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Microbial ecology is the study of microbes in the environment and their interactions with each other. Microbes are the smallest and most numerous living organisms on Earth. Despite their small size, natural microbial communities have a huge impact on our environment. They have a key role in biogeochemical cycles (e.g. nitrogen fixation, methane metabolism and sulphur metabolism) and in organic matter degradation. They harbour an amazing physiological versatility and catabolic potential for the breakdown of an enormous number of organic molecules, including xenobiotics, thanks to their great adaptability to different conditions. Natural microbial communities provide several regulating ecosystem services, maintaining soil and water quality1. Soil and water ecosystem recovery from contamination relies on the presence of abundant and diverse microbial communities with the ability to degrade contaminants. Finally, the microbial community structure reflects the impacts of environmental and anthropogenic factors on ecosystems. Even if most natural microbial populations provide benefits to humans (ecosystem services), some microbial processes can have deleterious effects on the conservation of cultural heritage owing to their biodeteriorative potential. This phenomenon is particularly evident in the case of outdoor cultural heritage, which is continuously exposed to abiotic and biotic factors; at the same time, some selected microorganisms and/or enzymes have recently been used for its bio-cleaning and bio-restoration. Microbial ecology makes it possible to study structure-function relationships between microbial communities and their environment at different levels through, for example, the evaluation of microbial abundance, diversity and activity. For example, the knowledge of species involved in the deterioration of cultural heritage can be very useful for managing their conservation, through suitable recovery measures. Trends in modern microbiology emphasize the need to know and understand the structure and function of complex microbial communities. Most types of microbes remain unknown. It is estimated that we know less than 1% of the microbial species on Earth. Yet microbes surround us everywhere - air, water, soil. An average gram of soil contains one billion (1,000,000,000) microbes representing probably several thousand species. Culture-independent molecular techniques are valuable tools for investigating the diversity and structure of bacterial communities. These techniques can be used on cultivable as well as non-cultivable bacteria.

Microorganisms such as Bacteria and Archaea can damage monuments and stone artworks through various mechanisms, including biofilm formation, chemical reaction with the substrate and production of pigments. The biogenic release of inorganic and/or organic corrosive acids is probably the best known and most commonly investigated biogeochemical damage mechanism concerning inorganic materials. For example, aerobic microorganisms producing respiratory carbon dioxide, which becomes carbonic acid, contribute to dissolving stone and forming soluble salts. Moreover, the production of organic acids, such as lactic, oxalic and acetic, has been associated with the dissolution of calcite in calcareous stones. For these reasons, the phylogenetic classification of microorganisms colonizing cultural heritage substrates is very important, in order to detect the taxa involved in biodeterioration and apply the appropriate countermeasures. Fluorescence in situ hybridization (FISH) technique is a rapid and highly valuable culture-independent molecular method for detecting and identifying individual microbial cells in environmental samples using rRNAtargeted oligonucleotide probes. When used for detecting and identifying Cultural Heritage biodeteriogens, the FISH method can be performed in two ways: on powdered samples from the surface studied or more recently on adhesive tape strips applied on the surface and immediately removed. In contrast to other identification approaches, the FISH method largely maintains the characteristics of the targeted microorganisms and makes it possible to have a mirror image of the whole microbial community of a sample.

Livestock and aquaculture activities involve the use of antibiotics. The use of manure and digestate in agricul-tural practices can introduce antibiotics and promote resistant bacteria into terrestrial and aquatic environ-ments. Sulfamethoxazole (SMX) is one of the most commonly prescribed and consumed sulfonamide antibiotic for its ability to inhibit both Gram-positive and Gram-negative bacteria. However, current knowledge about its persistence and effects on natural microbial communities are quite scarce. We investigated in microcosm laboratory experiments the effects on the structure and functioning of a soil natural microbial community treat-ed with cattle manure in presence/absence of the antibiotic SMX (20 mg/kg). At fixed times (0, 7, 13, 20, 60 days) microbiological abundance, viability, activity and SMX residual concentrations (ASE extraction and HPLC-UV detection) were analysed. Moreover, the microbial community diversity was investigated with NGS analyses. The results showed that although SMX had an initial acute negative effect on the cell abundance and viability, the antibiotic was then degraded in just a few days. The microbial community composition was influenced by the different treatments and the effect on different bacterial groups will be shown

