Applicazione di un formulato microbiologico probiotico per la protezione della salute delle api

La presente invenzione ha per oggetto un\u2019associazione probiotica per la protezione della salute delle api, in particolare per la protezione nei confronti di patologie batteriche, come ad esempio quella causata da Paenibacillus larvae, agente patogeno della Peste Americana

The rhizosphere of naturally selected plants shows the potential to sustain natural attenuation processes in an historical PCB polluted soil

Scientific Question and Context. The SIN Caffaro is an extended industrial polluted site of national priority located in Italy. Here the soil presents a mixed contamination of halogenated pollutants (PCBs, dioxins and furans), heavy metals and metalloids, uneven distributed and often largely exceeding the safety values. After the closure of the production plant, in the last 50 years only the process of natural attenuation occurred in the surrounding agricultural soils. We aimed to explore the natural attenuation potential of the SIN Caffaro soil microbiota, focusing on the interactions between bacteria and the plants naturally selected in the area. Methods. We studied the phylogenetic and functional diversity of the microbiome associated to three plant species growing in the most contaminated area of the SIN Caffaro. The bacterial community in the rhizosphere and the surrounding soil was described by Illumina sequencing of the 16S rRNA gene and qPCR quantification of the bphA gene, proxy of PCB aerobic degradation. A collection of cultivable rhizobacteria was tested for plant growth promotion (PGP) traits and biodegradation potential. Results. Both the soil niche and the plant species shaped the composition of the resident microbiota. The bacterial communities were more strongly influenced by the plant species in the rhizosphere than in the soil surrounding roots, leading to speculate that the \u201crhizosphere effect\u201d overlapped the high pollutant concentrations in shaping the community structure. The microbiota selected in all the soil niches showed nevertheless degrading potential. The cultivable microbiota in all the rhizospheres showed similar phylogenetic and functional composition. A large fraction of the strains harboured the bphA gene and displayed catechol 2,3-dioxygenase and ACC deaminase activities, the latter considered a PGP trait contributing to plant tolerance to environmental stress. Conclusions. Overall, this work highlighted that spontaneous plant species differentially affected the composition of the rhizosphere bacterial community and harboured the intrinsic potential to sustain natural attenuation processes, further exploitable for rhizoremediation interventions at the SIN Caffaro

Exploration of methods used to describe bacterial communities in silage of maize (Zea mays) cultivars

Different techniques to assess bacterial community structure and diversity were evaluated in silages prepared with four different maize cultivars, three conventional and one transgenic (cv. Tundra, event Bt-176). Plants were cultivated in the greenhouse and harvested after 30 days of growth. Silage samples were collected at successive times during fermentation and analyzed for bacterial counts and by various DNA-based fingerprinting techniques. Bacterial counts were similar between cultivars for the total culturable bacteria, sporeforming, and mesophilic and thermophilic lactic acid bacteria (LAB). Further analysis of the species composition of 388 LAB strains by intergenic transcribed spacer (ITS) PCR followed by sequencing of 16S rRNA gene did not reveal differences between cultivars. In contrast, molecular fingerprinting methods targeting whole bacterial communities, such as automated ribosomal intergenic spacers analysis (ARISA) and 16S rRNA gene length heterogeneity-PCR (LH-PCR), indicated that different maize silage batches or cultivars hosted different bacterial communities. Thus, ARISA and LH-PCR fingerprinting techniques offer a fast and sensitive method to compare bacterial communities, and to detect differences in silage bacterial communities

Diversity and phylogeny of culturable spore-forming Bacilli isolated from marine sediments

Members of the genus Bacillus and related genera are ubiquitous in nature. However, Bacillus species isolated from marine sediments have attracted less interest respect to their terrestrial relatives. Here, we report the phylogenetic diversity of a collection of 96 Bacilli, isolated from 17 distinct stations of 5 oceanographic campaigns. The diversity was analysed by phenotypic and molecular approaches based on the amplified rDNA restriction analysis (ARDRA), amplification of the internal transcribed spacers (ITS-PCR) and on 16S rRNA sequencing. Intraspecific polymorphism was efficiently detected by biochemical analysis and ARDRA while results of ITS-PCR were in agreement with 16S rRNA sequencing. The identification results assigned 68% of the isolates to the species B. subtilis, B. licheniformis, B. pumilus and B. cereus. Phylogenetic analysis allowed the separation of 9 isolates in a clade that may represent a group of obligate marine Bacillus since they clustered with B. firmus, B. foraminis and marine isolates with metal oxidation and bioaccumulation capabilities. The remaining isolates showed a close affiliation to the genera Virgibacillus, Gracilibacillus and Paenibacillus. The widespread of Bacilli and their high diversity level observed in this work point out the need of more extensive studies to understand their distribution and ecology in deep-sea environments

16S-23S rRNA internal transcribed spacers as molecular markers for the species of the 16s rRNA group I of the genes Bacillus

