Fungi Occurrence in Ready-to-Eat Hazelnuts (Corylus avellana) From Different Boreal Hemisphere Areas.

The present study evaluated the fungal contamination of ready-to-eat dried hazelnuts considering for the first time the application of the same condition drying process of several hazelnut cultivars from different boreal hemisphere areas. Fifty lots of hazelnuts (Corylus avellana), belonging to eight cultivars from seven regions in four countries, were analyzed for fungal microbiota, describing both load levels and species diversity. For this purpose, a polyphasic approach consisting of morphological examination (optical and scanning electron microscope observation) and molecular characterization [PCR-DGGE analysis and sequence analyses of the internal transcribed spacer (ITS)] was performed. The results show that different fungal populations occur in dried hazelnuts regardless of their geographical area of production. Although some varieties appear to be relatively less susceptible, species related to Aspergillus, such as A. commune and A. ochraceus, Penicillium, including P. commune, P. solitum, and P. expansum, and Rhizopus, for instance, R. stolonifer and R. oryzae, have generally been found. A related character "hazelnut cultivar-fungi" was found for species related to the genera Trichoderma and Fusarium, including F. oxyxporum, F. solani, and F. falciforme. All 14 species found are known to host pathogenic strains. Therefore, their presence in a ready-to-eat product, such as dried hazelnuts, can pose a real danger to the consumer. Based on these considerations, the development of new protective strategies seems highly desirable. The species-level description of the contaminating fungal community acquired through this study is the starting point for the development of tailor-made protective biotechnologies.

Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses.

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis-CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

Low-Fat and High-Quality Fermented Sausages.

The present study, considering for the first time microbiological concerns due to the use of lemon albedo as a fat replacer, aimed at the selection of an anti-Listeria strain to be used as protective culture in low-fat southern Italian fermented sausages. In fact, these kinds of products require appropriate bio-protective strategies to avoid risks due to Listeria monocytogenes. Sixty-seven Lactiplantibacillus plantarum strains isolated from diverse sources were screened for their antimicrobial activity and their interaction with starter strains (Latilactobacillus sakei 152 and Staphylococcus xylosus MVS9). Lactiplantibacillus plantarum Lpls100, highlighting both listericidal activity and the ability to promote Staphylococcus xylosus MVS9 growth, was used as a protective strain in low-fat fermented sausages prepared with lemon albedo as a fat replacer. The effect of the albedo and the protective strain on the fermentation process and the final quality was ascertained. Results highlighted that the use of the albedo did not affect the growth of starter strains and enhanced some quality features, such as fatty acid profiles and certain sensory attributes. However, the albedo also produced a slow decrease in water activity, compromising the microbial quality. The anti-Listeria strain, enhancing coagulase negative cocci growth and exerting antimicrobial activity, avoided the inconveniences caused by the use of the albedo. Moreover, the anti-Listeria effectiveness was assessed through a challenge test using a Listeria cocktail. The study revealed that Lactiplantibacillus plantarum Lpls100, regardless of the presence of the albedo, assures a prompt inhibition of Listeria spp. Therefore, its use could be an important contribution to the quality of low-fat fermented sausages.

Unraveling the Composition of the Root-Associated Bacterial Microbiota of Phragmites australis and Typha latifolia.