Although the use of DDTs has been banned in most Countries over 20 years ago, its residues (DTTr) still persist in the environment. DDE (1,1-dichloro-2,2-bis(4- chlorophenyl)ethylene) is the most toxic DDT metabolite and it is reported to be more persistent than its parental compound (Ortìz and Velasco, 2013). Plant-assisted bioremediation can be a promising and cost-effective clean-up technology to contaminated soil remediation. It relies on synergistic action between plant rhizosphere where microorganisms support toxic substance removal. Solanum lycopersicum is known to be able to accumulate DDTs in roots, owing to exudate production, which make these contaminants more bioavailable for soil microorganisms. Moreover, several natural bacterial strains have been shown to degrade organochlorine pesticide through their enzymes able to break-down complex molecules (Verma et al., 2014; Qu et al., 2015). In this context, a greenhouse experiment was performed to study the effects of tomato plant presence on DDE persistence in an agricultural soil. The experimental set up consisted of pots filled with contaminated soil (1ppm of DDE) in presence/absence of tomato plants and control soils (with/without plant and/or DDE). The plots were sampled 40 days from DDE exposure. The effects of DDE and/or plant presence on the natural microbial community were evaluated in terms of microbial abundance, viability, structure, dehydrogenase activity and DDE residual concentration. The overall results showed that although the plant presence stimulated the soil microbial community activity, it did not increase DDE degradation. On the other hand DDE presence not only did not negatively affect the natural microbial community, but the latter was able to degrade DDE independently from the plant presence, showing a preacquired capability presumably due to a soil previous contact with this pesticide.

Grapevine pinot gris virus (GPGV) has been originally found in Northern Italy associated to symptoms of stunting, chlorotic mottling and leaf deformations in grapevine plants. Successively, its presence has been reported in many countries even on asymptomatic plants. The study of this virus with regard to its biological and molecular characterization, its etiological role and spread by Eryophid mites vectors Colomerus vitis, the symptoms on different grapevine varieties cannot disregard the use of reliable, robust and validated diagnostic methods to implement possible prevention strategies. To this aim, a test performance study (TPS) was carried out to evaluate, harmonize and validate four diagnostic protocols used in the detection of GPGV in symptomatic and asymptomatic grapevine material. Specifically, two end point RT-PCR (Glasa et.al. 2014 - protocol 1; Saldarelli et al. 2015 - protocol 2) and two real time RT-PCR (Bianchi et al. 2015 - protocol 3; Ratti personal communication 2015- protocol 4) protocols have been compared. Obtained performance data from the four molecular protocols are reported and evaluated in the present Abstract. Both diagnostic methods, (end point and real time RT-PCR) were proven to be highly effective, although analytical sensitivity was more efficient for real time protocols, as expected. No statistically significant differences were observed for the other validation parameters. Moreover, all protocols gave satisfactory results even if six samples with low virus concentration were selected to stress the tests. Despite the large number of laboratories that performed the TPS and the different instruments and extraction methods (Nitrogen or different mechanic grinding system), no statistically significant differences were observed in the analytical specificity using the non target samples infected by other viruses than GPGV. All protocols showed good accuracy and robustness as proven using a large number of samples in a high number of laboratories. In conclusion, harmonized and validated reference protocols for the diagnosis of GPGV are, for the first time, available.

