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Among the hypogeous ectomycorrhizal fungi, the white truffle Tuber magnatum Picco is the species of greatest interest, both from an ecological and economic point of view. The increasing market demand of the precious white truffle along with the fall in its natural production led to a growing interest in cultivation techniques and encouraged truffle growers and researchers to deeper investigate factors that could affect and improve T. magnatum productivity. In this context, microbial communities play a central role. Indeed, in the last few years, the hypothesis of a potential link between microbial community composition and truffle orchard productivity is arousing a greater attention. Moreover, since the value of the prized T. magnatum can vary in relation to its provenience, the need to define a reliable tracking system is also emerging and bacteria appear to be a promising tool. Accordingly, the present mini-review summarises the knowledge currently available on T. magnatum microbial communities, focusing on the role of truffle-associated bacteria and highlighting similarities and differences between samples of different origin, to address the following issues: (i) Is there a correlation between microbial taxa and truffle ground productivity? (ii) Can bacteria actually be used as markers of T. magnatum geographic origin? The identification of microorganisms able to promote T. magnatum formation may represent an important advance in the field of truffle farming. Similarly, the detection of bacterial taxa that can be used as markers of T. magnatum origin could have a considerable impact on truffle industry and trade, even at local scale.

Several studies focused on Tuber magnatum, the most valuable truffle species, with a limited geographical distribution. However, no comprehensive information on the thickness of its peridium (the external surface of fruiting bodies) is available. Accordingly, to fill this lack of information and in order to provide a more in-depth morphological characterisation of white truffle populations from Molise region (one of the Italian richest areas of truffles), the peridium thickness of twenty-one T. magnatum ascomata collected from two different study sites was measured by light microscope observations. A considerable variability within the analysed populations emerged, with values ranging from 271.25 µm (minimum) to 1231.25 µm (maximum), and an average peridium thickness of 622.33 µm. Interestingly, significant differences were observed between the two groups, with truffles harvested in an inner area of the region showing a peridium significantly thinner than those collected on the border with Abruzzo region. No linear correlation between peridium thickness and other morphological parameters (ascoma weight and maturity) emerged. It is likely that the differences observed between the two T. magnatum populations could be related to factors such as soil characteristics, site-specific features, genetic traits, as well as truffle collection period, which should be properly investigated., Italian Journal of Mycology, Vol. 50 (2021)

The aim of this study was to evaluate, at site scale, the influence of freezing and freeze/thaw cycles on the survival of fecal coliforms and fecal enterococci in soil, in a climate change perspective. Viable cells of both fecal indicators were counted in ten different soil profiles and at different depths, before and after a winter season. Before the winter period and during grazing, viable cells of fecal coliforms and fecal enterococci were detected only in the first 10 cm below ground, while, after the winter period and before the new seasonal grazing, a lower number of viable cells of both fecal indicators was detected only in some of the soil profiles, and within the first 5 cm. Thus, a significant decrease in viable cells was observed in all soil profiles, due to cold shock, freezing, prolonged exposure to subzero temperatures, and alternating freezing and thawing periods, even though this decrease was not uniform at site scale. Taking into consideration the results of specific investigations, we hypothesised that the non-uniform spatial distribution of grass roots within the studied soil ccould play an important role in influencing this phenomenon, while several abiotic factors, such as organic matter, grain size and specific weight of particles, total porosity, and relative density, did not play any significant role. In a climate change perspective, taking into account also the local trend in air temperature, a different distribution of microbial pollution over time is expected in spring waters, and a higher risk of transmission of infections is expected throughout the year. The progressive increase in air temperature, with emphasis on the minimum one, will cause a progressive decrease in freezing and freeze/thawing at higher altitudes, therefore minimising cold shocks on microbial cells, and causing spring water pollution also during winter, differently from the actual observations.

