Yield and Nutraceutical Value of Lettuce and Basil Improved by a Microbial Inoculum in Greenhouse Experiments.

Members of Bacillus spp. have been widely used to enrich the soil/root interface to provide plant growth promoting activities. A new isolate, namely to Bacillus sp. VWC18, has been tested under greenhouse conditions in lettuce (Lactuca sativa L.) pots at different concentrations (103, 105, 107, and 109 CFU·mL-1) and application time (single inoculum at transplant and multiple inoculum every ten days) to evaluate the best application dose and frequency. Analysis of foliar yield, main nutrients, and minerals evidenced a significant response for all applications. The lowest (103 CFU·mL-1) and the highest doses (109 CFU·mL-1), applied every ten days until harvest, had the greatest efficacy; the nutrient yield (N, K, P, Na, Ca, Fe, Mg, Mn, Cu, and B) increased more than twice. A new randomized block design with three replicates was then performed in lettuce and basil (Ocinum basilicum L.), with the two best performing concentrations applied every ten days. In addition to previous analysis, root weight, chlorophyll, and carotenoids were also examined. Both experiments confirmed the previous results: inoculation of the substrate with Bacillus sp. VWC18 promoted plant growth, chlorophyll, and mineral uptake in both crop species. Root weight duplicated or triplicated compared to control plants, and chlorophyll concentration reached even higher values. Both parameters had a dose-dependent increase.

Environment or genetic isolation? An atypical intestinal microbiota in the Maltese honey bee Apis mellifera spp. ruttneri.

Introduction: Apis mellifera evolved mainly in African, Asian, and European continents over thousands of years, leading to the selection of a considerable number of honey bees subspecies that have adapted to various environments such as hot semi-desert zones and cold temperate zones. With the evolution of honey bee subspecies, it is possible that environmental conditions, food sources, and microbial communities typical of the colonized areas have shaped the honey bee gut microbiota. Methods: In this study the microbiota of two distinct lineages (mitochondrial haplotypes) of bees Apis mellifera ruttneri (lineage A) and Apis mellifera ligustica and carnica (both lineage C) were compared. Honey bee guts were collected in a dry period in the respective breeding areas (the island of Malta and the regions of Emilia-Romagna and South Tyrol in Italy). Microbial DNA from the honey bee gut was extracted and amplified for the V3-V4 regions of the 16S rRNA gene for bacteria and for ITS2 for fungi. Results: The analyses carried out show that the Maltese lineage A honey bees have a distinctive microbiota when compared to Italian lineage C honey bees, with the most abundant genera being Bartonellaceae and Lactobacillaceae, respectively. Lactobacillaceae in Maltese Lineage A honey bees consist mainly of Apilactobacillus instead of Lactobacillus and Bombilactobacillus in the lineage C. Lineage A honey bee gut microbiota also harbors higher proportions of Arsenophonus, Bombella, Commensalibacter, and Pseudomonas when compared to lineage C. Discussion: The environment seems to be the main driver in the acquisition of these marked differences in the gut microbiota. However, the influence of other factors such as host genetics, seasonality or geography may still play a significant role in the microbiome shaping, in synergy with the environmental aspects.

Characterization of Lactic Acid Bacteria Isolated from Spontaneously Fermented Sausages: Bioprotective, Technological and Functional Properties.

Fermentation is one of the most ancient strategies to improve safety and extend shelf-life of the products. Starter cultures are mainly represented by lactic acid bacteria (LAB), which may also be bioprotective agents controlling the fermentation process, the native microbiota and pathogen outgrowth. This work aimed to select new LAB strains from spontaneously fermented sausages produced in different areas of Italy, which can be effective as starter cultures and bioprotective agents in fermented salami. The strains, mainly belonging to the Latilactobacillus sakei species, were characterized for their ability to inhibit major meat pathogens, the presence of antibiotic resistances and amine production. Moreover, technological performances, such as growth and acidification kinetics at increasing NaCl concentrations, were studied. As a result, new autochthonous Lat. sakei strains were obtained, lacking antibiotic resistance, possessing antimicrobial activity against Clostridium sporogenes, Listeria monocytogenes, Salmonella and Escherichia coli and with high growth performance under osmotic pressure. These strains have the potential for future application to improve the safety of fermented meats, even under conditions in which chemical preservatives are reduced or eliminated. Moreover, studies on autochthonous cultures are pivotal for guaranteeing specific characteristics of traditional products that represent an important cultural heritage.