The internal transcribed spacers between the 16S and the 23S ribosomal RNA genes were used to discriminate species of the 16S rRNA group I of the genus Bacillus by PCR. The spacer-PCR fingerprints clearly discriminated the different species, except those closely related like the members of the `B. cereus group' (B. cereus, B. thuringiensis and B. mycoides) and the species of the `B. subtilis group' (B. amyloliquefaciens and B. licheniformis). Examining in more detail the shortest internal transcribed spacers, B. subtilis group species were distinguished by single-strand conformation polymorphism analysis, whereas B. mycoides was differentiated from B. cereus/B. thuringiensis by restriction analysis

Biotransformations of cinnamic and ferulic acid with actinomycetes

Biotransformations of cinnamic and ferulic acid catalysed by actinomycetes have been studied.Aprimary screeningwas carried out using 535 actinomycetes isolated from soil and able to grow in the presence of cinnamic (500) or ferulic acid (35). A mininiaturized procedure of biotransformation in solid medium showed that, among the strains able to metabolise the substrates, the more frequent behaviours were the degradation of cinnamic acid (often via benzoic acid formation) or transformation in cinnamamide (22 strains), while ferulic acid was mostly degraded with transient accumulation of vanillic acid in few cases. A more restricted screening (57 representative strains) was carried out in submerged cultures: strain GE 107678, identified as Streptomyces halstedii, converted cinnamic acid in cinnamamide (95% molar conversion from 2 g/l of substrate) and ferulic acid in vanillic acid (80% molar conversion from 1 g/l of substrate) with transient formation of low amounts of vanillin (0.10\u20130.15 g/l)

Bacteria associated to spontaneous plants in a historical PCB polluted soil show potential to sustain natural attenuation

The SIN Caffaro is a large polluted site of national priority located in Italy, originated by the activities of a former chemical factory. The site is highly contaminated by chlorinated persistent organic pollutants, mainly constituted by polychlorobyphenils (PCBs), together with heavy metals and metalloids, in variable concentrations and uneven distribution. We aimed to explore the diversity of root-associated bacterial communities driven by different plant species spontaneously established in abandoned agricultural soils within the most polluted area in the site. The overall structure of the non-vegetated and root-associated soil fractions bacterial communities was described by high-throughput sequencing of the 16S rRNA gene, and a collection of rhizobacteria was established and tested for plant growth promotion (PGP) traits and bioremediation potential. The results showed that the recruitment of specific bacterial communities in the root-associated soil fractions was driven by both soil fractions and plant species. PCR-based detection in the soil metagenome of bacterial bphA gene, encoding for the biphenyl dioxygenase \u3b1 subunit, indicated that the soil in the site possesses metabolic traits linked to PCB degradation. The cultivable microbiota in the rhizosphere of the different plant species showed highly similar phylogenetic and functional composition, in terms of PGP and bioremediation potential. 72% of the strains harbored the bphA gene and displayed catechol 2,3-dioxygenase and 46% showed ACC deaminase activities, the latter considered a PGP trait contributing to plant tolerance to environmental stress. Overall, this work highlighted that spontaneous plant species differentially affect the rhizosphere bacterial community composition and harbor the intrinsic potential to sustain natural attenuation processes, further exploitable for rhizoremediation intervention at the SIN Caffaro

Characterization of urease genes cluster of Streptococcus thermophilus

Aims: The milk acidification rate of Streptococcus thermophilus strains can be affected by several factors, one of which is the hydrolysis of urea by the urease complex. To evaluate the technological suitability of S. thermophilus strains deprived of urease activity in milk fermentation, the genetic cluster related to urease enzymatic activity has been characterized in the type strain DSM 20167(T). Methods and Results: Amplification of the urease genes of S. thermophilus DSM 20167(T) was developed on the basis of the urease gene cluster of the phylogenetically related S. salivarius. Nucleotide sequencing revealed the presence of eight open reading frames, which were most homologous to ureABC (structural genes) and ureI, ureEFGD (accessory genes) of S. salivarius and other ureolytic bacteria. Reverse transcriptase PCR experiments were in agreement with an operon organization for the eight genes (ureIABCEFGD). A food grade mutant A16 (DeltaureC3) with a 693 bp in-frame deletion in ureC gene exhibited a urease negative (Ure(-)) phenotype. Unlike the wild-type strain, the acidification rate of the mutant in reconstituted skimmed milk was not affected by the presence of urea or nickel ions. A small-scale yoghurt fermentation trials were carried out using the wild-type or the Ure(-) mutant A16 (DeltaureC3) in co-culture with Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 in presence of urea. The result obtained underlines that when the Ure(-) mutant was used as a co-starter the acidification rate was higher than that obtained using the wild-type strain. Conclusions: The study provides the first genetic characterization and the technological implication of S. thermophilus DSM 20617(T) urease activity. Significance and Impact of the Study: The detrimental effect of ureolytic activity on the rate of milk acidification was evaluated and superseded using a food-grade Ure(-) recombinant strain. Small-scale yoghurt production trials highlighted the positive role of a Ure(-)S. thermophilus mutant as a co-starter in milk fermentations. Moreover, the vector pMI108 developed for the construction of the Ure(-) strain, should be considered as a potential tool for the generation of Ure(-) dairy S. thermophilus strains selected for other relevant technological properties but characterized by the undesirable ureolytic phenotype