions and/or strict climatic parameters.