Biostimulants for Sustainable Management of Sport Turfgrass.

Research on the efficacy of innovative, ecofriendly biostimulants in sport turf management is scarce, with less information available from open-field experiments, and even less pertaining to thatch control-related problems. The objective was to investigate the open-field effectiveness of a commercial product, EM-1, and two newly developed products, ExpA and ExpB, in improving both rhizosphere and turfgrass, Agrostis stoloniferous L., characteristics on a golf green. ExpA and ExpB, identical in microbial composition, were equally effective in significantly increasing chlorophyll synthesis and visual turf quality, as well as in resistance to tearing out, compared to the untreated control 56 days after treatment (DAT). EM-1 showed intermediate trends between the control and novel biostimulants. The inclusion of humic acids and mycorrhizal fungi to the microbial composition in ExpB significantly improved some rhizosphere properties 56 DAT relative to the control. Results on ExpB evidenced a significant decrease in the thatch layer thickness and fresh leaf weight, associated with a significant increase in the humus thickness, organic matter decomposition and evapotranspiration efficiency. An increased dry leaf biomass was also shown. ExpA and EM-1 showed either marginal or intermediate improvements relative to the control. ExpB represents a promising alternative to alleviate negative environmental impacts associated with turf maintenance-related activities.

Valorization of coffee wastes as plant growth promoter in mulching film production: A contribution to a circular economy.

Food waste valorization, considered as energy and/or chemicals source, via biorefinery or biotechnology, gained great attention in recent years, because of the fast depletion of primary resources, increased waste generation and landfilling worldwide. Coffee by-products for example (i.e. coffee pulp, coffee husks, silver skin, spent coffee, etc.) have been investigated in different forms either as a source of antioxidant and valuable chemicals and as a filler in composites. A new valorization route for coffee silver skin (CSS), up to now just sent to damping, is here investigated: particulate bio-composites based on poly(butylene succinate-co-adipate) (PBSA), an aliphatic biodegradable polyester commercially available, have been formulated with up to a 30 wt% of CSS, in order to prepare mulching films for agriculture. The bacterial analysis of the filler indeed, has underlined the presence of potential Plant Growth-Promoting Bacteria species, mainly ascribed to the Bacillus genus, which can survive both the roasting and the compounding processes. The obtained composites have been characterized mechanically and thermally and their hydrophilic nature has been investigated by measuring their contact angle. Eventually, the bacteria release from the composite films has been examined by means of in-vitro tests. The plant growth promoting capability of the films was preliminarily evaluated in pot experiments using lettuce as a model crop. The composite films were able to release the endogenous bacteria in the soil and to stimulate plant and root growth of the assayed crop. The possibility to produce functionalized biodegradable mulching films by recycling agricultural wastes can thus be forecast, highlighting potential multiple advantages in terms of soil preservation/fertilization, decrease of polymeric materials in mulching products, exploitation of a waste.

Selection of Acetic Acid Bacterial Strains and Vinegar Production From Local Maltese Food Sources.

This study investigates the isolation, identification, and fermentation performance of autochthonous acetic acid bacteria (AAB) from local niche habitats on the Island of Gozo (Malta) and their further use for vinegar production, employing local raw materials. The bacteria were isolated from grapevine berries and vinegar produced in the cottage industry. Following phenotype and genotype identification, the AAB were ascribed to the genera Acetobacter, Gluconobacter, and Komagataeibacter. A mixture of selected AAB was tested as an inoculum for vinegar production in bench fermenters, under different conditions and substrates, namely, grapes, honey, figs, onions, prickly pear, and tomatoes. The bench fermenters were operated under semi-continuous fermentation where working volumes were maintained by discharging and subsequent recharging accordingly to maintain the acidity in fermenters by adding 30-50 g/l of acetic acid for optimal Acetobacteraceae performance. Finally, the vinegar products obtained from the different substrates were evaluated for their quality, including organoleptic properties, which showed the superior quality of wood-treated vinegar samples with respect to neat vinegar samples.