L'obiettivo della ricerca è stato quello di analizzare l'influenza di un suolo di origine piroclastica sulla contaminazione microbiologica delle acque sotterranee in un acquifero carbonatico dell’Appennino meridionale. La ricerca è stata effettuata attraverso prove in colonna in laboratorio su campioni di suolo indisturbati prelevati nei siti sperimentali. Tutti gli esperimenti sono stati effettuati utilizzando ceppi batterici antibiotico resistenti, per distinguerli dal background batterico naturalmente presente nel suolo. In una prima fase della ricerca è stata analizzata la ritenzione, nel suolo, dei principali indicatori di contaminazione microbiologica di origine fecale, Escherichia coli ed Enterococcus faecalis. Da tale fase della ricerca è emerso che il suolo analizzato è in grado di ritenere in modo significativo sia E. coli, sia E. faecalis. Risultati delle prove in colonna suggeriscono che E. coli è ritenuto maggiormente rispetto a E. faecalis, anche se questa differenza è risultata statisticamente significativa solo in due dei sei campioni di suolo analizzati. In una seconda fase della ricerca è stato effettuato uno studio comparativo sull’adsorbimento delle cellule di E. coli in due diversi suoli piroclastici prelevati in due siti sperimentali dell’Appennino carbonatico dell’Italia meridionale. Sono stati utilizzati due diversi surfattanti, sodio dodecil solfato, (SDS, surfattante anionico), e octylphenoxypolyethoxyethanol, (Triton X100, surfattante non ionico). Entrambe le tipologie di suolo sono ricche di materia organica (fino al 35%), ma solo uno contiene una significativa percentuale di argilla (2-5%). Gli esperimenti hanno dimostrato che cellule di E. coli sono significativamente adsorbite alla frazione argillosa presente nel suolo, mentre il contenuto di materia organica non gioca un ruolo significativo. In una terza fase della ricerca è stata analizzata l'influenza del pH delle acque di precipitazione sul trasporto delle cellule di E. coli e delle spore di Bacillus subtilis. Le prove in colonna sono state effettuate a 3 differenti valori di pH (1.5, 5.6, 8.0). Nonostante le diverse condizioni sperimentali, sono stati ottenuti risultati comparabili in tutte le prove effettuate. E’ stato osservato che la ritenzione di entrambi gli indicatori, cellule di E. coli e spore di B. subtilis, è indipendente dal pH delle acque piovane. Questo fenomeno è dovuto ad un’alta capacità tampone del suolo studiato, a sua volta funzione di un elevato contenuto in carbonati. The aim of the research was to analyse the influence of a topsoil of pyroclastic origin on microbial contamination of groundwater in a carbonate aquifer in southern Italy. The research was carried through column tests in soil blocks collected randomly in experimental sites. All experiments were performed using antibiotic-resistant bacterial strains, to differentiate them from background bacteria naturally present in topsoil. In a first phase of the research the retention of Escherichia coli and Enterococcus faecalis through the topsoil was analyzed. Both microorganisms were significantly retained, but retention of E. coli cells was more efficient than that of E. faecalis cells in some of the analyzed soil blocks. The second phase of the research demonstrated that retention of E. coli cells in pyroclastic soil is not significantly influenced by adsorption of cells to organic matter, despite the high organic matter content of such soil (up to 35%). Conversely, E. coli cells were significantly adsorbed to the clay fraction of the same soil. These results were obtained through column test that were carried out using different surfactants: sodium dodecyl sulphate, (SDS, anionic), and octylphenoxypolyethoxyethanol, (Triton X100, non-ionic). The third phase of the research demonstrated that transport of E. coli cells and spores of Bacillus subtilis is not significantly influenced by rainwater pH. This is due to the high buffering capacity of the soil. Dottorato di ricerca in Ambiente e Territorio (XXII ciclo)

L’obiettivo del lavoro è stato quello di analizzare le comunità microbiche in acquiferi caratterizzati da differenti peculiarità idrochimiche (acque oligominerali fredde; acque saline ed ipersaline fredde; acque saline calde) e da differenti fattori di condizionamento del circuito delle acque sotterranee. Ciò al fine ultimo di verificare sperimentalmente se le suddette comunità possono essere utilizzate efficacemente come nuovi traccianti naturali per studiare alcune dinamiche idrogeologiche in contesti a differente grado di complessità. L’obiettivo è stato conseguito mediante l’utilizzo di due differenti tecniche di fingerprinting genetico: DGGE e T-RFLP. La DGGE permette di risolvere, separare frammenti di DNA della stessa dimensione, basandosi esclusivamente sulle differenze nella sequenza nucleotidica. La T-RFLP è basata sulla digestione con endonucleasi di restrizione di prodotti di PCR; uno o entrambi i primers utilizzati sono legati ad una molecola fluorescente che permette di rilevare, mediante elettroforesi capillare, solo la presenza del frammento di restrizione terminale. I risultati principali conseguiti nel corso del dottorato possono essere sintetizzati come segue. All’interno degli ambienti acquatici analizzati è stata riscontrata la presenza di comunità microbiche “sito-specifiche”, piuttosto che “sistema-specifiche”, ed il carattere “sito-specifico” di tali comunità è apparso fortemente influenzato anche da fattori ambientali arealmente poco rappresentativi nell’ambito dei sistemi acquiferi esaminati. Ciò porta a ritenere che, contrariamente a quanto riportato nella recente letteratura, non esista necessariamente un’unica comunità microbica autoctona acquatica all’interno di sistemi naturali che possono essere considerati un “unicum” dal punto di vista idrogeologico. Dal punto di vista metodologico è emerso che l’attesa frammentazione e diversificazione delle comunità microbiche all’interno di un sistema acquatico può essere accertata e caratterizzata soprattutto mediante il prelievo di campioni in punti ubicati anche all’interno del sistema stesso e non esclusivamente presso il collettore principale (ad esempio, una sorgente) del corpo idrico sotterraneo. The aim of the research was to characterize microbial communities in aquifers with different hydrochemical properties (oligomineral cold waters; saline and hypersaline cold waters; saline hot waters) and different factors affecting the hydrogeological behaviour of the aquifer systems. The goal of the work was to experimentally verify if microbial communitites can be used as new effective natural tracers to analyze hydrogeological phenomena within different aquifer systems, characterized by different degrees of complexity. Two fingerprinting techniques have been used: DGGE and T-RFLP. DGGE is based on the separation of PCR amplicons of the same size but with different sequences. T-RFLP is based on the digestion with restriction endonucleases of PCR products: one or both the primers used in the PCR reaction are labeled with a fluorescent dye which allow to detect, through capillary electrophoresis, only the presence of the terminal restriction fragment. The main results obtained can be summerized as follows. In the aquatic environments analyzed, “site-specific” microbial communities were detected, instead of “system-specific” communities. The character “site-specific” of these communities is strongly influenced even by environmental factors little representative in the general context of the aquifers examined. Differently from the existing knowledge, this leads to the possible absence of single autochtonous microbial communities also in natural systems that can be considered as an “unicum” from a hydrogeological point of view. From a methodological point of view, the fragmentation and diversification of microbial communities in an aquatic system can be determined and characterized especially sampling at different points inside the system itself and not solely at the principal collector (for example, a spring) of the groundwater system. Dottorato di ricerca in Ambiente e Territorio (XXII ciclo)