Livestock raising in most countries involves the use of antibiotics in veterinary practices and to increase animal productivity ("production-purpose uses") (1. These compounds are designed to be quickly excreted from the threated body, through animal feces, either unaltered or as metabolites. The use of livestock manure or of the digestate (anaerobic digestion of the manure) as amending and fertilizer in agricultural practices, implicates the introduction of antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in terrestrial ecosystem (2. Due to their continual input into the environment and antimicrobial nature, antibiotics are classified as emerging pseudo-persistent organic pollutants (pseudo-POPs) (3. Unlike the other conventional POPs, antibiotics not only can kill or inhibit natural bacterial population involved in ecosystem functioning, but also cause the appearance and rapid spread of resistant bacteria in the environment (4. Indeed antibiotics, ARB and ARGs are recently included in the contaminant list of emerging concern (5. The synthetic compound sulfamethoxazole (SMX) is one of the most popularly prescribed and consumed antibiotics in human and veterinary medicines leading to its presence in aquatic and terrestrial ecosystems. SMX is bacteriostatic, inhibiting the synthesis of folic acid metabolism and causing bone marrow suppression. This compound is among the top ten drugs with high priority identified in the European assessment of pharmaceuticals and personal care compounds (6. The current knowledge about the persistence and the potential effects of SMX addition on the natural microbial community of an agricultural soil are still scarce. The aim of the present study was to evaluate the possible degradation of SMX and the response of the microbial community of a cattle manure amended and SMX-treated soil. Microcosm biodegradation experiments were performed by using microbiologically active (containing the natural microbial community) or sterilized cattle manure amended soil. The role of the natural microbial community on the degradation of the antibiotic and the effects of the latter on the microbial structure and functioning were investigated. Furthermore, a molecular marker for antibiotic resistance (7, the intI 1 gene was also analyzed.

Many recent studies have reported the ubiquitous occurrence of antibiotics in aquatic ecosystems. Antibiotics can both kill natural microbial populations and act as a pressure for the selection of resistant bacteria. Following administration, antibiotics are only partially metabolized and, therefore, a large amount is excreted unaltered or as active metabolites via urine and faeces; consequently, human antibiotics reach wastewater treatment plants. The latter are not specifically designed for antibiotic removal, and consequently these molecules are released directly into the receiving environments. The fate and effects on health ecosystem of these compounds is not still well known. Sulfamethoxazole (SMX) is one of the most widely synthesized sulfonamides, prescribed to treat urinary infections; it is also used in veterinary practice, aquaculture and livestock breeding. Its mechanism of action is based on the inhibition of folic acid synthesis in bacteria. SMX has been detected in groundwater, in effluents of WWTPs and in surface water, including drinkable water. SMX has been reported to be not readily biodegradable, resistant to hydrolysis and the photo-degradation as a possible degradation process in surface waters. Data on biodegradation in natural water ecosystem are quite scarce. In this context, the aim of the present work was to investigate the persistence of the antibiotic sulphametoxazole in river water, focusing on both the biodegradation and photodegradation processes. For this purpose, two different microcosm experiments were set up using river water treated with 500 µg/L of SMX in the presence or absence (river water sterilized) of the natural microbial community. In order to evaluate SMX biodegradation, the first experiment was performed in the dark. In the second one, river water microcosms were incubated under UV-light, in order to evaluate the possible photodegradation of SMX. At fixed times, water sample were collected from microcosms for measuring SMX residual concentrations over time by-using HLPC-UV. The disappearance time of 50% of the initial SMX concentration (DT50) was evaluated in both experiments. Moreover, the effects of the antibiotic on the natural microbial community were assessed in terms of cell vitality, abundance and phylogenetic diversity comparing the treated river water microcosms with those no treated one.

Contamination by Persistent Organic Pollutants such as DDTs (p´p´-DDT and its metabolites p´p´-DDE and p´p´-DDD) is an important threat to soil ecosystem quality. Plant-based clean up technologies are as sustainable solutions to contaminated soil remediation. They rely on the physiological abilities of selected plants to decrease pollutants from soil through different processes occurring in the rhizosphere involving symbiotic and non-symbiotic interactions with natural microorganisms. Solanum lycopersicum is known to be able to accumulate DDTs in roots, owing to exudate production, which makes this contaminant bioavailable in soil. In this context, a greenhouse experiment was performed using pots filled with DDE-contaminated soil and watered with different kinds of dissolved organic carbon (DOC). Control soils (without plant and/or DDE) were also performed. Finally, we obtained 12 different conditions. Soil and plant samples were collected 40 days after the DDE exposure. The effects of the different treatments on the natural microbial community of the rhizosphere were evaluated in terms of microbial abundance, viability, structure, dehydrogenase activity and possible DDE degradation. Moreover, roots and shoots of the tomato plants were also sampled and analysed for the determination of the pesticide bioconcentration. The main results from the different experimental conditions will be discussed.

