Identification and characterization of lactic acid bacteria and yeasts of PDO Tuscan bread sourdough by culture dependent and independent methods

Sourdough fermentation has been increasingly used worldwide, in accordance with the demand of consumers for tasty, natural and healthy food. The high diversity of lactic acid bacteria (LAB) and yeast species, detected in sourdoughs all over the world, may affect nutritional, organoleptic and technological traits of leavened baked goods. A wide regional variety of traditional sourdough breads, over 200 types, has been recorded in Italy, including special types selected as worthy of either Protected Geographical Indication (PGI) or Protected Designation of Origin (PDO), whose sourdough microbiota has been functionally and molecularly characterized. As, due to the very recent designation, the microbiota of Tuscan bread sourdough has not been investigated so far, the aim of the present work was to isolate and characterize the species composition of LAB and yeasts of PDO Tuscan bread sourdough by culture-independent and dependent methods. A total of 130 yeasts from WLN medium and 193 LAB from both mMRS and SDB media were isolated and maintained to constitute the germplasm bank of PDO Tuscan bread. Ninety six LAB from mMRS medium and 68 yeasts from WLN medium were randomly selected and molecularly identified by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and PCR-RFLP analysis of the ITS region, respectively, and sequencing. The yeast identity was confirmed by 26S D1/D2 sequencing. All bacterial isolates showed 99% identity with Lactobacillus sanfranciscensis, 65 yeast isolates were identified as Candida milleri, and 3 as Saccharomyces cerevisiae. Molecular characterization of PDO Tuscan bread sourdough by PCR-DGGE confirmed such data. The distinctive tripartite species association, detected as the microbiota characterizing the sourdough used to produce PDO Tuscan bread, encompassed a large number of L. sanfranciscensis and C. milleri strains, along with a few of S. cerevisiae. The relative composition and specific physiological characteristics of such microbiota could potentially affect the nutritional features of PDO Tuscan bread, as suggested by the qualitative functional characterization of the isolates. Investigations on the differential functional traits of such LAB and yeast isolates could lead to the selection of the most effective single strains and of the best performing strain combinations to be used as starters for the production of baked goods

Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices

Arbuscular Mycorrhizal Fungi (AMF) live in symbiosis with most crop plants and represent essentialelements of soil fertility and plant nutrition and productivity, facilitating soil mineral nutrient uptakeand protecting plants from biotic and abiotic stresses. These beneficial services may be mediated bythe dense and active spore-associated bacterial communities, which sustain diverse functions, such asthe promotion of mycorrhizal activity, biological control of soilborne diseases, nitrogen fixation, and the supply of nutrients and growth factors. In this work, we utilised culture-dependent methods to isolate and functionally characterize the microbiota strictly associated to Rhizophagus intraradices spores,and molecularly identified the strains with best potential plant growth promoting (PGP) activities by16S rDNA sequence analysis. We isolated in pure culture 374 bacterial strains belonging to different functional groups—actinobacteria, spore-forming, chitinolytic and N2-fixing bacteria—and screened 122 strains for their potential PGP activities. The most common PGP trait was represented by P solubilization from phytate (69.7%), followed by siderophore production (65.6%), mineral P solubilization (49.2%) and IAA production (42.6%). About 76% of actinobacteria and 65% of chitinolytic bacteria displayed multiple PGP activities. Nineteen strains with best potential PGP activities, assigned to Sinorhizobium meliloti, Streptomyces spp., Arthrobacter phenanthrenivorans, Nocardiodes albus, Bacillus sp. pumilus group, Fictibacillus barbaricus and Lysinibacillus fusiformis, showed the ability to produce IAA and siderophores and to solubilize P from mineral phosphate and phytate, representing suitable candidates as biocontrol agents,biofertilisers and bioenhancers, in the perspective of targeted management of beneficial symbionts and their associated bacteria in sustainable food production systems

Diverse bacterial communities are recruited on spores of different arbuscular mycorrhizal fungal isolates

