Role and potentialities of bacteria associated with Tuber magnatum: A mini-review.

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.

Identifying plant genes shaping microbiota composition in the barley rhizosphere.

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here, we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plant's capacity of shaping rhizosphere microbes.

Chemical Profile, In Vitro Biological Activity and Comparison of Essential Oils from Fresh and Dried Flowers of Lavandula angustifolia L.

The chemical composition of essential oils (EOs) from dried and fresh flowers of Lavandula angustifolia L. (lavender), named LA 2019 and LA 2020, respectively, grown in central Italy was analyzed and compared by GC and GC-MS. For both samples, 61 compounds were identified, corresponding to 97.9% and 98.1% of the total essential oils. Explorative data analysis, performed to compare the statistical composition of the samples, resulted in a high level of global similarity (around 93%). The compositions of both samples were characterized by 10 major compounds, with a predominance of Linalool (35.3-36.0%), Borneol (15.6-19.4%) and 1,8-Cineole (11.0-9.0%). The in vitro antibacterial activity assay by disk diffusion tests against Bacillus subtilis PY79 and Escherichia coli DH5α showed inhibition of growth in both indicator strains. In addition, plate counts revealed a bactericidal effect on E. coli, which was particularly noticeable when using oil from the fresh lavender flowers at the highest concentrations. An in vitro antifungal assay showed that the EOs inhibited the growth of Sclerotium rolfsii, a phytopathogenic fungus that causes post-harvest diseases in many fruits and vegetables. The antioxidant activity was also assessed using the ABTS free radical scavenging assay, which showed a different antioxidant activity in both EOs. In addition, the potential application of EOs as a green method to control biodeterioration phenomena on an artistic wood painting (XIX century) was evaluated.

Heterogeneity of the white truffle Tuber magnatum in a limited geographic area of Central-Southern Italy.

Molise region (Central-Southern Italy) is one of the Italian richest areas of truffles and contributes significantly to the national production of the precious Tuber magnatum. Nevertheless, Molise truffle has received little scientific attention. Accordingly, in the present study, two T. magnatum populations collected in two different sites of Molise region were characterised from a morphological, genetic and microbiological point of view. A considerable variability between and within the two analysed groups emerged, suggesting an interesting heterogeneity of Molise white truffle populations. Ascocarps of the two groups significantly differed in size and maturation degree, although no linear correlation between weight and maturity was found. Genetic investigations focused on the Sequence-Characterised Amplified Region SCAR A21-inf. Three haplotypes, randomly distributed within the two truffle groups regardless of their collection sites, were detected. The 16S rRNA gene amplicon high-throughput sequencing provided an overview of the composition of the ascocarp-associated bacterial communities. A predominance of α-Proteobacteria was observed, with Bradyrhizobium among the main genera. However, some truffles showed unusual microbial profiles, with Pedobacter, Polaromonas and other bacterial genera as dominant taxa.

The rhizosphere of Salix viminalis plants after a phytostabilization process assisted by biochar, compost, and iron grit: chemical and (micro)-biological analyses.

Amendments, such as biochar, compost, and iron grit, used in phytostabilization studies, showed positive effects on soil physico-chemical properties, plant growth, and the microbial community. However, assisted phytostabilization studies do not always focus on the rhizosphere area where soil, plants, and microorganisms are affected by the amendments and plants and microorganisms can also interact with each other. The aims of this study were to evaluate the effects of amendment application on the exudation of organic acids by Salix viminalis plant roots, as well as the effects of amendments and plant development on the soil CHNS contents and the microbial community activity and diversity, assessed by measuring enzyme activities and using Biolog EcoPlatesTM tests and next-generation sequencing analyses. The results of the mesocosm experiment showed that soil C, H, and N contents were increased by amendment application, especially biochar and compost, while the one of S decreased. Enzyme activities, microbial activity, and diversity were also increased by the addition of amendments, except iron grit alone. Finally, the quantity of organic acids exuded by roots were little affected by amendments, which could in part explain the reduced effect of plant development on soil chemical and microbiological parameters. In conclusion, this study showed in particular that biochar and compost were beneficial for the soil CHN contents and the microbial community while affecting poorly Salix viminalis root exudates.

Evaluation of direct and biochar carrier-based inoculation of Bacillus sp. on As- and Pb-contaminated technosol: effect on metal(loid) availability, Salix viminalis growth, and soil microbial diversity/activity.

Phytomanagement manipulates the soil-plant system to lower the risk posed by contaminated soils. In this process, the addition of amendments, such as biochar, and bacteria can improve the fertility of poor contaminated soils and consequently ameliorate plant growth. A number of studies based on the inoculation of soil with microorganisms of the genus Bacillus, previously isolated from contaminated sites, revealed positive effects on soil properties and plant growth. Furthermore, when the Bacillus isolates were used in association with biochar, better results were obtained, as biochar can ameliorate soil properties and serve as habitat for microorganisms. Accordingly, a mesocosm study was set-up using a mining technosol amended with biochar and inoculated with an endogenous Bacillus isolate, to evaluate the effect of inoculation on soil properties, metal(loid) immobilization, and Salix viminalis growth. Two inoculation methods were compared: (1) direct inoculation of bacteria (Bacillus sp.) and (2) inoculation using biochar as a carrier. Results showed that the Bacillus isolate modified soil properties and ameliorated plant growth, while having a reduced effect on metal(loid) accumulation. The microbial activity was also stimulated, and the community composition was shifted, more importantly when biochar was used as a carrier. In conclusion, this research revealed an improvement of the plant growth and microbial activity after the addition of the endogenous bacterium to the analyzed former mining soil, with better results recorded when a carrier was used.

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.

Winter survival of microbial contaminants in soil: an in situ verification.

The aim of the research was to evaluate, at site scale, the influence of freezing and freeze/thaw cycles on the survival of faecal coliforms and faecal enterococci in soil, in a climate change perspective. Before the winter period and during grazing, viable cells of faecal coliforms and faecal 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 faecal indicators was detected only in some of the investigated soil profiles, and within the first 5 cm. Taking into consideration the results of specific investigations, we hypothesise that the non-uniform spatial distribution of grass roots within the studied soil can play an important role in influencing this phenomenon, while several abiotic factors do not play any significant role. Taking into account the local trend in the increase of air temperature, a different distribution of microbial pollution over time is expected in spring waters, in future climate scenarios. The progressive increase in air temperature will cause a progressive decrease in freeze/thaw cycles at higher altitudes, minimising cold shocks on microbial cells, and causing spring water pollution also during winter.

Potential role of Bacillus endospores in soil amended by olive mill wastewater.

The main aim of this work was to know how spread is laccase activity in spores of Bacillus species isolated from a soil where Italian law allows olive mill wastewater (OMW) spreading, and to investigate the potential role of such autochthonous soil microorganisms in degradation of OMW phenols, and prevention of groundwater pollution. Laccase activity was detected for the first time in spores of wild-type Bacillus pumilus, B. cereus sensu lato, and B. amyloliquefaciens strains. Because B. pumilus, B. cereus sensu lato, and B. amyloliquefaciens, together with B. subtilis account for a total of 93% of Bacillus isolates at the study site, the nearly totality of Bacillus spores reveals laccase activity. Thus, taking also into consideration that Bacillus spores are more abundant (about 100-fold) than white-rot fungi (that possess a well known extracellular, radical-based ligninolytic enzyme system capable of degrading OMW phenols) in the studied soil, these spores may contribute to in-situ degradation of OMW phenols. This role is further emphasized by dilution of crude OMW during infiltration of rainwater through soil that allows to minimize the antibacterial activity of phenols. The widespread presence of Bacillus spores in soils indicates a potential detoxifying role of these spores in a broader context.

Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity.

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

Role of organic matter and clay fraction on migration of Escherichia coli cells through pyroclastic soils, southern Italy.

A comparative study on the adsorption of Escherichia coli cells to two different pyroclastic soils collected in southern Italy (carbonate Apennines) was performed in laboratory using surfactant-free solutions and solutions with the surfactants sodium dodecyl sulphate (SDS, anionic) and octylphenoxypolyethoxyethanol (Triton X-100, non-ionic). Both soils are rich in organic matter (up to 35%), but only one contains a clay fraction (2-5%). The experiments demonstrated that E. coli cells are significantly adsorbed to the clay fraction of the soil, while the organic matter content does not play a significant role. The pore size exclusion phenomenon is another factor to consider when analyzing the retention of E. coli cells within such soils. However, despite the existence of different factors that enhance bacterial cells retention, a high percent of E. coli cells is transported through soil media. The not absolute protection of such soils against microbial pollution is supported not only by the results of the column experiments at lab scale, but also by the findings of a field monitoring at site scale.

