Aridity modulates belowground bacterial community dynamics in olive tree.

Aridity negatively affects the diversity and abundance of edaphic microbial communities and their multiple ecosystem services, ultimately impacting vegetation productivity and biotic interactions. Investigation about how plant-associated microbial communities respond to increasing aridity is of particular importance, especially in light of the global climate change predictions. To assess the effect of aridity on plant associated bacterial communities, we investigated the diversity and co-occurrence of bacteria associated with the bulk soil and the root system of olive trees cultivated in orchards located in higher, middle and lower arid regions of Tunisia. The results indicated that the selective process mediated by the plant root system is amplified with the increment of aridity, defining distinct bacterial communities, dominated by aridity-winner and aridity-loser bacteria negatively and positively correlated with increasing annual rainfall, respectively. Aridity regulated also the co-occurrence interactions among bacteria by determining specific modules enriched with one of the two categories (aridity-winners or aridity-losers), which included bacteria with multiple PGP functions against aridity. Our findings provide new insights into the process of bacterial assembly and interactions with the host plant in response to aridity, contributing to understand how the increasing aridity predicted by climate changes may affect the resilience of the plant holobiont.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.

Improved plant resistance to drought is promoted by the root-associated microbiome as a water stress-dependent trait.

Although drought is an increasing problem in agriculture, the contribution of the root-associated bacterial microbiome to plant adaptation to water stress is poorly studied. We investigated if the culturable bacterial microbiome associated with five grapevine rootstocks and the grapevine cultivar Barbera may enhance plant growth under drought stress. Eight isolates, over 510 strains, were tested in vivo for their capacity to support grapevine growth under water stress. The selected strains exhibited a vast array of plant growth promoting (PGP) traits, and confocal microscopy observation of gfp-labelled Acinetobacter and Pseudomonas isolates showed their ability to adhere and colonize both the Arabidopsis and grapevine rhizoplane. Tests on pepper plants fertilized with the selected strains, under both optimal irrigation and drought conditions, showed that PGP activity was a stress-dependent and not a per se feature of the strains. The isolates were capable of increasing shoot and leaf biomass, shoot length, and photosynthetic activity of drought-challenged grapevines, with an enhanced effect in drought-sensitive rootstock. Three isolates were further assayed for PGP capacity under outdoor conditions, exhibiting the ability to increase grapevine root biomass. Overall, the results indicate that PGP bacteria contribute to improve plant adaptation to drought through a water stress-induced promotion ability.

Are drought-resistance promoting bacteria cross-compatible with different plant models?

The association between plant and plant growth promoting bacteria (PGPB) contributes to the successful thriving of plants in extreme environments featured by water shortage. We have recently shown that, with respect to the non-cultivated desert soil, the rhizosphere of pepper plants cultivated under desert farming hosts PGPB communities that are endowed with a large portfolio of PGP traits. Pepper plants exposed to bacterial isolates from plants cultivated under desert farming exhibited a higher tolerance to water shortage, compared with untreated control. This promotion was mediated by a larger root system (up to 40%), stimulated by the bacteria, that enhanced plant ability to uptake water from dry soil. We provide initial evidence that the nature of the interaction can have a limited level of specificity and that PGPB isolates may determine resistance to water stress in plants others than the one of the original isolation. It is apparent that, in relation to plant resistance to water stress, a feature of primary evolutionary importance for all plants, a cross-compatibility between PGPB and different plant models exists at least on a short-term.

Plant growth promotion potential is equally represented in diverse grapevine root-associated bacterial communities from different biopedoclimatic environments.

Plant-associated bacteria provide important services to host plants. Environmental factors such as cultivar type and pedoclimatic conditions contribute to shape their diversity. However, whether these environmental factors may influence the plant growth promoting (PGP) potential of the root-associated bacteria is not widely understood. To address this issue, the diversity and PGP potential of the bacterial assemblage associated with the grapevine root system of different cultivars in three Mediterranean environments along a macrotransect identifying an aridity gradient were assessed by culture-dependent and independent approaches. According to 16S rRNA gene PCR-DGGE, the structure of endosphere and rhizosphere bacterial communities was highly diverse (P = 0.03) and was associated with a cultivar/latitudinal/climatic effect. Despite being diverse, the bacterial communities associated with Egyptian grapevines shared a higher similarity with the Tunisian grapevines than those cultivated in North Italy. A similar distribution, according to the cultivar/latitude/aridity gradients, was observed for the cultivable bacteria. Many isolates (23%) presented in vitro multiple stress resistance capabilities and PGP activities, the most frequent being auxin synthesis (82%), insoluble phosphate solubilisation (61%), and ammonia production (70%). The comparable numbers and types of potential PGP traits among the three different environmental settings indicate a strong functional homeostasis of beneficial bacteria associated with grape root.