Sulfonamide antibiotics are currently prescribed to treat urinary infections in human and veterinary therapeutics. Following administration, they are only partially metabolized and, therefore, a large amount is excreted unaltered or as active metabolites via urine and faeces; in the case of human administration, the antibiotic residues can reach wastewater treatment plants and, through effluents, the receiving waterbodies. Currently, there is little known about the fate and effects on health ecosystem of these compounds. Among Sulfonamides, sulfamethoxazole (SMX) is one of the most detected antibiotics in river waters. SMX disrupts the folate biosynthetic pathway in bacteria, which is identical to that of plants, raising concerns about toxicity effects on non-target organisms. Therefore, improving our knowledge on the persistence of SMX, especially in natural water ecosystems, is particularly needed. In this framework, macrophyte aquatic plants, such as Lemna minor L, growing in wetlands and contaminated water bodies, can be a useful tool to evaluate the interaction between aquatic plants and organic pollutants. Moreover, the plant-natural microbial community interactions on the antibiotics biodegradations have to take into accounts. In this context, the aim of this study was to improve our knowledge on the biodegradability of SMX in river water, focusing on the effect of the antibiotic on both the natural microbial community and plants. Lab-scale experiments were set up by using microcosms containing river water treated with 500 ug/L of SMX both in the presence/absence of the plant L. minor and in presence/absence (sterilized river water) of the natural microbial community. Other microcosms (Controls) were set up with no-treated river water (without the antibiotic). The experimental set was kept in a growth chamber under controlled conditions. Water samples were collected at fixed times and the SMX residual concentrations were measured over time, by a chromatographic method (HPLC-UV). The effects of the antibiotic on the natural microbial community were assessed in terms of cell vitality, abundance and phylogenetic diversity. Moreover, physiological and biochemical determinations were performed on plants. SMX residual concentrations in plants were also analyzed at the end of the experiment; in this case, Accelerated Solvent Extraction (ASE) followed by Solid Phase Extraction (SPE) purification methods were used.

Plant-based clean up technologies are gaining popularity as sustainable solutions to contaminated soil remediation. In particular, plant-assisted bioremediation or phyto-assisted bioremediaton exploits the synergistic action between plant root system and natural microorganisms (bacteria and fungi) to remove, convert or contain toxic substances in soil and water. It relies on the use of a selected appropriate plant for stimulating in the rhizosphere (e.g. through root exudates production, oxygen transport) the biodegradation activity of natural soil microorganisms. In this context, a poplar-assisted bioremediation strategy has been applying for three years to a multi-contaminated (PCBs and heavy metals) area in Southern Italy using a specific poplar clone (Monviso). It was chosen thanks to its capabilities previous tested for promoting hexachlorocyclohexane degradation. At selected times (0, 420, 900 days) PCB and heavy metal (HMs: V, Cr, Sn, Pb) concentrations were assessed on soil samples at different depths and distance from tree trunks inside some contaminated plots. Similarly, microbial analyses were performed on soil samples to assess total microbial abundance, cell viability, dehydrogenase activity and the phylogenetic composition of the autochthonous microbial community. Three years after the poplar planting a significant decrease in PCB and HMs concentrations was observed. Currently, the values of all PCBs detected are under the Italian legislation limits in the plots investigated. The microbiological analysis show an overall improvement in soil quality in terms of an increase in microbial abundance, cell viability and organic carbon content in the rhizosphere soil samples. Moreover, the phylogenetic analysis of the microbial community showed a higher percentage of Bacteria in the rhizosphere than in the bulk soil. In particular, a significant increase in Actinobacteria and Alpha-Gamma-Proteobacteria, which include several speciesable to degrade PCBs, was observed. Overall results show that the poplar-assisted bioremediation strategy was able to promote both the persistent organic contaminant degradation and the phytostabilization of the inorganic ones.