Phragmites australis and Typha latifolia are two macrophytes commonly present in natural and artificial wetlands. Roots of these plants engage in interactions with a broad range of microorganisms, collectively referred to as the microbiota. The microbiota contributes to the natural process of phytodepuration, whereby pollutants are removed from contaminated water bodies through plants. The outermost layer of the root corpus, the rhizoplane, is a hot-spot for these interactions where microorganisms establish specialized aggregates designated biofilm. Earlier studies suggest that biofilm-forming members of the microbiota play a crucial role in the process of phytodepuration. However, the composition and recruitment cue of the Phragmites, and Typha microbiota remain poorly understood. We therefore decided to investigate the composition and functional capacities of the bacterial microbiota thriving at the P. australis and T. latifolia root-soil interface. By using 16S rRNA gene Illumina MiSeq sequencing approach we demonstrated that, despite a different composition of the initial basin inoculum, the microbiota associated with the rhizosphere and rhizoplane of P. australis and T. latifolia tends to converge toward a common taxonomic composition dominated by members of the phyla Actinobacteria, Firmicutes, Proteobacteria, and Planctomycetes. This indicates the existence of a selecting process acting at the root-soil interface of these aquatic plants reminiscent of the one observed for land plants. The magnitude of this selection process is maximum at the level of the rhizoplane, where we identified different bacteria enriched in and discriminating between rhizoplane and rhizosphere fractions in a species-dependent and -independent way. This led us to hypothesize that the structural diversification of the rhizoplane community underpins specific metabolic capabilities of the microbiota. We tested this hypothesis by complementing the sequencing survey with a biochemical approach and scanning electron microscopy demonstrating that rhizoplane-enriched bacteria have a bias for biofilm-forming members. Together, our data will be critical to facilitate the rational exploitation of plant-microbiota interactions for phytodepuration.

Cd and Cu accumulation, translocation and tolerance in Populus alba clone (Villafranca) in autotrophic in vitro screening.

The present study investigated accumulation, translocation and tolerance of autotrophic Populus alba clone "Villafranca" in response to excess concentrations of cadmium (Cd) and copper (Cu) provided to the plants. For this purpose, increasing concentrations of Cd (0, 5, 50 and 250 µM) and Cu (0, 5, 50, 250 and 500 µM) were administered to the growth medium in which micropropagated poplar plantlets were exposed to metal treatments for 15 days. Filter bags, instead of the conventional in vitro screening, were applied to improve the experimental design. Results showed that Cd and Cu increased in shoots and roots at increasing metal concentration in the medium. The highest Cd content was found in leaves, while the highest Cu content was found in roots. In "Villafranca", Cu showed toxic effects on the development of the seedlings, especially at the highest concentrations, reducing plant dry mass. However, the tolerance index (Ti) indicated good tolerance in this clone under exposure to excess metal concentrations, whereas plants had higher translocation factor (Tf). We recommend in vitro selection of tolerant genotypes, aimed at providing early indication on accumulation potentiality and tolerance capability in research on plant sensitivity to excess heavy metal concentrations.

Survival of Coliform Bacteria in Virgin Olive Oil.

Coliform bacteria consist of both nonpathogen commensal and human opportunistic pathogen species isolated from different habitats like animals, man, vegetables, and water. Olives normally carry natural nonpathogenic epiphytic bacteria, but during growth, harvest, and processing, one of the final products, represented by virgin olive oil, can be contaminated with coliform. Present study showed that coliform bacteria can survive and reproduce in virgin olive oil containing low level of phenolic compounds. The laboratory inoculation trials demonstrated that when the bacterium Escherichia coli, isolated from the olives carposphere, was transferred in olive oil containing high polar phenols content, equal to 372 mg caffeic acid equivalent per kg, the survival was completely inhibited after 15 days of storage. On the contrary, the bacterium reproduced quickly when it was inoculated in virgin olive oil samples containing lower concentration of polar phenols. The SDS-PAGE analysis of the E. coli proteins showed different electrophoretic patterns when the bacterium was inoculated in the virgin olive oil with high phenolic compounds content, confirming the strong interaction between the olive oil phenols content and the bacterial wall proteins. The SEM ultrastructural observations confirmed the presence of a more higher number of damaged microbial cells in virgin olive oil rich of polar phenols. This finding needs further studies since, in an era of antibiotic resistance, the development of new strategies to fight unwanted food bacteria is promising way for the future.

Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool to improve the shelf life of fresh black truffles.

