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Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

Legume shrubs have great potential for rehabilitation of semi-arid degraded soils in Mediterranean ecosystems as they establish mutualistic symbiosis with Nfxing rhizobia. Eighty-eight symbiotic rhizobia were isolated from seven wild legume shrubs native of Sicily (Southern Italy) and grouped in operational taxonomic units (OTU) by analysis of the ribosomal internal transcribed spacer (ITS) polymorphism. Partial sequencing of 16S rRNA gene of representative isolates of each OTU revealed that most Genisteae symbionts are related to Bradyrhizobium canariense, B. japonicum and B. elkanii. Teline monspessulana was the only Genistea nodulated by Mesorhizobium strains, and Anagyris foetida (Thermopsideae) was promiscuosly nodulated by Rhizobium, Mesorhizobium, Agrobacterium and Bradyrhizobium strains. Analysis of the nodulation gene nodA assigned most Mediterranean Genisteae bradyrhizobia to clade II but also to clades IV, I and III, which included, so far, sequences of (sub)tropical and Australian isolates. The high diversity and low host speciWcity observed in most wild legumes isolates suggest that preferential associations may establish in the Weld depending on diVerences in the beneWts conferred to the host and on competition ability. Once identifed, these benefcial symbiosis can be exploited for rehabilitation of arid, low productive and human-impacted soils of the Mediterranean countries.

In a desert, plants as holobionts quickly respond to resource pulses like precipitation. However, little is known on how environment and plants modulate the rhizosphere-associated microbiome. As a model species to represent the Atacama Desert bloom, Cistanthe longiscapa (Montiaceae family) was selected to study the influence of abiotic and biotic environment on the diversity and structure of the microbiota associated to its rhizosphere. We analyzed the rhizosphere and soil microbiome along a North-South precipitation gradient and between a dry and rainy year by using Illumina high−throughput sequencing of 16S rRNA gene fragments and ITS2 regions for prokaryotes and fungi, respectively. In the rhizosphere of C. longiscapa the microbiota clearly differs in composition and structure from the surrounding bulk soil. The fungal and bacterial communities respond differently to environmental conditions. The diversity and richness of fungal OTUs were negatively correlated with aridity, as predicted. The community structure was predominantly influenced by other soil characteristics (pH, organic matter content) but not by aridity. In contrast, diversity, composition, and structure of the bacterial community were not influenced by aridity or any other evaluated soil parameter. These findings coincide with the identification of mainly site-specific microbial communities, not shared along the sites. These local communities contain a group of OTUs, which are exclusive to the rhizosphere of each site and presumably vertically inherited as seed endophytes. Their ecological functions and dispersal mechanisms remain unclear. The analysis of co-occurrence patterns highlights the strong effect of the desert habitat over the soil- and rhizosphere-microbiome. The site-independent enrichment of only a small bacterial cluster consistently associated with the rhizosphere of C. longiscapa further supports this conclusion. In a rainy year, the rhizosphere microbiota significantly differed from bulk and bare soil, whereas in a dry year, the community structure of the former rhizosphere approximates to the one found in the bulk. In the context of plant–microbe interactions in desert environments, our study contributes new insights into the importance of aridity in microbial community structure and composition, discovering the influence of other soil parameters in this complex dynamic network, which needs further to be investigated.

A Gram-negative bacterium, strain HA7T, was isolated from the microhabitat of common hazel (Corylus avellana L.) pollen. HA7T was found to be an aerobic, rod-shaped, catalase-positive, oxidase-negative bacterium with an optimum growth temperature of 25 °C and pH of 7. The nearly complete 16S rRNA gene sequence of HA7T strain showed the closest similarities to Spirosoma linguale DSM 74T (97.4 %) and Spirosoma fluviale DSM 29961T (97.43 %). The major fatty acids (>5 %) were C16 : 1ω5c, summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were an unidentified aminophospholipid and phosphatidylethanolamine. The major respiratory quinone detected was menaquinone MK-7 (95 %). The draft genome sequence included 8 794 837 bases, which contained 3665 predicted coding sequences and had a G+C content of 47.9 mol%. The genome-based comparison between HA7T and S. linguale DSM 74T and S. fluviale DSM 29961T with pairwise average nucleotide identity indicated a clear distinction, between 76.2–76.3 %. Moreover, the digital DNA–DNA relatedness of HA7T to these strains was 26.5 and 25.1 %. Based on the differential genotypic, phenotypic and chemotaxonomic properties to closely related type strains, strain HA7T ought to be assigned with the status of a new species, for which the name Spirosoma pollinicola sp. nov. is proposed. The type strain is HA7T (DSM 105799T=LMG 30282T).

Mangroves, dominating tropical intertidal zones and estuaries, are among the most salt tolerant plants, and propagate through reproductive units called propagules. Similarly to plant seeds, propagules may harbor beneficial bacteria. Our hypothesis was that mangroves, being able to grow into seawater, should harbor bacteria able to interact with the host and to exert positive effects under salt stress, which could be exploited to improve crop production. Therefore, we isolated bacterial endophytes from mangrove propagules with the aim to test whether these bacteria have a beneficial potential on their natural host and on different crops such as barley and rice, cultivated under salt stress. The 172 bacterial isolates obtained were screened for plant growth promotion (PGP) activities in vitro, and the 12 most promising isolates were tested on barley under non-axenic conditions and salt stress. Gordonia terrae KMP456-M40 was the best performing isolate, increasing ear weight by 65%. Based on the in vivo PGP activity and the root colonization ability, investigated by fluorescence in situ hybridization and confocal microscopy, three strains were additionally tested on mangrove propagule germination and on rice growth. The most effective strain was again G. terrae KMP456-M40, which enhanced the root length of mangrove seedlings and the biomass of salt-stressed rice under axenic conditions up to 65% and 62%, respectively. We demonstrated that propagules, the reproductive units of mangroves, host beneficial bacteria that enhance the potential of mangrove seedlings establishment and confer salt tolerance to cereal crops.