The anionic surfactant sodium lauryl ether sulphate (SLES) is the main component of most commercial products used for soil conditioning in the excavation industry, in particular as lubricants for mechanized tunnelling. Its use during the excavation processes can result in either the subsequent possible re-use of the huge amount of soil debris as by products (e.g. land covering) or its discharge as waste. Currently, there are neither SLES soil threshold limits in European legislation, nor comprehensive studies on the environmental risk for soil ecosystems in these exposure scenarios. Although SLES is generally reported to be biodegradable in standard tests, data on its biodegradation in environmental studies are quite scarce. Consequently, assessing SLES biodegradation rates in field conditions is crucial for evaluating if in residual concentrations it can or not be a potential hazard for terrestrial and water organisms. In this context, a set of microcosms was set up using soil samples from a construction site. Soil samples were treated separately with one of two commercial products (P1 and P2) in absence/presence of lime at the concentrations used for the mechanized drill. Lime treatment is used because chemically transforms unstable soils into usable materials, making it possible their appropriate use for land covering. At selected times (0, 7, 14, 21, 28 d) soil samples were collected for assessing SLES concentration in the different conditions. Moreover, microbiological analysis were performed in order to assess microbial abundance, cell viability, dehydrogenase activity and microbial structure. The SLES degradation rates were higher when SLES was present in the P1 (DT50=11 d) foaming agent than in the P2 (DT50= 17d) one. Lime addition slowed down SLES halving in P1 (DT50= 31 d), while inhibited its degradation in P2. In line with these results, the bacterial community was differently affected by the foaming agent P1 or P2 both in presence and absence of the lime. The overall results show SLES as a biodegradable compound and that the different formulation of each specific foaming agents (P1 or P2) can affect its persistence.

Plant-assisted bioremediation, the use of plants to promote the extraction, the sequester or detoxification of pollutants through biological processes is a green, effective, in situ, non intrusive, low cost, aesthetically pleasing and, socially accepted technology to remediate contaminated soil. Potential for plant-assisted bioremediation depends upon the interactions among, soil, contaminants, microorganisms (bacteria and microfungi) and plants occurring in the rhizosphere where, the release of root exudates and oxygen with circadian rhythm can stimulate the biodegradation activity of natural soil microorganisms. This technology has been applying for three years to a multi-contaminated (PCBs and heavy metals) area in Southern Italy using as plant a selected poplar clone (Monviso).

I protocolli diagnostici descritti in questo volume sono il prodotto dell'attività effettuata nell'ambito del Progetto Finalizzato 'ARON-ARNADIA', finanziato dal Ministero per le Politiche Agricole, Alimentari e Forestali (DM 19738/7303/08 del 29/12/2008). I protocolli forniscono le linee guida per la diagnosi e l'identificazione di patogeni delle piante (funghi, batteri, fitoplasmi, virus e viroidi) nei laboratori preposti alla diagnosi degli organismi di qualità e/o quarantena, presenti sul territorio italiano. L'uso di protocolli diagnostici armonizzati è alla base di un'efficiente applicazione delle misure fitosanitarie e consente il confronto dei risultati ottenuti da diversi laboratori in diverse condizioni operative. Le metodologie di laboratorio riportate nei protocolli di questo volume sono state selezionate sulla base del valore, ottenuto per ciascuna di esse, dei parametri di sensibilità diagnostica (inclusività per le colture batteriche), specificità diagnostica (esclusività per le colture batteriche), selettività, accuratezza, sensibilità analitica (limite di rilevabilità), specificità analitica, ripetibilità e riproducibilità (ISO/IEC standard 16140:2003; ISO/IEC standard 17025:2005; EPPO standard PM7/98).

