Microrganismi probiotici per le piante: una strategia di riduzione degli input nella coltura del pomodoro col metodo biologico

In the recent years, consumers became more aware and sensible in respect to environment and food safety matters. They are more and more interested in organic agriculture and markets and tend to prefer ‘organic’ products more than their traditional counterparts. To increase the quality and reduce the cost of production in organic and low-input agriculture, the 6FP-European “QLIF” project investigated the use of natural products such as bio-inoculants. They are mostly composed by arbuscular mycorrhizal fungi and other microorganisms, so-called “plant probiotic” microorganisms (PPM), because they help keeping an high yield, even under abiotic and biotic stressful conditions. Italian laws (DLgs 217, 2006) have recently included them as “special fertilizers”. This thesis focuses on the use of special fertilizers when growing tomatoes with organic methods in open field conditions, and the effects they induce on yield, quality and microbial rhizospheric communities. The primary objective was to achieve a better understanding of how plant-probiotic micro-flora management could buffer future reduction of external inputs, while keeping tomato fruit yield, quality and system sustainability. We studied microbial rhizospheric communities with statistical, molecular and histological methods. This work have demonstrated that long-lasting introduction of inoculum positively affected micorrhizal colonization and resistance against pathogens. Instead repeated introduction of compost negatively affected tomato quality, likely because it destabilized the ripening process, leading to over-ripening and increasing the amount of not-marketable product. Instead. After two years without any significant difference, the third year extreme combinations of inoculum and compost inputs (low inoculum with high amounts of compost, or vice versa) increased mycorrhizal colonization. As a result, in order to reduce production costs, we recommend using only inoculum rather than compost. Secondly, this thesis analyses how mycorrhizal colonization varies in respect to different tomato cultivars and experimental field locations. We found statistically significant differences between locations and between arbuscular colonization patterns per variety. To confirm these histological findings, we started a set of molecular experiments. The thesis discusses preliminary results and recommends their continuation and refinement to gather the complete results.

Mechanism of colonization in the Mediterranean-sea. Asperarca nodulosa: morphological and genetic analysis

Il mar Mediterraneo è un bacino acquifero peculiare per la recente colonizzazione di specie aliene, per l’evento geologico legato alla Crisi di salinità del Messiniano e per l’ampio range di salinità. L’individuazione dei meccanismi di colonizzazione si è incentrata sullo studio morfologico, istologico e molecolare delle specie Asperarca nodulosa ed Anadara demiri (Arcidae-Bivalvia-Mollusca). La ricerca si è basata sulla caratterizzazione morfologica, con utilizzo del microscopio elettronico a scansione, al fine di individuare il tipo di sviluppo larvale. Successivamente i dati rilevati al S.E.M. sono stati supportati dall’indagine istologica che ha evidenziato la presenza di gonadi a sessi distinti e la non incubazione larvale. L’ulteriore analisi filogenetica ha permesso di evidenziare la netta suddivisione tra le tre popolazioni studiate, indagine effettuata tramite marcatori arbitrari (RAPDs) e nucleari specifici (ITS). I risultati ottenuti trovano supporto da quanto noto su base morfologica. I dati, nel complesso, mostrano una perdita delle capacità di diffusione della specie tramite sviluppo larvale plantotrofico a favore di quello lecitotrofico o diretto; tale tesi è ulteriormente supportata dai dati molecolari che mostrano una netta separazione delle popolazioni prese in esame ed un conseguente isolamento tra individui appartenenti a zone di profondità del Mediterraneo (sub-bacini abissali). La ricerca ha, inoltre,esaminato i meccanismi di introduzione attuali nel bacino acquifero che è soggetto ad una nuova invasione da parte specie aliene dovuta all’apertura del canale di Suez. L’analisi si è focalizzata sullo studio per l’ individuazione dell’origine della specie aliena A. demiri , di presunta derivazione Indo-Pacifica, ma rivelatasi, nei dati preliminari, di origine Atlantica.

