Bioprospecting for plant resilience to climate change: mycorrhizal symbionts of European and American beachgrass (Ammophila arenaria and Ammophila breviligulata) from maritime sand dunes.

Climate change and global warming have contributed to increase terrestrial drought, causing negative impacts on agricultural production. Drought stress may be addressed using novel agronomic practices and beneficial soil microorganisms, such as arbuscular mycorrhizal fungi (AMF), able to enhance plant use efficiency of soil resources and water and increase plant antioxidant defence systems. Specific traits functional to plant resilience improvement in dry conditions could have developed in AMF growing in association with xerophytic plants in maritime sand dunes, a drought-stressed and low-fertility environment. The most studied of such plants are European beachgrass (Ammophila arenaria Link), native to Europe and the Mediterranean basin, and American beachgrass (Ammophila breviligulata Fern.), found in North America. Given the critical role of AMF for the survival of these beachgrasses, knowledge of the composition of AMF communities colonizing their roots and rhizospheres and their distribution worldwide is fundamental for the location and isolation of native AMF as potential candidates to be tested for promoting crop growth and resilience under climate change. This review provides quantitative and qualitative data on the occurrence of AMF communities of A. arenaria and A. breviligulata growing in European, Mediterranean basin and North American maritime sand dunes, as detected by morphological studies, trap culture isolation and molecular methods, and reports on their symbiotic performance. Moreover, the review indicates the dominant AMF species associated with the two Ammophila species and the common species to be further studied to assess possible specific traits increasing their host plants resilience toward drought stress under climate change.

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 reaction-denaturing 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.

Storage time and temperature affect microbial dynamics of yeasts and acetic acid bacteria in a kombucha beverage.

Kombucha is a mildly sweet, slightly acidic fermented beverage, commercially available worldwide, that has attracted increasing consumers' interest due to its potential health benefits. Kombucha is commonly prepared using sugared black or green tea, but also other plant substrates are frequently utilised. Kombucha is obtained by fermentation using a symbiotic culture of bacteria and yeasts, whose composition varies depending on inoculum origin, plant substrates and environmental conditions. After fermentation, kombucha drinks are usually refrigerated at 4 °C, in order to maintain their biological and functional properties. There are no reports on the fate of microbial communities of kombucha in relation to long-term storage time and temperature. Here, for the first time, we monitored the diversity and dynamics of the microbial communities of a kombucha beverage fermented with different herbs during storage at 4 °C and at room temperature, for a period of 90 days, utilising culture-dependent and independent approaches. Moreover, cultivable yeasts and acetic acid bacteria (AAB) were isolated from the beverage, inoculated in pure culture, identified by molecular methods, and yeasts assessed for their functional properties. Total yeast counts were not affected by storage temperature and time, although their community composition changed, as Saccharomyces species significantly decreased after 45 days of storage at room temperature, completely disappearing after 90 days. On the other hand, Dekkera anomala (Brettanomyces anomalus), representing 52 % of the yeast isolates, remained viable up to 90 days at both storage temperatures, and was able to produce high levels of organic acids and exopolysaccharides. Data from DGGE (Denaturing Gradient Gel Electrophoresis) band sequencing confirmed that it was the dominant yeast species in all samples across storage. Other yeast isolates were represented by Saccharomyces and Zygosaccharomyces species. Among AAB, Gluconobacter oxydans, Novacetimonas hansenii and Komagataeibacter saccharivorans represented 46, 36 and 18 % of the isolates, whose occurrence remained unchanged across storage at 4 °C and did not vary up to 20 days of storage at room temperature. This work showed that the combination of culture-dependent and independent approaches is important for obtaining a complete picture of the distinctive core microbial community in kombucha beverages during storage, elucidating its diversity and composition, and preliminary characterizing yeast strains with putative functional activities.

Impact of sheep wool residues as soil amendments on olive beneficial symbionts and bacterial diversity.

In recent years the use of organic matter soil amendments, such as agricultural by-products, has been implemented with the aim of increasing soil fertility, while minimizing the environmental impact of agriculture. Sheep wool residues (SWR) have shown beneficial effects on plant nutrition and soil properties, while only few works assessed their impact on soil microbial communities. The main aim of this work was to investigate the possible valorization of two SWR types (scoured residues, white wool, WW, and carbonized scoured residues, black wool, BW) as organic soil amendments, in pot-grown olive trees, by evaluating their impact on soil bacterial communities and mycorrhizal symbionts. The two SWR types did not negatively impact on the diversity and composition of soil bacterial communities, as revealed by PCR-denaturating gradient gel electrophoresis (PCR-DGGE) of partial 16S rRNA gene, and on the activity of native arbuscular mycorrhizal fungi (AMF), while positively affecting plant growth. Only the highest doses of one SWR type (2% BW) caused a decrease in bacterial diversity and native AMF ability to colonize olive roots. DGGE bands sequencing allowed the identification of the major bacterial taxa. Sequences corresponding to Ohtaekwangia spp., Beta proteobacterium, Blastocatella sp., Ramlibacter monticola and Massilia frigida/rubra, Dongia sp. and Chloroflexi were mainly represented in SWR-amended soils, while those represented by Chryseolinea soli and Acidobacteria were abundant in control soil. Overall, this work showed that SWR may be valorized as organic soil amendments, as soil bacteria and AMF, representing key factors of biological soil fertility, were not negatively affected, while the activity of bacterial genera and species known for their ability to decompose complex compounds was boosted. Further studies will investigate the biodegradation efficiency of the diverse bacterial taxa developing in SWR-amended soils.