Il Ministero per le Politiche Agricole, Alimentari e Forestali (MiPAAF) nel 2009 ha finanziato il Progetto "ARNADIA" finalizzato alla produzione di protocolli diagnostici, armonizzati e validati, che fossero di riferimento per il controllo e il monitoraggio dei patogeni vegetali di interesse fitosanitario. In questo ambito, è stato costituito il "Gruppo di Lavoro ARNADIA - virus della vite" composto da 8 Università ed Enti di Ricerca, 3 Laboratori privati accreditati, un Laboratorio dei Servizi Fitosanitari e un'Associazione di vivaisti viticoli. L'obiettivo del Gruppo di Lavoro è stato quello di produrre protocolli diagnostici di riferimento, sia sierologici che molecolari, calcolandone i parametri di validazione al fine di ottenere l'armonizzazione della diagnosi di 8 virus vite: Grapevine leafroll-associated virus-1, -2, -3, (GLRaV 1, 2, 3 ) Grapevine virus A (GVA), Grapevine virus B (GVB), Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) e Grapevine fleck virus (GFkV). La validazione di un protocollo consiste nella valutazione di specifici parametri volti a determinare la loro idoneità per identificare la presenza di un "target" specifico. I parametri che influenzano la capacità di un protocollo di definire accuratamente lo stato sanitario del campione in esame sono: la sensibilità diagnostica (capacità del metodo utilizzato di rilevare la presenza del patogeno nei campioni veramente infetti dal patogeno in questione - veri positivi) e la specificità diagnostica (capacità del metodo utilizzato di non per rilevare la presenza del patogeno in campioni non infetti dal patogeno in questione - veri negativi). Altri parametri che devono essere considerati e che determinano l'efficienza di un protocollo sono: la sensibilità analitica (la più piccola quantità di entità infettiva che può essere identificata dal protocollo), la ripetibilità o accordanza (grado di conformità dei risultati ottenuti nella replicazione del protocollo, in intervalli di tempo brevi, utilizzando gli stessi campioni di riferimenti nelle stesse condizioni di lavoro cioè apparecchiature, operatore, laboratorio) e riproducibilità o concordanza (grado di conformità dei risultati ottenuti con lo stesso metodo con gli stessi campioni di riferimento in diversi laboratori). Quest'ultimo parametro è stato definito con la collaborazione di altri 5 laboratori dei Servizi Fitosanitari Regionali. Nello specifico, 122 campioni di vite (varietà, portainnesti e campioni "pool" composti da 5 piante, di cui solo una infetta) sono stati analizzati mediante ELISA, utilizzando 25 antisieri di tre società commerciali (Agritest, Bioreba e Sediag) e multiplex RT-PCR. Per l'ELISA i test sono stati condotti seguendo le indicazioni fornite dalle Ditte; per la multiplex RT-PCR è stato utilizzato il protocollo descritto da Gambino e Gribaudo, (2006). I test sono stati eseguiti in 18 laboratori utilizzando gli stessi campioni (analizzati in condizioni di anonimato) e reagenti; i risultati sono stati ottenuti utilizzando lo stesso valore di soglia calcolato sulla base delle letture spettrofotometriche per l'ELISA e analizzando i gel elettroforetici per la multiplex RT-PCR. L'elaborazione dei risultati (circa 24.000 dati) ha portato alla definizione dei parametri di validazione secondo le norme UNI / EN / ISO 16140 e 17025 e gli standard EPPO PM7/76 e PM7/98.L'ELISA ha dimostrato di essere una tecnica molto efficace, paragonabile al metodo molecolare, anche se quest'ultimo si è rivelato, come previsto, più efficiente e sensibile per alcuni virus e su alcuni campioni specifici (portainnesti e "pool"). L'efficienza e l'affidabilità dei protocolli sono stati provati e dimostrati, per la prima volta per quanto riguarda la diagnosi dei virus della vite, utilizzando un gran quantità di campioni in un ampio panorama di laboratori. Sulla base dell'attività svolta e dei risultati ottenuti, si può affermare che sia l'ELISA che la multiplex RT-PCR si sono dimostrati altamente validi, e il loro impiego ottimale è in funzione delle diverse esigenze e scopi diagnostici.

The Italian Ministry of Agriculture funded in 2009 the "ARNADIA" Project, aimed at producing validated reference diagnostic protocols for the control and monitoring of plant pathogens of phytosanitary interest and, among them, grapevine viruses. In this framework, the "Working group ARNADIA-grapevine viruses (WG)", composed of eight universities and research bodies, three accredited private laboratories, one plant health service and one association of grapevine nurseries, was established.