Biomimetica per l'ingegneria tissutale dell'osso

Reconstruction of bone is needed for high bone loss due to congenital deformities, trauma or neoplastic diseases. Commonly, orthopaedic surgical treatments are autologus or allogenic bone implant or prosthetic implant. A choice to the traditional approaches could be represented by tissue engineering that use cells (and/or their products) and innovative biomaterials to perform bone substitutes biologically active as an alternative to artificial devices. In the last years, there was a wide improvement in biology on stem cells potential research and in biomedical engineering through development of new biomaterials designed to resemble the physiological tissues. Tissue engineering strategies and smart materials aim together to stimulate in vivo bone regeneration. This approaches drive at restore not only structure integrity and/or function of the original tissue, but also to induce new tissue deposition in situ. An intelligent bone substitute is now designed like not only a scaffold but also as carrier of regeneration biomolecular signals. Biomimetics has helped to project new tissue engineered devices to simulate the physiological substrates architecture, such extracellular matrix (ECM), and molecular signals that drive the integration at the interface between pre-existing tissue and scaffold. Biomimetic strategies want to increase the material surface biological activity with physical modifications (topography) o chemical ones (adhesive peptides), to improve cell adhesion to material surface and possibly scaffold colonization. This study evaluated the effects of biomimetic modifications of surgical materials surface, as poly-caprolattone (PCL) and titanium on bone stem cells behaviour in a marrow experimental model in vitro. Two biomimetic strategies were analyzed; ione beam irradiation, that changes the surface roughness at the nanoscale, and surface functionalization with specific adhesive peptides or Self Assembled Monolayers (SAMs). These new concept could be a mean to improve the early (cell adhesion, spreading..) and late phases (osteoblast differentiation) of cell/substrate interactions.

Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Nuove biotenologie per la produzione di piante micorrizate con tartufo

Il lavoro svolto durante questa tesi di dottorato pone le basi per lo sviluppo di nuove biotecnologie della micorrizazione di piante forestali con tartufi pregiati ed in particolare con Tuber magnatum. Durante questa tesi è stato possibile isolare e mantenere in coltura pura il micelio di T. magnatum, ad ottenere e descrivere le sue micorrize e quelle di altri tartufi “bianchi” (T. oligospermum, T. borchii) e a seguire l’evoluzione del micelio nel suolo utilizzando la tecnica della real time PCR. Sono stati disegnati primer specie specifici in grado di identificare T. oligospermum ed è stata verificata la possibiltà di utilizzare questi primers in PCR multiplex con quelli specifici di T. magnatum e di T. borchii già presenti in bibliografia, al fine di “scovare” sia frodi nella commercializzaione degli ascomi sia eventuali contaminazioni nelle piante micorrizate. Per migliorare lo sviluppo miceliare di tartufo abbiamo si è cercato di migliorare il mezzo nutritivo per la crescita del micelio utilizzando: fonti di carbonio diverse, estratti radicali di nocciolo e singole frazioni separate dagli stessi. Infine sono stati sviluppati protocolli di crioconservazione per miceli di tartufo. Gli estratti radicali sono in grado di stimolare le crescita miceliare del tartufo modello T. borchii e dimodificarne la morfologia ifale. Questo risultati sono stati confermati anche dall’aumento dell’espressione di geni CDC42 e Rho-GDI, due geni legati alla crescita apicale polarizzata delle ife dei funghi filamentosi. Inoltre è stato dimostrato che il mantenimento in coltura per numerosi anni dei miceli di tartufo provoca una perdita della capacità d’infettare le radici delle piante e quindi il loro potenziale utilizzo sia a scopo sperimentale sia a scopo colturale. Questo pone in risalto l’importanza della conservazione a lungo termine del materiale biologico a disposizione ed è stato dimostrato che la crioconservazione è applicabile con successo anche alle specie del genere Tuber.

Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.