Honeybee Exposure to Veterinary Drugs: How Is the Gut Microbiota Affected?

Several studies have outlined that a balanced gut microbiota offers metabolic and protective functions supporting honeybee health and performance. The present work contributes to increasing knowledge on the impact on the honeybee gut microbiota of the three most common veterinary drugs (oxytetracycline, sulfonamides, and tylosin). The study was designed with a semi-field approach in micro-hives containing about 500 honeybees. Micro-hives were located in an incubator during the day and moved outdoors in the late afternoon, considering the restrictions on the use of antibiotics in the open field but allowing a certain freedom to honeybees; 6 replicates were considered for each treatment. The absolute abundance of the major gut microbial taxa in newly eclosed individuals was studied with qPCR and next-generation sequencing. Antimicrobial resistance genes for the target antibiotics were also monitored using a qPCR approach. The results showed that the total amount of gut bacteria was not altered by antibiotic treatment, but qualitative variations were observed. Tylosin treatment determined a significant decrease of α- and ß-diversity indices and a strong depletion of the rectum population (lactobacilli and bifidobacteria) while favoring the ileum microorganisms (Gilliamella, Snodgrassella, and Frischella spp.). Major changes were also observed in honeybees treated with sulfonamides, with a decrease in Bartonella and Frischella core taxa and an increase of Bombilactobacillus spp. and Snodgrassella spp. The present study also shows an important effect of tetracycline that is focused on specific taxa with minor impact on alfa and beta diversity. Monitoring of antibiotic resistance genes confirmed that honeybees represent a great reservoir of tetracycline resistance genes. Tetracycline and sulfonamides resistance genes tended to increase in the gut microbiota population upon antibiotic administration. IMPORTANCE This study investigates the impact of the three most widely used antibiotics in the beekeeping sector (oxytetracycline, tylosin, and sulfonamides) on the honeybee gut microbiota and on the spread of antibiotic resistance genes. The research represents an advance to the present literature, considering that the tylosin and sulfonamides effects on the gut microbiota have never been studied. Another original aspect lies in the experimental approach used, as the study looks at the impact of veterinary drugs and feed supplements 24 days after the beginning of the administration, in order to explore perturbations in newly eclosed honeybees, instead of the same treated honeybee generation. Moreover, the study was not performed with cage tests but in micro-hives, thus achieving conditions closer to real hives. The study reaches the conclusion that the most common veterinary drugs determine changes in some core microbiota members and that incidence of resistance genes for tetracycline and sulfonamides increases following antibiotic treatment.

Honeybees Exposure to Natural Feed Additives: How Is the Gut Microbiota Affected?

The role of a balanced gut microbiota to maintain health and prevent diseases is largely established in humans and livestock. Conversely, in honeybees, studies on gut microbiota perturbations by external factors have started only recently. Natural methods alternative to chemical products to preserve honeybee health have been proposed, but their effect on the gut microbiota has not been examined in detail. This study aims to investigate the effect of the administration of a bacterial mixture of bifidobacteria and Lactobacillaceae and a commercial product HiveAliveTM on honeybee gut microbiota. The study was developed in 18 hives of about 2500 bees, with six replicates for each experimental condition for a total of three experimental groups. The absolute abundance of main microbial taxa was studied using qPCR and NGS. The results showed that the majority of the administered strains were detected in the gut. On the whole, great perturbations upon the administration of the bacterial mixture and the plant-based commercial product were not observed in the gut microbiota. Significant variations with respect to the untreated control were only observed for Snodgrassella sp. for the bacterial mixture, Bartonella sp. in HiveAliveTM and Bombilactobacillus sp. for both. Therefore, the studied approaches are respectful of the honeybee microbiota composition, conceivably without compromising the bee nutritional, social and ecological functions.

Heterodimeric GW7604 Derivatives: Modification of the Pharmacological Profile by Additional Interactions at the Coactivator Binding Site.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) ß (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERß)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERß)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 µM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.

A prospective longitudinal study on the microbiota composition in amyotrophic lateral sclerosis.