Refrigeration alone or in combination with other technologies represents the main tool used in the last decades to preserve the freshness of black truffles. This is principally due to the delicateness and vulnerability of this edible hypogeous fungus, so that other invasive preservation practices cannot be adopted. However, the proliferation of some microbial species during the cold storage still represents an unsolved problem. Pseudomonads are among the main spoiler bacteria responsible for the deterioration of refrigerated black truffles. Their growth ability at low temperatures requires the use of additional hurdles to prolong the shelf-life of truffles without altering their major features. The use of natural compounds may represent an alternative system for the biocontrol of this kind of product. Specifically, gallic acid (GA) is a phenolic acid naturally present in different foods, whose effectiveness was in vitro demonstrated against Pseudomonas spp. In our study, we reported the antimicrobial activity expressed by GA not only in vitro, using as target bacteria Pseudomonas putida DSMZ 291T, P. fluorescens DSMZ 50090T, P. fragi DSMZ 3456T and Pseudomonas spp. P30-4, previously isolated from black truffles, but also in situ on fresh black truffles stored at 4°C for 28days. Our results showed Minimum Inhibitory Concentrations (MIC) of 2.5mg/mL GA for all tested strains, except for P. fluorescens DSMZ 50090T, having a MIC corresponding to 5mg/mL GA. The Minimum Bactericidal Concentration (MBC) was 10mg/mL for all strains. The analysis of kinetic parameters showed that the survival declined passing from 2.5 to 10mg/mL GA concentrations, with P. fluorescens confirmed to be the most resistant strain. Moreover, images obtained from Scanning Electron Microscopy revealed that Pseudomonas cells were strongly injured by the treatment with GA at 2.5mg/mL concentration, displaying visible pores on the cellular surfaces, absence of flagella and lysis with loss of cytoplasmic material. The storage test performed on fresh black truffles confirmed in situ the GA antimicrobial activity observed in vitro, with a drastic reduction not only of Pseudomonas spp., but also of the other assessed microbial groups, including Enterobacteriaceae and Eumycetes. Finally, sensory analysis established the absence of off-flavours and the preservation of positive features in black truffles treated with 2.5mg/mL GA and stored for 28 d at 4°C. The results obtained in this study suggest that GA is a potential biocontrol tool to decontaminate and preserve fresh black truffles during refrigerated storage.

Detection of different microenvironments and Lactobacillus sakei biotypes in Ventricina, a traditional fermented sausage from central Italy.

The present study evaluated the physico-chemical and microbiological features of Ventricina, considering for the first time the presence of different compartments deriving from the technology of production. In fact meat pieces (pork muscle and fat cut into cubes of about 10-20cm3), mixed with other ingredients and then stuffed into pig bladder, are still distinguishable at the end of the ripening. They appear delimited on the outside by the casing and inside by thin layers consisting of spices (mainly red pepper powder), salt and meat juices. Our results showed that the exterior (portion of the product in contact with the casing), the interstice (area between the different cubes of meat or fat) and the heart (the inner portion of meat cubes) had distinctive values of pH and aw, and a typical microbial progression, so that they can be considered as different ecological niches, here called microenvironments. The study of lactic acid bacteria population, performed with PCR-DGGE and sequence analysis targeting the V1-V3 region of the 16S rRNA gene (rDNA), highlighted the presence of a few species, including Lactobacillus sakei, Lb. plantarum, Weissella hellenica and Leuconostoc mesenteroides. The RAPD-PCR analysis performed on Lb. sakei, recognised as the predominant species, allowed the differentiation into three biotypes, with that characterised by the highest acidifying and proteolytic activities and the highest ability to grow in the presence of sodium chloride prevailing. This leading biotype, detectable in the interstice during the entire ripening period, was isolated in the microenvironments exterior and heart starting from the 30th d of ripening, and it was the sole biotype present at the end of the ripening. The analysis of microenvironments through the scanning electron microscopy (SEM) evidenced the presence of micro-channels, which could favour the microbial flow from the interstice to the exterior and the heart. Moreover, the SEM analysis allowed the detection of biofilms, recognised as responsible for the correct colonisation of the different meat niches.

Variation in Ecophysiological Traits and Drought Tolerance of Beech (Fagus sylvatica L.) Seedlings from Different Populations.