A drought resistance-promoting microbiome is selected by root system under desert farming.

BACKGROUND: Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of "reverse desertification". Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (Capsicum annuum L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. Bacillus spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by Klebsiella spp. (61% and 44% respectively). Most of the isolates (95%) presented in vitro multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for Bacillus and the rhizobacteria strains. We show that the C. annuum rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress. CONCLUSIONS/SIGNIFICANCE: Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a "resource island" able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.

Cyanobacteria cause black staining of the National Museum of the American Indian Building, Washington, DC, USA.

Microbial deterioration of stone is a widely recognised problem affecting monuments and buildings all over the world. In this paper, dark-coloured staining, putatively attributed to microorganisms, on areas of the National Museum of the American Indian Building, Washington, DC, USA, were studied. Observations by optical and electron microscopy of surfaces and cross sections of limestone indicated that biofilms, which penetrated up to a maximum depth of about 1 mm, were mainly composed of cyanobacteria, with the predominance of Gloeocapsa and Lyngbya. Denaturing gradient gel electrophoresis analysis revealed that the microbial community also included eukaryotic algae (Trebouxiophyceae) and fungi (Ascomycota), along with a consortium of bacteria. Energy-dispersive X-ray spectroscopy analysis showed the same elemental composition in stained and unstained areas of the samples, indicating that the discolouration was not due to abiotic chemical changes within the stone. The dark pigmentation of the stone was correlated with the high content of scytonemin, which was found in all samples.

The stability and degradation of dietary DNA in the gastrointestinal tract of mammals: implications for horizontal gene transfer and the biosafety of GMOs.

The fate of dietary DNA in the gastrointestinal tract (GIT) of animals has gained renewed interest after the commercial introduction of genetically modified organisms (GMO). Among the concerns regarding GM food, are the possible consequences of horizontal gene transfer (HGT) of recombinant dietary DNA to bacteria or animal cells. The exposure of the GIT to dietary DNA is related to the extent of food processing, food composition, and to the level of intake. Animal feeding studies have demonstrated that a minor amount of fragmented dietary DNA may resist the digestive process. Mammals have been shown to take up dietary DNA from the GIT, but stable integration and expression of internalized DNA has not been demonstrated. Despite the ability of several bacterial species to acquire external DNA by natural transformation, in vivo transfer of dietary DNA to bacteria in the intestine has not been detected in the few experimental studies conducted so far. However, major methodological limitations and knowledge gaps of the mechanistic aspects of HGT calls for methodological improvements and further studies to understand the fate of various types of dietary DNA in the GIT.

Microbial deterioration of artistic tiles from the façade of the Grande Albergo Ausonia & Hungaria (Venice, Italy).

The Grande Albergo Ausonia & Hungaria (Venice Lido, Italy) has an Art Nouveau polychrome ceramic coating on its façade, which was restored in 2007. Soon after the conservation treatment, many tiles of the façade decoration showed coloured alterations putatively attributed to the presence of microbial communities. To confirm the presence of the biological deposit and the stratigraphy of the Hungaria tiles, stereomicroscope, optical and environmental scanning electron microscope observations were made. The characterisation of the microbial community was performed using a PCR-DGGE approach. This study reported the first use of a culture-independent approach to identify the total community present in biodeteriorated artistic tiles. The case study examined here reveals that the coloured alterations on the tiles were mainly due to the presence of cryptoendolithic cyanobacteria. In addition, we proved that the microflora present on the tiles was generally greatly influenced by the environment of the Hungaria hotel. We found several microorganisms related to the alkaline environment, which is in the range of the tile pH, and related to the aquatic environment, the presence of the acrylic resin Paraloid B72® used during the 2007 treatment and the pollutants of the Venice lagoon.

Molecular studies of microbial community structure on stained pages of Leonardo da Vinci's Atlantic Codex.