BACKGROUND: A connection between amyotrophic lateral sclerosis (ALS) and altered gut microbiota composition has previously been reported in animal models. This work is the first prospective longitudinal study addressing the microbiota composition in ALS patients and the impact of a probiotic supplementation on the gut microbiota and disease progression. METHODS: Fifty patients and 50 matched controls were enrolled. The microbial profile of stool samples from patients and controls was analyzed via PCR-Denaturing Gradient Gel Electrophoresis, and the main microbial groups quantified via qPCR. The whole microbiota was then analyzed via next generation sequencing after amplification of the V3-V4 region of 16S rDNA. Patients were then randomized to receive probiotic treatment or placebo and followed up for 6 months with ALSFRS-R, BMI, and FVC%. RESULTS: The results demonstrate that the gut microbiota of ALS patients is characterized by some differences with respect to controls, regardless of the disability degree. Moreover, the gut microbiota composition changes during the course of the disease as demonstrated by the significant decrease in the number of observed operational taxonomic unit during the follow-up. Interestingly, an unbalance between potentially protective microbial groups, such as Bacteroidetes, and other with potential neurotoxic or pro-inflammatory activity, such as Cyanobacteria, has been shown. The 6-month probiotic treatment influenced the gut microbial composition; however, it did not bring the biodiversity of intestinal microbiota of patients closer to that of control subjects and no influence on the progression of the disease measured by ALSFRS-R was demonstrated. CONCLUSIONS: Our study poses the bases for larger clinical studies to characterize the microbiota changes as a novel ALS biomarker and to test new microbial strategy to ameliorate the health status of the gut. TRIAL REGISTRATION: CE 107/14, approved by the Ethics Committee of the "Maggiore della Carità" University Hospital, Italy.

Development of bivalent triarylalkene- and cyclofenil-derived dual estrogen receptor antagonists and downregulators.

Up to 80% of mammary carcinoma initially exhibit estrogen-dependent growth, which can be treated by aromatase inhibitors or SERMs/SERDs. To increase the options after failure of the hormonal therapy with these drugs, the search for alternatives with a different mode of action to prevent estrogen action is of high relevance. Therefore, this study focused on the inhibition of coactivator recruitment at the estrogen receptor (ER) by targeted attachment of bivalent compounds at the coactivator binding site besides the primary binding at the ligand binding domain. Eight homodimeric 4-[1-(4-hydroxyphenyl)-2-phenyl-1-butenyl]cinnamic acid (GW7604)- or cyclofenilacrylic acid-based ER ligands with diaminoalkane linkers (C2-C5) were synthesized and their effects on the ER subtypes were assessed in vitro. All compounds possessed full antagonistic potency at ERα/ß as determined in a transactivation assay. Furthermore, they exerted medium downregulatory effects dependent on the spacer length and did not stimulate the ER expression as observed for 4-hydroxytamoxifen. The cyclofenil-derived dimer with C4 spacer (15b) showed the highest binding affinity to ERα (RBA = 79.2%) and downregulated the ER content in MCF-7 cells with an efficiency of 38% at 1 µM.

Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract.

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from solitary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found. Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955T=LMG 30142T), reference strain XV16 and Bifidobacterium aemilianum sp. nov. whose type strain is XV10 (=DSM 104956T=LMG 30143T), reference strain XV4.

Kombucha Beverage from Green, Black and Rooibos Teas: A Comparative Study Looking at Microbiology, Chemistry and Antioxidant Activity.

Kombucha is usually obtained from the fermentation of black or green tea by a consortium of acetic acid bacteria and yeasts. In this study, kombucha was prepared from the same starter consortium using green and black teas as well as, for the first time, an infusion of rooibos leaves (Aspalathus linearis). Microbial diversity was analysed during fermentation both in the biofilm and in the corresponding kombuchas, using culture-dependent and -independent methods. Polyphenols, flavonoids, ethanol, and acids were quantified and anti-oxidant activities were monitored. All of the Kombuchas showed similarity in bacterial composition, with the dominance of Komagataeibacter spp. Beta diversity showed that the yeast community was significantly different among all tea substrates, between 7 and 14 days of fermentation and between biofilm and kombucha, indicating the influence of the substrate on the fermenting microbiota. Kombucha from rooibos has a low ethanol concentration (1.1 mg/mL), and a glucuronic acid amount that was comparable to black tea. Although antioxidant activity was higher in black and green kombucha compared to rooibos, the latter showed an important effect on the recovery of oxidative damage on fibroblast cell lines against oxidative stress. These results make rooibos leaves interesting for the preparation of a fermented beverage with health benefits.

