PROSIT - Plant micRObiomes in Sustainable vITiculture

The grapevine industry is an economically important sector for several Mediterranean Countries and the pressure of a growing market demand has led to an acceleration of the whole production processes. The intensification of grapevine cultivation is causing a progressive simplification of the agro-ecosystems associated with a progressive loss of biodiversity with possible negative consequences on the functions and ecosystem services connected to it. In this context it is necessary to define management protocols that promote sustainability by supporting the maintenance and improvement of biodiversity within the vineyard agro-ecosystem (Eurpean SDG 15: Life on land). In the field, plants are continuously exposed to severe abiotic stresses (e.g. high temperatures, drought) often associated with extreme weather events that are more frequent in the last decades as a consequence of the global climate changes. Water shortage is the most significant limiting factor of crop production worldwide, particularly in areas characterized by Mediterranean climates such as Southern Europe and Northern Africa. The water issue is the most important for the environmental sustainability of viticulture with a 60% presence in semi-arid areas. The annual average precipitation in many viticultural areas is generally lower than vineyard consumption (300 to 700 mm). In the Mediterranean regions, the water requirement for grape development is approximately 500 mm, reaching 800 mm along the whole season. Under unlimited water supply, the grapevine water use depends on evapotranspiration and leaf area. In dry areas, water use by irrigation scheduling can be a compromise for environmental sustainability of the crop and sometimes be a competition with other critical human uses. Moreover, water demand is expected to increase because of increased global temperature and intensity of climatic anomalies, such as droughts and heat waves. Several studies have shown that changes in grapevine water status at critical phenological stages have a direct effect on grape composition and quality attributes by influencing vegetative growth, yield, canopy microclimate and metabolism. For all these reasons, there is an urgent need to enhance grapevine tolerance to environmental stresses and in particular to drought. The use of microbial endophytes as biological control agents, or their bioactive compounds, encompass several advantages, as these microbial inoculants are environmentally safe, show a smaller risk for human and animal health and the environment, and allow a reduction of agrochemical inputs. Also, and importantly, beneficial microbial endophytes are effective for a targeted activity for plant diseases and stress control, thus allowing a reduced development of pathogen resistance and the balance between microbial ecosystems is preserved. The application of beneficial microbial endophytes is permitted in both conventional and integrated pest management practices.

PROSIT is a multidisciplinary research project combining agronomic field surveys, in vitro culture, genetics, plant physiology assays, microbiology tecniques, pathogen assessment, molecular biology tests, microscopy analysis and bioinformatics. The project starts by field observations of grapevine plants of four different local varieties, growing in arid regions in Sicily and Algeria. of therefore endowed with superior tolerance and resistance to drought. Plants will be visually assessed to estimate their sanitary status, presence of evident disease symptoms and to confirm the varietal identities. To that aim, surveys will be mainly conducted during the vegetative season when leaves and fruit are present. Then, collected material from selected Mother Plants (MPs) will be transferred from the field to the laboratory for microbiome characterization. Selected MPs from the field will be used as rootstock donors, sanitized Pinot Noir (PN) scions will be grafted on them to transfer the endophytes from rootstock to scion. These grafted plants will be subjected to drought stress treatment under controlled laboratory conditions and the plant material will be collected for molecular and physiological characterization. Microbiome characterization will be performed by metabarcoding of bacterial 16S and fungal ITS amplicons. This techniques bypasses the restrictions imposed by cultivation steps and provides a comprehensive list of microorganisms (Djemiel et al. (2017) Front Microbiol 8: 2052). The subset of culturable endophytes will be also isolated by successive rounds of subculturing, and used for direct inoculation tests at the end of the project. The culturable fraction of selected endophyte, will be inoculated in sanitized PN. These plants will be analysed using established protocols for an integrated ‘OMICs’ approach and an advanced bioinformatic platform in order to determine the molecular mechanisms triggered by microbial associations in response to water stress. Bioinformatic analysis of data will follow system biology approaches, aimed to identify robust differentially expressed genes, master regulators, main regulatory modules, metabolic pathways and markers involved in stress response. In parallel, transformed Pinot Noir lines harbouring fluorescent probes for the plant stress hormone abscisic acid (ABA) and the cell messenger calcium will be inoculated with bacterial consortia to determine at the cellular level, in vivo and non-invasively, the levels of the stress hormone ABA involved during the stress response in plants and the calcium signature associated to the stress response. Comparison among different grapevine/microbial associations will allow to identify the endophyte combinations involved in the grapevine tolerance to drought, representative of each variety.As a confirmation for the role of the selected microbial associations and to explore the possibility of commercial exploitation of our findings, we will perform pilot tests, in small scale, of the application of culturable microbial consortia in economically important grapevine varieties at international and local levels. To this aim the endophytes will be transferred to grapevine multiplication material by both grafting and direct inoculation of the cultivable fraction. We will then repeat the drought test and measure plant response to compare the effects of the original associations with the artificial consortia. PROSIT implies the engagement of different stekeholders (agronomist, winegrowers, wine producers and nurserymen) already during the project (see the attached endorsement letters). The different stakeholders will make their experience available for the project advance, helping to measure the exploitation level of the results obtained.

PROSIT will bring direct benefits in terms of reduction of the workload associated with irrigation operations, reduction of costs for the construction and maintenance of water supply and distribution systems, replacement of senescent plants due to the impact of water stress and indirect benefits by creating new market segments and new job opportunities (development of biotech laboratories and specialized personnel for in field application of the new technologies). In addition, PROSIT outcomes will allow wine producers to valorize their products responding to the requests of consumers who are increasingly sensitive not only to the quality of wine but also to managing environmentally friendly vineyards. As part of PROSIT, the transfer of the project outcomes to the grapevine industry (nurseries, field technicians, farmers, wine consortia) is envisaged to provide guidance for the application in the field of the new technologies. The transition to more sustainable and resilient farming systems will be investigated by identifying and measuring appropriate socio-economic and ecological performance indicators, such as savings on irrigation costs and dedicated working hours, percentage of the wine-growing area reached by innovation and number of field technicians/farmers formed, number and dimension of private companies/start up involved in the development of new bacterial formulations, water savings in terms of quantity of water not removed from environmental resources measured both at the level of single companies and on regional scale (Sicily and Algeria). The implementation of the use of endophyte will open a new vision for the agriculture in order to optimize the benefic natural resources in order to set up an agrobiological system by optimizing natural resources available in site. The use of endophyte for stress management is a new way to optimize grapevine capacity to struggle drought, pest and diseases. PROSIT will contribute to sustainable development by involving partners with different expertise, and also located in different Mediterranean area with agriculture practices linked to the local culture/tradition PROSIT also involve local stakeholders such as growers, agronomists, oenologists, wineries, wine consortia and final consumers. Some of them have been contacted and endorsed PROSIT with letters of interest (attached to this document).