EFFECTS OF NON-LETHAL CONCENTRATIONS OF BIOACTIVE COMPOUNDS ON PLANT-RELATED BIOFILMS

It has been estimated that at least 99 % of the world\u2019s microbial biomass exists in form of biofilm, a complex differentiated surface-associated community embedded in a self-produced polymeric matrix enabling microorganisms to develop coordinated and efficient survival strategies. Biofilm formation is a dynamic and cyclical process involving attachment, maturation and a final dispersal phase, and these steps are initiated by a variety of signals. Despite their positive effects in some cases, biofilms can be detrimental in different environmental domains since microorganisms are able to colonize almost all types of surfaces both abiotic and biotic, leading to consequences in terms of social and economic impact. These include human tissues, implantable medical devices, natural aquatic systems, plants, food and industrial lines. Once biofilm is formed, its eradication becomes difficult because its resilience to environmental stresses, disinfectants, and antimicrobial treatments. Plants support a diverse array of microorganisms that exist in form of biofilms. Even if in some cases the association with plants leads to beneficial interactions promoting plant growth, inducing plant defense mechanisms and preventing the deleterious effects of pathogenic microorganisms, in other cases they have a significant negative impact. For instance, in agriculture, plant colonization of fungi and bacteria in form of biofilm is a cause of plant diseases, affecting crop quality and productivity. Indeed, despite the planktonic growth, biofilm lifestyle improves microbial resistance to antimicrobials up to several orders of magnitude, often reducing the possibility of treating biofilm effectively. In addition, due to the worrisome consequences related to the use of these substances on human health and on their persistence in the environment, increasingly regulations are arising to limit antimicrobial application. Furthermore, in addition to the principles of integrated pest management (IPM) embraced by the worldwide legislation aims to recommend alternative approaches to the application of pesticides, an innovative approach could be the use of biocide-free bioactive compounds characterized by novel targets, unique modes of action and properties that are separate from those currently highlighted in the use of antimicrobials. Indeed, the application of non-lethal doses of bio-inspired molecules able to interfere with specific key-steps involved in the biofilm formation process has been suggested as a complementary/alternative strategy to hinder biofilm formation. In addition, this approach also lead to deprive microorganisms of their virulence factors without affecting their viability and decreasing the selection pressure for biocides resistance. In this PhD thesis, the in vitro effects of non-lethal concentrations of several bioactive compounds were evaluated on the biofilm formation of different plant-associated microorganisms. Specifically, the aim of this work was to provide new effective preventive or integrated solutions against bacterial and fungal biofilm formation. In chapter III, the methanol extracts obtained by different plant portions of three seagrass species collected in Vietnam and in India (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) were investigated for their effects in mediating non-lethal interactions on sessile Escherichia coli and Candida albicans cultures taken as models of bacterial and fungal biofilms respectively. The study was focused on anti-biofilm activities of seagrass extracts, without killing cells. Seagrass extracts appeared to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. Results revealed that E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an anti-biofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes. Methanolic extracts were characterized and major phenolic compounds were identified by MS/MS analysis, showing the unique profile of the E. acoroides leaf extract. In chapter IV, two essential oils (PK and PK-IK) derived from two cultivars of Perilla frutescens, an annual short day plant widely used in therapeutics in the traditional medicine as well as in food preparations in Asian countries. Essential oils were extracted from the leaves and were characterized. Subsequeltly, their ability to affect biofilm formation of the phytopathogenic model fungi Colletotrichum musae, Fusarium dimerum and F. oxysporum have been studied. PK and PK-IK neither inhibited fungal growth nor were they utilized as a carbon energy source. In addition, PK and PK-IK essential oils showed excellent anti-biofilm performances inhibiting conidia germination and reducing conidia adhesion. Furthermore, they revealed a magnificent anti-biofilm effect even during biofilm maturation, affecting biofilm structural development, with a reduction of dried weight, extracellular polysaccharides and proteins. In all cases PK-IK displayed better activity than PK. Thus, the anti-biofilm effects were exploited with a non-lethal mechanism. This research supported the spreading of PK and PK-IK essential oils as biocide-free agents suitable for a preventive or integrative approach for sustainable crop protection. Lastly, in chapter V, a non-lethal concentration of N-Acetylcysteine (NAC) was evaluated on the biofilm formation of Xylella fastidiosa, a phytopathogen bacterium that causes a range of economically important plant diseases worldwide and that has been recently found in Italy in olive plants, where it causes the olive quick decline syndrome (OQSD). NAC is a naturally occurring compound found in several vegetables (including garlic, onion, peppers and asparagus) and it is mostly known in clinical area, in which it is employed at lethal concentrations in the treatment of human diseases due to its ability to reduce bacterial adhesion, inhibit the production of extracellular polysaccharides and promote the dispersion of pre-formed mature biofilms. In this study, N-Acetylcysteine (NAC) was tested for its ability to affect biofilm response of X. fastidiosa CoDiRO strain, mimicking a preventive, a curative and a combination of both approaches. The not-lethal dose 0.08 mg/ml was chosen as representative of plant concentration after its application. NAC did not alter planktonic bacterial growth but promoted biofilm formation in terms of biofilm biomass (above 62 %) and matrix polysaccharides (above 53%) through a ROS-mediated mechanism. Additionally, NAC was not able to destroy X. fastidiosa biofilm when already established on the surface but rather, it was suitable to contain the biofilm infection limiting biofilm dispersal. On the contrary, a combination of both preventive and curative approach has been found promising in biofilm dissolving making it more vulnerable