Frequency and intensity of heat waves and drought events are expected to increase in Europe due to climate change. European beech (Fagus sylvatica L.) is one of the most important native tree species in Europe. Beech populations originating throughout its native range were selected for common-garden experiments with the aim to determine whether there are functional variations in drought stress responses among different populations. One-year old seedlings from four to seven beech populations were grown and drought-treated in a greenhouse, replicating the experiment at two contrasting sites, in Italy (Mediterranean mountains) and Germany (Central Europe). Experimental findings indicated that: (1) drought (water stress) mainly affected gas exchange describing a critical threshold of drought response between 30 and 26% SWA for photosynthetic rate and Ci/Ca, respectively; (2) the Ci to Ca ratio increased substantially with severe water stress suggesting a stable instantaneous water use efficiency and an efficient regulation capacity of water balance achieved by a tight stomatal control; (3) there was a different response to water stress among the considered beech populations, differently combining traits, although there was not a well-defined variability in drought tolerance. A combined analysis of functional and structural traits for detecting stress signals in beech seedlings is suggested to assess plant performance under limiting moisture conditions and, consequently, to estimate evolutionary potential of beech under a changing environmental scenario.

Enhancing phytoextraction of Cd by combining poplar (clone "I-214") with Pseudomonas fluorescens and microbial consortia.

The plant-microorganism combinations may contribute to the success of phytoextraction of heavy metal-polluted soil. The purpose of this study was to investigate the effects of cadmium (Cd) soil concentration on selected physiological parameters of the poplar clone "I-214" inoculated at root level with a strain (BT4) of Pseudomonas fluorescens and a commercial product based on microbial consortia (Micosat F Fito®). Plants were subjected to Cd treatment of 40 mg kg(-1) in greenhouse. The effects of plant-microbe interactions, plant growth, leaf physiology, and microbial activity were periodically monitored. Metal concentration and translocation factors in plant tissues proved enhanced Cd uptake in roots of plants inoculated with P. fluorescens and transfer to shoots in plants inoculated with Micosat F Fito®, suggesting a promising strategy for using microbes in support of Cd uptake. Plant-microbe integration increased total removal of Cd, without interfering with plant growth, while improving the photosynthetic capacity. Two major mechanisms of metal phytoextraction inducted by microbial inoculation may be suggested: improved Cd accumulation in roots inoculated with P. fluorescens, implying phytostabilization prospective and high Cd transfer to shoots of inoculated plants, outlining enhanced metal translocation.

Lactobacillus plantarum 29 inhibits Penicillium spp. involved in the spoilage of black truffles (Tuber aestivum).

The effect of an antifungal culture of Lactobacillus plantarum to be used in the storage at refrigeration temperature of fresh black truffles was examined. The strain was selected among 29 lactobacilli isolated from foods and evaluated for their viability and acidification activity at 4 °C, as well as for their inhibitory activity against 11 Penicillium strains isolated from truffles stored at refrigeration temperature. Lb. plantarum 29 showed the ability to hold not only the growth of Penicillium isolated from truffles, but also that of P. digitatum DSM 2750, a green mold involved in the spoilage of truffles. The antifungal activity was observed in vitro and in situ, and the sensory characteristics of truffles were preserved during the cold storage.

Irradiation treatments to improve the shelf life of fresh black truffles (truffles preservation by gamma-rays).

The aim of this study was to individuate a method to improve the shelf life of fresh truffles, hypogeous fungi highly prized but very perishable as well. Microbial ecology of fresh products was investigated and the effect of irradiation at different doses on microbial and sensorial parameters was assayed. Data showed that fresh truffles are strongly contaminated from different groups of microorganisms. Irradiation was able to influence their growth, determining an impressive reduction. Treatments at 5 and 10 kGy doses produced the highest microbial decontamination but negatively influenced sensorial parameters. Irradiation at a 1.5 kGy dose was the best solution to improve the shelf life of truffles.