Therapeutic Microbiology: The Role of Bifidobacterium breve as Food Supplement for the Prevention/Treatment of Paediatric Diseases.

The human intestinal microbiota, establishing a symbiotic relationship with the host, plays a significant role for human health. It is also well known that a disease status is frequently characterized by a dysbiotic condition of the gut microbiota. A probiotic treatment can represent an alternative therapy for enteric disorders and human pathologies not apparently linked to the gastrointestinal tract. Among bifidobacteria, strains of the species Bifidobacterium breve are widely used in paediatrics. B. breve is the dominant species in the gut of breast-fed infants and it has also been isolated from human milk. It has antimicrobial activity against human pathogens, it does not possess transmissible antibiotic resistance traits, it is not cytotoxic and it has immuno-stimulating abilities. This review describes the applications of B. breve strains mainly for the prevention/treatment of paediatric pathologies. The target pathologies range from widespread gut diseases, including diarrhoea and infant colics, to celiac disease, obesity, allergic and neurological disorders. Moreover, B. breve strains are used for the prevention of side infections in preterm newborns and during antibiotic treatments or chemotherapy. With this documentation, we hope to increase knowledge on this species to boost the interest in the emerging discipline known as "therapeutic microbiology".

Potential Role of Gut Microbiota in ALS Pathogenesis and Possible Novel Therapeutic Strategies.

BACKGROUND: Recent preclinical studies suggest that dysfunction of gastrointestinal tract may play a role in amyotrophic lateral sclerosis (ALS) pathogenesis through a modification of the gut microbiota brain axis. Our study is the first focused on microbiota analysis in ALS patients. AIM: Our aim was to study the main human gut microbial groups and the overall microbial diversity in ALS and healthy subjects. Moreover we have examined the influence of a treatment with a specific bacteriotherapy composed of Lactobacillus strains (Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus salivarius) acting on the gastrointestinal barrier. METHODS: We enrolled 50 ALS patients and 50 healthy controls, matched for sex, age, and origin. Fecal samples were used for total genomic DNA extraction. Enterobacteria, Bifidobacterium spp., Lactobacillus spp., Clostridium sensu stricto, Escherichia coli and yeast were quantified using quantitative polymerase chain reaction approach. Denaturing gradient gel electrophoresis analyses were performed to investigate total eubacteria and yeasts populations. Patients were randomized to double-blind treatment either with microorganisms or placebo for 6 months and monitored for clinical progression and microbiota composition. RESULTS: The comparison between ALS subjects and healthy group revealed a variation in the intestinal microbial composition with a higher abundance of E. coli and enterobacteria and a low abundance of total yeast in patients. Polymerase chain reaction denaturing gradient gel electrophoresis analysis showed a cluster distinction between the bacterial profiles of ALS patients and the healthy subjects. The complexity of the profiles in both cases may indicate that a real dysbiosis status is not evident in the ALS patients although differences between healthy and patients exist. The effects of the progression of the disease and of the bacteriotherapy on the bacterial and yeast populations are currently in progress. CONCLUSIONS: Our preliminary results confirm that there is a difference in the microbiota profile in ALS patients.

Evidence of Campylobacter jejuni reduction in broilers with early synbiotic administration.