Filtration of Bacillus subtilis and Bacillus cereus spores in a pyroclastic topsoil, carbonate Apennines, southern Italy.

A comparative study on the filtration of Bacillus subtilis and Bacillus cereus spores in a pyroclastic topsoil was performed in laboratory using surfactant-free solutions and solutions with the surfactant sodium dodecyl sulphate (SDS) (anionic). The results of the column experiments demonstrate that the SDS does not significantly influence the retention of both B. subtilis and B. cereus spores. Since the SDS is adsorbed through hydrophobic interaction with the organic matter of soil media, these results suggest that hydrophobic interaction between spores and organic matter does not play a significant role on filtration processes within the studied topsoil. This statement is of utmost importance taking into consideration the hydrophobic nature of Bacillus spores and the very high organic matter content in the studied topsoil (20-34%). Conversely, the retention of the analyzed spores seems to be influenced by the pore size exclusion phenomenon.

Expression of the heat shock gene clpL of Streptococcus thermophilus is induced by both heat and cold shock.

BACKGROUND: Heat and cold shock response are normally considered as independent phenomena. A small amount of evidence suggests instead that interactions may exist between them in two Lactococcus strains. RESULTS: We show the occurrence of molecular relationships between the mechanisms of cold and heat adaptations in Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentation, where it undergoes both types of stress. We observed that cryotolerance is increased when cells are pre-incubated at high temperature. In addition, the production of a protein, identified as ClpL, a member of the heat-shock ATPase family Clp A/B, is induced at both high and low temperature. A knock-out clpL mutant is deficient in both heat and cold tolerance. However lack of production of this protein does not abolish the positive effect of heat pre-treatment towards cryotolerance. CONCLUSION: Dual induction of ClpL by cold and heat exposure of cells and reduced tolerance to both temperature shocks in a clpL mutant indicates that the two stress responses are correlated in S. thermophilus. However this protein is not responsible by itself for cryotolerance of cells pre-treated at high temperature, indicating that ClpL is necessary for the two phenomena, but does not account by itself for the relationships between them.

Influence of precipitation and soil on transport of fecal enterococci in fractured limestone aquifers.

Limestone aquifers provide the main drinking water resources of southern Italy. The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading. The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness. This study was carried out in order to analyze the interaction between Enterococcus faecalis and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination. E. faecalis was chosen because, in the study area involved, it represents a better indicator than Escherichia coli. The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks). The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area. Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring. The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale.

Impaired temperature stress response of a Streptococcus thermophilus deoD mutant.

An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20 degrees C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.

Alteration of cell morphology and viability in a recA mutant of Streptococcus thermophilus upon induction of heat shock and nutrient starvation.

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis.

BACKGROUND: Applications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods. Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH. Thus the isolation of new bacteriocins active in foods is desirable. RESULTS: We isolated a Bacillus licheniformis thermophilic strain producing a bacteriocin with some novel features, named here bacillocin 490. This bacteriocin was inactivated by pronase E and proteinase K and was active against closely related Bacillus spp. both in aerobic and in anaerobic conditions. Bactericidal activity was kept during storage at 4 degrees C and was remarkably stable in a wide pH range. The bacteriocin was partially purified by elution after adhesion to cells of the food-isolated strain Bacillus smithii and had a rather low mass (2 KDa). Antimicrobial activity against B. smithii was observed also when this organism was grown in water buffalo milk. CONCLUSIONS: Bacillocin 490 is a novel candidate as a food anti-microbial agent since it displays its activity in milk, is stable to heat treatment and during storage, is active in a wide pH range and has bactericidal activity also at high temperature. These features may allow the use of bacillocin 490 during processes performed at high temperature and as a complementary antimicrobial agent of nisin against some Bacillus spp. in non-acidic foods. The small size suggests its use on solid foods.