Use of chitosan and tannins as alternatives to antibiotics to control mold growth on PDO Pecorino Toscano cheese rind.

The fungal microbiota usually growing on the cheese surface during ripening processes promote rind formation and the development of organoleptic characteristics, imparting positive sensory attributes to cheeses. As cheese contamination may also occur by undesirable molds, specific actions for preventing their growth are usually realized in dairy industries by using the antibiotic natamycin, which may represent a risk factor for human health and environmental sustainability. Here, agroindustrial by-products with natural antimicrobial properties, i.e. tannins and chitosan, were tested in a cheese-making trial producing PDO Tuscan pecorino cheese. Morphological and molecular methods revealed that the main components of rind fungal communities of PDO Tuscan pecorino cheese were represented by P. solitum, P. discolour and P. verrucosum. The use of chitosan on cheese rinds did not significantly affect the composition of rind fungal communities developing during the whole ripening process compared with controls treated with natamycin, whose numbers ranged from 3.4 ± 1.3 × 103 to 3.2 ± 1.8 × 104 and from 6.3 ± 3.5 × 102 to 4.0 ± 1.5 × 104, respectively. Overall, grape marc tannins and chitosan did not significantly affect the number and composition of fungal communities developing during PDO Pecorino Toscano cheese ripening, as well as its physical, chemical and nutritional profiles, showing that they may represent effective alternatives to the antibiotic natamycin.

Beneficial Plant Microorganisms Affect the Endophytic Bacterial Communities of Durum Wheat Roots as Detected by Different Molecular Approaches.

This study aimed at characterising the endophytic bacterial communities living in durum wheat roots, as affected by wheat cultivar and inoculation of the Arbuscular mycorrhizal fungus Funneliformis mosseae IMA1 and the wheat root endophytic bacterium Lactobacillus plantarum B.MD.R.A2. These microorganisms were inoculated, alone or in combination, in durum wheat (cultivars Odisseo and Saragolla). Non-inoculated plants of each cultivar represented the controls. Forty-three days after sowing, roots were deprived of the epiphytic microbiota and subjected to DNA extraction. The DNA was used as template in PCR-DGGE analysis of the 16S rRNA gene (variable region V3-V5) and 16S (region V1-V3) metagenetics. Odisseo and Saragolla root endophytic bacterial biotas differed for number of OTUs and composition. In detail, Pseudomonas was higher in Odisseo than in Saragolla. The inoculation of F. mosseae and L. plantarum increased the abundance of Pseudomonas, some Actinobacteria (e.g., Streptomyces, Microbacterium, two genera including several plant growth promoting (PGP) strains) and Bacteroidetes in both cultivars. However, the endophytic bacterial biota of Saragolla roots inoculated just with lactobacilli did not differ from that of the control. The inoculation of Saragolla with F. mosseae, alone or in combination with lactobacilli, led to higher abundance of Rhodococcus, belonging to Actinobacteria and encompassing PGP strains. First, this work showed that F. mosseae and L. plantarum shape the endophytic bacterial biota of durum wheat roots. Abundance of some OTUs was affected by the microbial inoculation, depending on the cultivar. This result represents a starting point for exploitation of beneficial endophytes of wheat roots.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.

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.

Bacteria Associated With a Commercial Mycorrhizal Inoculum: Community Composition and Multifunctional Activity as Assessed by Illumina Sequencing and Culture-Dependent Tools.