C. jejuni is considered a food safety concern to both public health authorities and consumers since it is the leading bacterial cause of food-borne gastroenteritis in humans. A high incidence of C. jejuni in broiler flocks is often correlated to pathogen recovery in retail poultry meat, which is the main source of human infection. In this work broiler chickens were fed with a synbiotic product mixed with conventional feed using two different administration strategies. The synbiotic was formulated with the microencapsulated probiotic Bifidobacterium longum PCB133 and a xylo-oligosaccharide (XOS). 1-day old chicks were infected with C. jejuni strain M1 (105 cells) and the synbiotic mixture was then administered starting from the first and the 14th day of chicken life (for animal groups GrpC and GrpB respectively). The goal of this study was to monitor C. jejuni load at caecum level at different sampling time by real-time PCR, identifying the best administration strategy. The microbiological analysis of the caecal content also considered the quantification of Campylobacter spp., Bifidobacterium spp. and B. longum. The supplemented synbiotic was more successful in reducing C. jejuni and Campylobacter spp. when administered lifelong, compared to the shorter supplementation (GrpB). Bifidobacterium spp. quantification did not show significant differences among treatments and B. longum PCB133 was detected in both supplemented groups evidencing the successful colonization of the strain. Moreover, the samples of the control group (GrpA) and GrpC were analysed with PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to compare the caecal microbial community profiles at the beginning and at the end of the trial. Pattern analysis evidenced the strong influence of the early synbiotic supplementation, although a physiological change in the microbial community, occurring during growth, could be observed. Experimental results demonstrate that the synbiotic approach at farm level can be an effective strategy, combined with biosecurity measures, to improve the safety of poultry meat.

Beneficial microorganisms for honey bees: problems and progresses.

Nowadays, honey bees are stressed by a number of biotic and abiotic factors which may compromise to some extent the pollination service and the hive productivity. The EU ban of antibiotics as therapeutic agents against bee pathogens has stimulated the search for natural alternatives. The increasing knowledge on the composition and functions of the bee gut microbiota and the link between a balanced gut microbiota and health status have encouraged the research on the use of gut microorganisms to improve bee health. Somehow, we are assisting to the transfer of the "probiotic concept" into the bee science. In this review, we examine the role of the honey bee gut microbiota in bee health and critically describe the available applications of beneficial microorganisms as pest control agents and health support. Most of the strains, mainly belonging to the genera Lactobacillus, Bifidobacterium and Bacillus, are isolated from honey bee crop or gut, but some applications involve environmental strains or formulation for animal and human consumption. Overall, the obtained results show the favourable effect of applied microbial strains on bee health and productivity, in particular if strains of bee origin are used. However, it is actually not yet possible to conclude whether this strategy will ever work. In particular, many aspects regarding the overall setup of the experiments, the dose, the timing and the duration of the treatment need to be optimized, also considering the microbiological safety of the hive products (i.e. pollen and honey). In addition, a deep investigation about the effect on host immunity and physiology is envisaged. Lastly, the final users of the formulations, i.e. beekeepers, should be taken into account for the achievement of high-quality, cost-effective and easy-to-use products.

Microbial inoculants for the biocontrol of Fusarium spp. in durum wheat.

BACKGROUND: Fusarium head blight (FHB) is a severe disease caused by different Fusarium species, which affects a wide range of cereal crops, including wheat. It determines from 10 to 30% of yield loss in Europe. Chemical fungicides are mainly used to reduce the incidence of FHB, but low environmental impact solutions are looked forward. Applications of soil/rhizobacteria as biocontrol agents against FHB in wheat are described in literature, whereas the potential use of lactobacilli in agriculture has scarcely been explored. RESULTS: The aim of this work was to study the inhibitory effect of two bacterial strains, Lactobacillus plantarum SLG17 and Bacillus amyloliquefaciens FLN13, against Fusarium spp. in vitro and to assess their efficacy in field, coupled to the study of the microbial community profile of wheat seeds. Antimicrobial assays were performed on agar plates and showed that the two antagonistic strains possessed antimicrobial activity against Fusarium spp. In the field study, a mixture of the two strains was applied to durum wheat i) weekly from heading until anthesis and ii) at flowering, compared to untreated and fungicide treated plots. The FHB index, combining both disease incidence and disease severity, was used to evaluate the extent of the disease on wheat. A mixture of the two microorganisms, when applied in field from heading until anthesis, was capable of reducing the FHB index. Microbial community profile of seeds was studied via PCR-DGGE, showing the presence of L. plantarum SLG17 in wheat seeds and thus underlining an endophytic behavior of the strain. CONCLUSIONS: L. plantarum SLG17 and B. amyloliquefaciens FLN13, applied as biocontrol agents starting from the heading period until anthesis of wheat plants, are promising agents for the reduction of FHB index.

Validation of candidate reference genes in Bifidobacterium adolescentis for gene expression normalization.

Normalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress.

Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment.

BACKGROUND: Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results. RESULTS: This work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns. CONCLUSIONS: The developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis.