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

Agronomic strategies to enhance the early vigor and yield of maize. Part I: the role of seed applied biostimulant, hybrid and starter fertilization on rhizosphere bacteria profile and diversity

The sustainable intensification of maize-based systems may reduce greenhouse-gas emissions and the excessive use of non-renewable inputs. Considering the key role that the microbiological fertility has on crop growth and resilience, it is worth of interest studying the role of cropping system on the rhizosphere bacterial communities, that affect soil health and biological soil fertility. In this work we monitored and characterized the diversity and composition of native rhizosphere bacterial communities during the early growth phases of two maize genotypes of different early vigor, using a nitrogen (N)-phosphorus (P) starter fertilization and a biostimulant seed treatment, in a growth chamber experiment, by polymerase chain reactiondenaturing gradient gel electrophoresis of partial 16S rRNA gene and amplicon sequencing. Cluster analyses showed that the biostimulant treatment affected the rhizosphere bacterial microbiota of the ordinary hybrid more than that of the early vigor, both at plant emergence and at the 5-leaf stage. Moreover, the diversity indices calculated from the community profiles, revealed significant effects of NP fertilization on richness and the estimated effective number of species (H2) in both maize genotypes, while the biostimulant had a positive effect on plant growth promoting community of the ordinary hybrid, both at the plant emergence and at the fifth leaf stage. Our data showed that maize genotype was the major factor shaping rhizosphere bacterial community composition suggesting that the root system of the two maize hybrids recruited a different microbiota. Moreover, for the first time, we identified at the species and genus level the predominant native bacteria associated with two maize hybrids differing for vigor. These results pave the way for further studies to be performed on the effects of cropping system and specific crop practices, considering also the application of biostimulants, on beneficial rhizosphere microorganisms

Effects of pavements on diversity and activity of mycorrhizal symbionts associated with urban trees

This study investigated the impact of different types of soil sealing on the communities of a group of beneficial plant symbionts, arbuscular mycorrhizal fungi (AMF), colonizing the roots of two shade trees, Celtis australis and Fraxinus ornus, frequently grown in urbanized sites. Such plants were grown in an experimental site, in northern Italy, established in November 2011 and subjected to four different pavement treatments: impermeable monolithic asphalt, permeable pavers, permeable concrete and unpaved soil. The diversity and composition of root AMF communities were assessed by PCR denaturating gradient gel electrophoresis of partial 18S rRNA gene, AMF taxa were identified by amplicon sequencing and mycorrhizal colonization was evaluated after root clearing and staining. For the first time, our molecular work revealed that impermeable pavements induced shifts in the composition of AMF communities associated to the roots of C. australis and F. ornus and impacted on the percentage of mycorrhizal root length. When the root-zone was covered with permeable pavements, a similar AMF community as that observed in the unpaved soil was detected, providing novel information to be utilised for reducing the disturbance caused by specific types of soil sealing on AMF symbionts, which play a key role in plant nutrition and health. A total of 45 AMF sequence types were detected, with Sclerocystis and Septoglomus as the most abundant phylotypes, accounting for 84% of the sequences. The predominance of Sclerocystis species in the roots of both tree species under impermeable pavements indicated their high and unforeseen tolerance towards harsh environmental conditions. Such species could be utilized as AMF inocula specifically selected for their proven resilience in paved sites, in order to exploit their ability to boost biogeochemical processes fundamental for energy fluxes and plant nutrition and health

Native mycorrhizal communities in maize roots as affected by plant genotype, starter fertilization and a seed-applied biostimulant

Background and aims: One of the most promising strategies for sustainable intensification of crop production involves the utilization of beneficial root-associated microorganisms, such as plant growth-promoting bacteria and arbuscular mycorrhizal fungi (AMF). The aim of this study was to investigate whether a seed-applied biostimulant, based on the bacterial strain Bacillus amyloliquefaciens IT-45 and a plant polysaccharide extract, and crop enhancement tools, such as hybrids with contrasting early vigor and nitrogen (N) plus phosphorus (P) starter fertilization, and their interactions, shape the communities of native root-colonizing AMF symbionts in maize. Methods: A factorial growth chamber experiment was set up with two maize genotypes in natural soil. Mycorrhizal colonization was evaluated after root staining. The diversity and composition of AMF communities were assessed by PCR-DGGE of the 18S rRNA gene and amplicon sequencing. Results: N and P fertilization determined a consistent reduction of AMF root colonization and, in combination of biostimulant, a reduction of AMF richness. The biostimulant alone generally did not affect AMF colonization or the community biodiversity. In addition the effect of the two factors were modulated by maize genotype. In all treatments, predominant AMF were represented by Glomus sp. and Funneliformis mosseae, while populations of the genus Rhizoglomus were rarely detected in biostimulant and NP fertilization treatments. Conclusion: The results of this study increase our understanding of how the biostimulant seed treatment may affect native AMF communities, depending on NP fertilization and maize genotype and may improve the implementation of innovative tools in sustainable and resilient agroecosystems