Arbuscular mycorrhizal fungi (AMF) establish mutualistic symbioses with the roots of most food crops, playing a key role in soil fertility and plant nutrition and health. The beneficial activity of AMF may be positively affected by bacterial communities living associated with mycorrhizal roots, spores and extraradical hyphae. Here, we investigated the diversity of bacterial communities associated with the spores of six AMF isolates, belonging to different genera and species and maintained for several generations in pot cultures with the same host plant, under the same environmental conditions and with the same soil. The occurrence of large bacterial communities intimately associated with spores of the AMF isolates was revealed by PCR denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of DGGE bands. Cluster and canonical correspondence analysis showed that the six AMF isolates displayed diverse bacterial community profiles unrelated with their taxonomic position, suggesting that each AMF isolate recruits on its spores a different microbiota. The 48 sequenced fragments were affiliated with Actinomycetales, Bacillales, Pseudomonadales, Burkholderiales, Rhizobiales and with Mollicutes-related endobacteria (Mre). For the first time, we report the occurrence of Mre in Funneliformis coronatum and Rhizophagus intraradices and sequences related to endobacteria of Mortierella elongata in F. coronatum and Funneliformis mosseae. The bacterial species identified are known to possess diverse and specific physiological characteristics and may play multifunctional roles affecting the differential performance of AMF isolates, in terms of infectivity and efficiency

Quorum sensing in rhizobia isolated from the spores of the mycorrhizal symbiont Rhizophagus intraradices

Most beneficial services provided by arbuscular mycorrhizal fungi (AMF), encompassing improved crop performance and soil resource availability, are mediated by AMF-associated bacteria, showing key-plant growth-promoting (PGP) traits, i.e., the production of indole acetic acid, siderophores and antibiotics, and activities increasing the availability of plant nutrients by nitrogen fixation and phosphate mobilization. Such functions may be affected by the ability of AMF-associated bacteria to communicate through the production and secretion of extracellular small diffusible chemical signals, N-acyl homoserine lactone signal molecules (AHLs), that regulate bacterial behavior at the community level (quorum sensing, QS). This work investigated the occurrence and extent of QS among rhizobia isolated from AMF spores, using two different QS reporter strains, Agrobacterium tumefaciens NTL4 pZRL4 and Chromobacterium violaceum CV026. We also assessed the quorum quenching (QQ) activity among Bacillus isolated from the same AMF spores. Most rhizobia were found to be quorum-signaling positive, including six isolates producing very high levels of AHLs. The results were confirmed by microtiter plate assay, which detected 65% of the tested bacteria as medium/high AHL producers. A 16S rDNA sequence analysis grouped the rhizobia into two clusters, consistent with the QS phenotype. None of the tested bacteria showed QQ activity able to disrupt the QS signaling, suggesting the absence of antagonism among bacteria living in AMF sporosphere. Our results provide the first evidence of the ability of AMF-associated rhizobia to communicate through QS, suggesting further studies on the potential importance of such a behavior in association with key-plant growth-promoting functions

Assessment of the Life Cycle Environmental Impact of the Olive Oil Extraction Solid Wastes in the European Union

There is an increasing interest in developing sustainable systems in the European Union (EU) to recover and upgrade the solid wastes of the olive oil extraction process, i.e. wet husk. A Life Cycle Environmental Impact Assessment (LCIA) of wet husk has been carried out aiming at facilitating an appropriate Life Cycle Management of this biomass. Three scenarios have been considered, i.e. combustion for domestic heat, generation of electric power, and composting. The Environmental Product Declaration and the ReCiPe method were used for Life Cycle Impact Assessment. Domestic heating and power generation were the most important impact factors in damaging human health, ecosystems, and natural resources depletion. Composting was 2-4 orders of magnitude less impacting than domestic heat and power generation. Considering human health, the impact of climate change, human toxicity and particulate matter formation represented the main impact categories. Considering ecosystems, climate change and natural land transformation were the main impact categories. Within natural resources, fossil fuel depletion was impacted three orders more than metal depletion. Within domestic heating and power generation scenarios, storage of wet husk along with the extraction by organic solvent, and the waste treatment were the most impacting phases for global warming potential, ozone layer depletion, acidification and non renewable fossil resources depletion. The results obtained for the waste disposal have been comparatively assessed with respect to the environmental impact of the olive oil production chain

Microbially-enhanced composting of olive mill solid waste (wet husk): Bacterial and fungal community dynamics at industrial pilot and farm level.