In 2006, after a visual inspection of the Leonardo da Vinci's Atlantic Codex by a scholar, active molds were reported to have been present on Codex pages showing areas of staining. In the present paper, molecular methods were used to assess the current microbiological risk to stained pages of the manuscript. Bacterial and fungal communities were sampled by a non-invasive technique employing nitrocellulose membranes. Denaturing gradient gel electrophoresis of 16 S rRNA gene and internal transcribed space regions were carried out to study the structure of the bacterial and fungal communities and band patterns were analyzed by the multivariate technique of principal component analysis. Any relationship between the presence of an active microbial community and staining was excluded. The presence of potential biodeteriogens was evaluated by constructing bacterial and fungal clone libraries, analyzing them by an operational taxonomic unit (OTU) approach. Among the bacteria, some OTUs were associated with species found on floors in clean room while others were identified with human skin contamination. Some fungal OTU representatives were potential biodeteriogens that, under proper thermo-hygrometric conditions, could grow. The retrieval of these potential biodeteriogens and microorganisms related to human skin suggests the need for a continuous and rigorous monitoring of the environmental conditions, and the need to improve handling procedures.

Scripta manent? Assessing microbial risk to paper heritage.

Paper, like all other cultural heritage materials, degrades over time, but conservation slows down the rate of its deterioration. There is a long history of cooperation between microbiologists and conservators of libraries and archival materials, but current approaches addressing paper deterioration need urgent reassessment to take full advantage of modern microbiological methodologies. The present article discusses what we believe are the current priority research areas in assessing microbial risk to paper heritage, and reports studies on a 13th century Italian manuscript and on Leonardo da Vinci's Atlantic Codex which illustrate the problems and challenges encountered when dealing with microbial investigations of paper artworks. The potential of using a more advanced microbiological approach is highlighted.

Groundwater remediation by an in situ biobarrier: a bench scale feasibility test for methyl tert-butyl ether and other gasoline compounds.

Most gasoline contains high percentages of methyl tert-butyl ether (MTBE) as an additive. The physico-chemical properties of this substance (high water solubility, low sorption in soil) result in high mobility and dissolved concentrations in soil. In situ permeable biological barriers (biobarriers, BBs) can remediate MTBE polluted groundwater by allowing pure cultures or microbial consortia to degrade MTBE when aerobic conditions are present, either by direct metabolism or cometabolism. Lab-scale batch and column tests were carried out to assess a selected microbial consortium in biodegrading MTBE and other gasoline compounds (benzene B, toluene T, ethylbenzene E, xylenes X) and to measure the parameters affecting the efficacy of a BB treatment of polluted groundwater. During the aerobic phase of the batch tests, the simultaneous biodegradation of MTBE, tert-butyl alcohol (TBA), B, T, E and o-X was observed. The rapid biodegradation of BTEXs resulted in decreased oxygen availability, but MTBE degradation was nevertheless measured in the presence of BTEXs. Stationary concentrations of MTBE and TBA were measured when anoxic conditions occurred in the systems. Values for a first order kinetic removal process were obtained for MTBE (0.031+/-0.001 d(-1)), B (0.045+/-0.002 d(-1)) and T (0.080+/-0.004 d(-1)) in the inoculated column tests. The estimate of the BB design parameters suggested that inoculation could significantly modify (double) the longitudinal dispersivity value of the biomass support medium. No effect was observed in the retardation factors for MTBE, B and T.

The effect of copper on the structure of the ammonia-oxidizing microbial community in an activated sludge wastewater treatment plant.

Molecular approaches based on both whole-cell and extracted DNA were applied to assess chronic and acute effects of copper on the ammonia oxidizing bacteria (AOB) community in an activated sludge system. The ammonia monooxygenase amoA gene was chosen as the functional marker to evaluate changes in the AOB community. Using in situ polymerase chain reaction, we were able to visualize the peripheric distribution of the amoA gene-possessing bacteria in activated sludge flocs. The AOB biomass content was constant in both chronic and acute toxicity experiments, but the ammonia oxidizing activity, measured as ammonia uptake rate, was different. The AOB community structural changes due to the copper presence were evaluated by multivariate analysis of the DGGE bands profiles. The chronic contamination caused a change in the AOB community compared to the control. In contrast, acute inputs led to a temporary change in the AOB community, after which the community was similar to the control. Recovery after acute intoxication was achieved after 72 h. The present study reports on the effects of chronic and acute copper contamination on the ammonia uptake ability of the AO microorganisms and the structure of the AOB community in a wastewater system and, as a consequence, gives indications on the response of wastewater plants under similar conditions.

Exploration of methods used to describe bacterial communities in silage of maize (Zea mays) cultivars.