The implementation of sustainable agriculture encompasses practices enhancing the activity of beneficial soil microorganisms, able to modulate biogeochemical soil cycles and to affect soil fertility. Among them, arbuscular mycorrhizal fungi (AMF) establish symbioses with the roots of most food crops and play a key role in nutrient uptake and plant protection from biotic and abiotic stresses. Such beneficial services, encompassing improved crop performances, and soil resources availability, are the outcome of the synergistic action of AMF and the vast communities of mycorrhizospheric bacteria living strictly associated with their mycelium and spores, most of which showing plant growth promoting (PGP) activities, such as the ability to solubilize phosphate and produce siderophores and indole acetic acid (IAA). One of the strategies devised to exploit AMF benefits is represented by the inoculation of selected isolates, either as single species or in a mixture. Here, for the first time, the microbiota associated with a commercial AMF inoculum was identified and characterized, using a polyphasic approach, i.e., a combination of culture-dependent analyses and metagenomic sequencing. Overall, 276 bacterial genera were identified by Illumina high-throughput sequencing, belonging to 165 families, 107 orders, and 23 phyla, mostly represented by Proteobacteria and Bacteroidetes. The commercial inoculum harbored a rich culturable heterotrophic bacterial community, whose populations ranged from 2.5 to 6.1 × 106 CFU/mL. The isolation of functional groups allowed the selection of 36 bacterial strains showing PGP activities. Among them, 14 strains showed strong IAA and/or siderophores production and were affiliated with Actinomycetales (Microbacterium trichotecenolyticum, Streptomyces deccanensis/scabiei), Bacillales (Bacillus litoralis, Bacillus megaterium), Enterobacteriales (Enterobacter), Rhizobiales (Rhizobium radiobacter). This work demonstrates for the first time that an AMF inoculum, obtained following industrial production processes, is home of a large and diverse community of bacteria with important functional PGP traits, possibly acting in synergy with AMF and providing additional services and benefits. Such bacteria, available in pure culture, could be utilized, individually and/or in multispecies consortia with AMF, as biofertilizers and bioenhancers in sustainable agroecosystems, aimed at minimizing the use of chemical fertilizers and pesticides, promoting primary production, and maintaining soil health and fertility.

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 essential elements of soil fertility and plant nutrition and productivity, facilitating soil mineral nutrient uptake and protecting plants from biotic and abiotic stresses. These beneficial services may be mediated by the dense and active spore-associated bacterial communities, which sustain diverse functions, such as the 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 by 16S 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.

Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis.

The effect of different sulphur dioxide concentrations on culturability and viability of seven strains of Brettanomyces bruxellensis was tested in a synthetic wine medium (SWM) and a different response to molecular SO(2) among strains was detected. Sulphur dioxide induced a viable but non culturable (VBNC) state in all the strains. The greater percentage of VBNC cells were identified for five strains at molecular SO(2) concentrations of 0.2mg/L and for two strains at the concentration of 0.4mg/L. Vinyl phenols were detected in media containing VBNC or not viable B. bruxellensis, suggesting that its spoilage metabolism could be maintained during wine storage. Overall, this study indicates that SO(2) is a chemical stressor inducing VBNC state in B. bruxellensis grown in synthetic wine medium. Further studies are needed to evaluate the effects of SO(2) on the metabolism of this yeast in wine spoilage.

Genetic and phenotypic diversity of geographically different isolates of Glomus mosseae.

In this work, we combined morphological taxonomy and molecular methods to investigate the intraspecific diversity of Glomus mosseae, whose global distribution has been reviewed by a survey of scientific literature and Web-available records from international germplasm collections (International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi and International Bank of Glomeromycota). We surveyed 186 publications reporting the occurrence of G. mosseae from at least 474 different sites from 55 countries throughout all continents, producing a geographical map of their distribution. The relationships among G. mosseae isolates originating from Europe (United Kingdom), the United States (Arizona, Florida, and Indiana), Africa (Namibia), and West Asia (Syria) were analyzed. The level of resolution of internal transcribed spacer (ITS) sequences strongly supports the morphological species definition of G. mosseae. An ITS - restriction fragment length polymorphism assay with the enzyme HinfI yielded a unique profile for all G. mosseae isolates, allowing a straightforward identification of this morphospecies. Genetic variability among G. mosseae isolates was revealed by the inter-simple-sequence repeat (ISSR) - polymerase chain reaction: the magnitude of genetic divergence shown by the investigated geographical isolates was higher than 50%, consistent with previous data on vegetative compatibility and functional diversity. The variability of ISSR patterns suggests that intraspecific diversity is much higher than that foreseen by morphology and rDNA regions, and should be further investigated by using other genes, such as those related to functional diversity.

Grapevine vitivirus A eradication in Vitis vinifera explants by antiviral drugs and thermotherapy.

Grapevine shoot cultures infected by Grapevine vitivirus A (GVA) were grown on Quorin-Lepoivre basic medium and submitted to in vitro chemotherapy and thermotherapy sanitation techniques. Ribavirin (Rb) at 20gml(-1), dihydroxypropyladenine (DHPA) at 60gml(-1) and their combination (RbDH) were added to the proliferating medium for three subsequent subcultures of 30 days each. Phytotoxicity was observed on drug-treated plantlets, which displayed a high percentage of mortality for each drug at doses higher than those aforementioned. Sequential ELISA were performed at the end of each subculture and ELISA-negative explants were submitted to RT-PCR. ELISA showed no antiviral activity following DHPA administration. Rb and RbDH treatment produced ELISA-negative explants which were assayed by RT-PCR and nested PCR. Biomolecular results showed no virus eradication in Rb treated explants but RbDH administration generated a percentage (40.0%) of GVA-free plantlets that permitted restoration of a new healthy generation of explants. Sixty percent (60%) of GVA eradication as confirmed by RT-PCR was obtained by in vitro thermotherapy at 36 degrees C for 57 days.

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.