Bacterial and fungal community dynamics during microbially-enhanced composting of olive mill solid waste (wet husk), used as a sole raw material, were analysed in a process carried out at industrial pilot and at farm level by the PCR-DGGE profiling of the 16 and 26S rRNA genes. The use of microbial starters enhanced the biotransformation process leading to an earlier and increased level of bacterial diversity. The bacterial community showed a change within 15 days during the first phases of composting. Without microbial starters bacterial biodiversity increased within 60 days. Moreover, the thermophilic phase was characterized by the highest bacterial biodiversity. By contrast, the biodiversity of fungal communities in the piles composted with the starters decreased during the thermophilic phase. The biodiversity of the microbial populations, along with physico-chemical traits, evolved similarly at industrial pilot and farm level, showing different maturation times

Composition of health-promoting phenolic compounds in two extra-virgin olive oils and diversity of associated yeasts

Extra virgin olive oil (EVOO), a basic component of the Mediterranean diet, is an important functional food, for its content in health-promoting compounds, showing antioxidant, antiinflammatory and antiproliferative activities. Here, two Tuscan EVOOs were analyzed for the occurrence and concentrations of health-promoting phenols, such as tyrosol and hydroxytyrosol and the secoiridoid derivatives, oleocanthal and oleacein. Independently of the milling period, the two EVOOs showed different contents of oleocanthal and oleacein. During storage, the contents of oleocanthal and oleacein decreased, while those of simple phenols increased. In all oil samples oleacein displayed a higher rate of reduction than oleocanthal. Multivariate analyses of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles, revealed the occurrence and diversity of oil-borne yeast communities, which differed in the two EVOOs. Sequences of excised DGGE bands identified Candida adriatica, Eremothecium coryli and Lachancea fermentati as the main components of the oil-borne yeast community. Our work detected, for the first time, differences in the content of tyrosol, hydroxytyrosol, oleocanthal and oleacein between the two Tuscan EVOOs analyzed, consistent with the differences found in yeast community composition. Further studies could confirm whether oil-borne yeasts may affect the composition of health-promoting oil phenolic compounds

A coding region in Diaporthe helianthi reveals genetic variability among isolates of different geographic origin

A set of Diaporthe helianthi isolates collected in different geographic areas was studied in order to examine whether different genetic biotypes could be responsible for epidemiological differences shown by sunflower stem canker. D. helianthi causes serious losses in France and in former Yugoslavia, while the pathogen is only sporadically recorded in Italy in spite of conducive pedoclimatic conditions. Variability of a D. helianthi coding genomic region, evaluated by means of polymerase chain reaction (PCR), Southern blot hybridisation and restriction fragments length polymorphism (RFLP), showed a conserved homogeneous pattern shared by French and Yugoslavian isolates compared with the heterogeneous pattern of Italian isolates. These results are consistent with other investigations (IGS and ITS region variability) performed on the same set of isolates, allowing a correlation between D. helianthi biotypes, their geographic origin and sunflower stem canker epidemiology

Multitrophic interactions in the sporosphere of the plant beneficial symbiont Rhizoglomus intraradices

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) establish beneficial associations with the roots of most land plants, including important food crops, from cereals to legumes, vegetables and fruit trees, and contribute to key agroecosystem processes, such as nutrient uptake, soil aggregation and carbon sequestration. In addition, AMF protect plants from soilborne fungal pathogens and abiotic stresses, such as drought and salinity. AMF performance is affected by the diverse bacterial communities strictly associated with their spores, which differentially enhance the availability of nutrients, such as P, N, S, K, Ca, Cu and Zn. In our work we utilized: i) a culture-independent approach, PCR-DGGE, to identify the diverse bacterial species associated with Rhizoglomus intraradices IMA6 spores; ii) a culture-dependent approach to isolate such bacteria and detect their functionally important traits. Sequence analysis of the major DGGE bands showed the occurrence of Arthrobacter, Bacillus, Herbaspirillum, Massilia, Pseudomonas, Rhizobium, Streptomyces genera, possibly playing functional roles in the enhancement of nutrient availability to fungal mycelium and plant roots, in plant pathogens control and in plant growth promotion. Isolation in pure culture of the microbiota associated with R. intraradices spores showed the occurrence of 374 strains, which were functionally characterized for P-solubilizing, chitinase, nitrogen fixing activity and production of siderophores and indole acetic acid (IAA). Phosphatase and phytase activities were detected in 73% and 100% of Actinobacteria, in 74% and 83% of chitinolytic bacteria and in 44% and 52% of nitrogen-fixers. Variable combinations of such bacteria may be at the basis of the differential performance of AMF isolates, in terms of infectivity and efficiency. The isolation of bacteria strictly associated with AMF spores and the investigation of their functional significance represent an essential step, in order to select the best AMF/bacteria combinations to be used as biofertilisers and bioenhancers in sustainable agroecosystems