Different techniques to assess bacterial community structure and diversity were evaluated in silages prepared with four different maize cultivars, three conventional and one transgenic (cv. Tundra, event Bt-176). Plants were cultivated in the greenhouse and harvested after 30 days of growth. Silage samples were collected at successive times during fermentation and analyzed for bacterial counts and by various DNA-based fingerprinting techniques. Bacterial counts were similar between cultivars for the total culturable bacteria, sporeforming, and mesophilic and thermophilic lactic acid bacteria (LAB). Further analysis of the species composition of 388 LAB strains by intergenic transcribed spacer (ITS) PCR followed by sequencing of 16S rRNA gene did not reveal differences between cultivars. In contrast, molecular fingerprinting methods targeting whole bacterial communities, such as automated ribosomal intergenic spacers analysis (ARISA) and 16S rRNA gene length heterogeneity-PCR (LH-PCR), indicated that different maize silage batches or cultivars hosted different bacterial communities. Thus, ARISA and LH-PCR fingerprinting techniques offer a fast and sensitive method to compare bacterial communities, and to detect differences in silage bacterial communities.

Effects of rhizodeposition of non-transgenic and transplastomic tobaccos on the soil bacterial community.

The effect of root-released compounds of transplastomic tobacco (Nicotiana tabacum) on the soil bacterial community structure, and their potential to support horizontal gene transfer (HGT) to bacteria have been studied. Soil microcosms were exposed to root-released compounds collected from transplastomic and non-transgenic tobacco cultivars. Cluster analysis of automated ribosomal intergenic spacer analysis (ARISA) profiles of the soil bacterial community after 48 h incubation grouped the transgenic cultivar apart from the non-transgenic, indicating that it had a rhizodeposition pattern different from the parental plants. However, these differences were less than between the two non-transgenic tobacco cultivars studied. NMR characterization of the root-released compounds showed some differences in chemical fingerprinting pattern between the transplastomic and the parental cultivar. However, the effect on bacterial community structure was transient, and tended to disappear after 96 h of incubation. The potential of root-released compounds as a source of transforming DNA for bacteria was investigated by using four potential recipient species. No transformants were obtained following exposure of all the recipients to the root-released compounds. Root-released compounds amended to transgene donor DNA decreased the transformation frequency of Acinetobacter baylyi strain ADP1200, while Azospirillum, Agrobacterium, and Sinorhizobium strains failed to develop competence also in the presence of an external added transgene source. Detection of plastid sequences by PCR suggested that a very low amount of fragmented plastid donor DNA was present in the root-released compounds.

Strategy for in situ detection of natural transformation-based horizontal gene transfer events.

A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.

Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments.

This study compares two cleaning methods, one involving an ammonium carbonate-EDTA mixture and the other involving the sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, for the removal of black crust (containing gypsum) on marble of the Milan Cathedral (Italy). In contrast to the chemical cleaning method, the biological procedure resulted in more homogeneous removal of the surface deposits and preserved the patina noble under the black crust. Whereas both of the treatments converted gypsum to calcite, allowing consolidation, the chemical treatment also formed undesirable sodium sulfate.

Bacterial and fungal deterioration of the Milan Cathedral marble treated with protective synthetic resins.

Surfaces are continuously exposed to physical, chemical and biological degradation. Among the biological agents that cause deterioration, microorganisms are of critical importance. This work is part of a research programme for the characterisation of the alterations of the Milan Cathedral (Italy). Four stone samples of the Milan Cathedral were chemically analysed and the microbiological growth assessed. X-ray diffraction (XRD) showed that calcite was always present in each sample and one sample was also characterised by the chemical form of alteration gypsum. Using Fourier Transform Infrared Spectroscopy (FTIR) together with Scanning Electron Microscopy (SEM), it was possible to prove that the samples were consolidated with the synthetic acrylics and epoxy resins. The green-black biological patinas of the specimens were studied using cultivation, microscope observations and a method for single-cell detection. Sampling for fluorescent in-situ hybridisation (FISH), with ribosomal RNA targeted oligonucleotide probes, was also performed using adhesive tapes. The bulk of the prokaryotes were Bacteria but some Archaea were also found. The bacterial cells were further characterised using specific probes for Cyanobacteria, and alpha-, beta-and gamma-Proteobacteria. In addition, black fungi isolated from the stone and the fungi of the standard ASTM G21-96(2002) method were employed to test if the detected synthetic resins could be used as the sole source of carbon and energy. One isolated Cladosporium sp. attacked the freshly dried acrylic resin. Results show that the detected bacteria and fungi can cause severe damage both to the stone monument and its synthetic consolidants.

Synthetic consolidants attacked by melanin-producing fungi: case study of the biodeterioration of Milan (Italy) cathedral marble treated with acrylics.

Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.