BACTERIAL PLAYERS IN CROP SYSTEMS: BENEFITS AND RISKS IN THE " ONE HEALTH" PERSPECTIVES.

In recent years, agri-food ecosystems have recognized as crucial environment in the One Health approach, which deals with the intersections between human, animal and environmental health, recognising them as a unique interconnected system. The research conducted in the frame of this PhD thesis dealt with the investigation on the effect of bacteria entrance in agri-food ecosystems. In compliance with the ‘One Health’ concept, both the benefits and the risks given by the introduction, voluntary or accidental, of bacterial players in environmental habitats related to food production were considered. An example of deliberate release of microbes in agricultural systems is the use of plant beneficial strains (i.e., biofertilizer and biostimulants). Experiments were designed to better clarify the interactions occurring between bacteria inoculants and the recipient plant microbiome and, additionally, the research was focused on the design of novel delivery system for the administration of plant probiotics. The knowledge improvement regarding these two aspects has the potential to promote the transition from the lab to the field of microbial based solution for sustainable agriculture, such as the replacement of chemical fertilizers or the use of strains to improve plant tolerance to biotic and abiotic stresses. We characterized for Plant Growth Promotion (PGP) activities a collection of endophytic strains established from grapevine and lettuce plants collected in the field. Among the most promising strains, Rhizobium sp. GR12 and Kosakonia sp. VR04 were tagged with genes coding for fluorescent proteins and used to inoculate micropropagated grapevine cuttings. Plantlets were grown in vitro under optimal growth conditions or on a diluted medium to mimic nutritional deficit. The measurement of plant biomass showed that Rhizobium sp. GR12 displayed PGP activity, when the plantlest were grown under nutritional deficit. The colonization of the plant tissues was confirmed for both Rhizobium sp. GR12 and Kosakonia sp. VR04 through qPCR amplification of the GFP and m-Scarlet protein, respectively. However, though both the strains were able to colonize root and lead endosphere of the treated plantlets, metataxonomic analyses revealed that the composition of the endophytic community was differently modulated by Rhizobium sp. GR12 and Kosakonia sp. VR04. The former had a minor impact on the endophytic bacterial community while Kosakonia sp. VR04 caused a major change in community composition, suggesting an opportunistic colonization pattern. The results of this study corroborate the importance of preserving the native endophytic community structure and functions during plant microbiome engineering. Moreover, for the first time, the structure of bacterial endophytic community of micropropagated grapevine cuttings was described, showing that it clearly differed from those generally associated to this plant species in the field. Rhizobium sp. GR12 strain was included in a panel of plant-associated bacterial strains isolated from different host species and selected based on their PGP properties, abiotic stress tolerance and polysaccharides degrading activity. These strains were used to test the possibility to provide beneficial bacterial inoculant through the functionalization of a bio-based polymer, derived from food industry waste and designed with the final aim to obtain biodegradable nursery pots. Cell viability within the biopolymer was evaluated by embedding the strains into the liquid material and by isolating them after its solidification, drying and storage. The results showed that the approach was suitable only for the tested spore-forming Bacillus strains, which were able to cope the desiccation step during the production of the biopolymeric film. Thus, to improve the survival of non-spore-forming strains, we changed strategy and tested food waste derived materials, and other different molecules, as cryoprotective agents to obtain a lyophilised PGP inoculum. Using as model bacterium Rhizobium sp. GR12 strain, interesting results were observed using a residual fraction obtained from the extraction of proteins from okara, a soy-milk by-product, in comparison with other cryoprotectants. The second pillar of the present PhD thesis concern i) the investigation of the resistome profiling of water-dwelling microbiomes in environments associated with water reuse practices in agriculture and ii) the studies of Horizontal Gene Transfer (HGT) on lettuce phylloplane. On one hand, we collected freshwater samples along a gradient of increasing distance from the effluent releasing point of a WWTP located in the Cremona municipality to the Po River. From these samples, bacterial collection was established on selective media for Enterobacteriaceae and Enterococcaceae, aiming to characterize the resistome in this natural environment and applying different techniques, like Minimum Inhibitory Concentration (MIC) analyses and disk diffusion test. Taxonomic identification showed that the established collection encompasses, besides faecal-indicator-bacteria, several genera known to be able to interact with plants (e.g., Acinetobacter, Klebsiella) and previously reported as potential human and animal opportunistic pathogens. The results of MIC analyses showed the presence of 5 multi-drug-resistant strains. Based on the antibiotic resistance profiling, different strains were selected for genome sequencing. Moreover, we tested the possible introduction and spread of ARB in the microbiome of plants of agricultural interest, by administering some of the detected multi-drug-resistant strains to lettuces plants under greenhouse conditions. Eight bacterial strains belonging to Acinetobacter, Klebsiella and Escherichia genera were tested for colonization ability and were able to establish in the lettuce plants’ rhizosphere with a concentration ranging between 104 to 106 CFU/g of soil. The conducted antibiotic resistance survey showed how a high percentage of isolates important under the ‘One Health’ frame carried AR traits and/or were MDR strains. Rhizospheric colonization of a plant of agricultural interest by MDR strains, could represent an important trigger for HGT events related to AR transmission. The genomes of 15 isolates chosen among the MDR strains are currently under analyses to get more knowledge about AR traits, in particular ARGs localization and possible diffusion mechanisms. In this context, the possible acquisition of extracellular DNA (exDNA) on lettuce phylloplane by the model strain A. baylyi BD413 was characterized performing in planta experiments, to clarify the role of natural transformation as entrance pathway of ARGs into the epiphytic and/or endophytic bacterial communities associated to leafy vegetables. Furthermore, we investigated the effect of surfactants, which are chemicals widely used in agriculture, on exDNA acquisition by bacteria and plant tissues, and on the bacterium leaf endosphere penetration. Firstly, we demonstrated that A. baylyi BD413 was able to acquire extracellular DNA on lettuce leaf when DNA was provided at low quantity mimicking those detected in treated wastewater. Notably, natural transformation frequency in planta was comparable to that occurring under optimal conditions (i.e., temperature, nutrient provision, and absence of microbial competitors). The transformation frequency of A. baylyi BD413 was not enhanced by the presence of the surfactant, a data in agreement with no increase of the bacterial cell permeability when the surfactant was added in the growth medium. Interestingly, the concentrations of total and transformant A. baylyi BD413 colonies retrieved from the lettuce endosphere showed higher values in leaves treated with the surfactant and such difference was statistically significant for transformant colonies. In our experimental set, we could not totally exclude that the latter result could be related to a higher uptake of exDNA by plant tissues in the presence of surfactant resulting in the occurrence of transformation events directly in the endosphere. All in all, the work conducted on natural transformation events using A. bayily BD413 highlighted the importance to obtain more experimental data on HGT mechanisms directly in planta, since produces represent the possible entry point of ARGs and ARB into food production, especially if raw consumed. The results of this PhD thesis research led to an improved knowledge of beneficial bacterial invasion outcomes in planta, linking the response of all the involved players in the holobiont, and provided novel information to design technological solutions aimed at improving beneficial bacteria delivery to plants. On the other hand, the data collected on bacterial resistome and HGT events in relation to water reuse scenario confirmed the importance to assess the possible impact of agricultural practices under the framework of the One Health approach.