Flower pollen represents a unique microbial habitat, however the factors driving microbial assemblages and microbe–microbe interactions remain largely unexplored. Here we compared the structure and diversity of the bacterial–fungal microbiome between eight different pollen species (four wind-pollinated and four insect-pollinated) from close geographical locations, using high-throughput sequencing of the 16S the rRNA gene fragment (bacteria) and the internal transcribed spacer 2 (ITS2, fungi). Proteobacteria and Ascomycota were the most abundant bacterial and fungal phyla, respectively. Pseudomonas (bacterial) and Cladosporium (fungal) were the most abundant genera. Both bacterial and fungal microbiota were significantly influenced by plant species and pollination type, but showed a core microbiome consisting of 12 bacterial and 33 fungal genera. Co-occurrence analysis highlighted significant inter- and intra-kingdom interactions, and the interaction network was shaped by four bacterial hub taxa: Methylobacterium (two OTUs), Friedmanniella and Rosenbergiella. Rosenbergiella prevailed in insect-pollinated pollen and was negatively correlated with the other hubs, indicating habitat complementarity. Inter-kingdom co-occurrence showed a predominant effect of fungal on bacterial taxa. This study enhances our basic knowledge of pollen microbiota, and poses the basis for further inter- and intra-kingdom interaction studies in the plant reproductive organs.

The colonization behavior of the Xylella fastidiosa strain CoDiRO, the causal agent of olive quick decline syndrome (OQDS), within the xylem of Olea europaea L. is still quite controversial. As previous literature suggests, even if xylem vessel occlusions in naturally infected olive plants were observed, cell aggregation in the formation of occlusions had a minimal role. This observation left some open questions about the whole behavior of the CoDiRO strain and its actual role in OQDS pathogenesis. In order to evaluate the extent of bacterial infection in olive trees and the role of bacterial aggregates in vessel occlusions, we tested a specific fluorescence in situ hybridization (FISH) probe (KO 210) for X. fastidiosa and quantified the level of infection and vessel occlusion in both petioles and branches of naturally infected and non-infected olive trees. All symptomatic petioles showed colonization by X. fastidiosa, especially in the larger innermost vessels. In several cases, the vessels appeared completely occluded by a biofilm containing bacterial cells and extracellular matrix and the frequent colonization of adjacent vessels suggested a horizontal movement of the bacteria. Infected symptomatic trees had 21.6 ± 10.7% of petiole vessels colonized by the pathogen, indicating an irregular distribution in olive tree xylem. Thus, our observations point out the primary role of the pathogen in olive vessel occlusions. Furthermore, our findings indicate that the KO 210 FISH probe is suitable for the specific detection of X. fastidiosa.

The importance of the plant microbiome for host fitness has led to the concept of the "plant holobiont". Seeds are reservoirs and vectors for beneficial microbes, which are very intimate partners of higher plants with the potential to connect plant generations. In this study, the endophytic seed microbiota of numerous barley samples, representing different cultivars, geographical sites and harvest years, was investigated. Cultivation-dependent and -independent analyses, microscopy, functional plate assays, greenhouse assays and functional prediction were used, with the aim of assessing the composition, stability and function of the barley seed endophytic bacterial microbiota. Associations were consistently detected in the seed endosphere with Paenibacillus, Pantoea and Pseudomonas spp., which were able to colonize the root with a notable rhizocompetence after seed germination. In greenhouse assays, enrichment with these bacteria promoted barley growth, improved mineral nutrition and induced resistance against the fungal pathogen Blumeria graminis. We demonstrated here that barley, an important crop plant, was consistently associated with beneficial bacteria inside the seeds. The results have relevant implications for plant microbiome ecology and for the holobiont concept, as well as opening up new possibilities for research and application of seed endophytes as bioinoculants in sustainable agriculture.

Sphagnum mosses serve as a unique habitat for microorganisms, which play an important role both for the host plants and the peatland ecosystems. The aim of the present study was to isolate endophytic bacteria from the tissues of Sphagnum mosses and to screen them for strains promising for further application in agricultural microbiology. About 50 samples of Sphagnum fallax (H. Klinggr.) H. Klinggr. and Sphagnum magellanicum Brid. were collected in the Austrian Alps and the Lenindgrad Region of Russia in 2009–2010. Endophytic bacteria were detected inside the moss plants using fluorescent in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM). Altogether, 283 isolates were obtained by cultivation on the nutrient media. Examination of the isolates for the antagonistic activity revealed that more than 50% of them could suppress the growth of phytopathogenic and toxigenic fungi. More than 30% of isolates showed some antagonistic activity against microbial phytopathogens. The isolated strains could colonize crops and promote their growth. Molecular-genetic identification coupled with physiological/biochemical characterization showed that the dominant endophytic groups belonged to the genera Burkholderia, Pseudomonas, Flavobacterium, Serratia and Collimonas. The isolated endophytes were shown to be promising objects for the development of effective growth-promoting and protective microbiological preparations to be used in agriculture.

Plant protection via disease-suppressive bacteria in desert farming requires specific biological control agents (BCAs) adapted to the unique arid conditions. We performed an ecological study of below-ground communities in desert farm soil and untreated desert soil, and based on these findings, selected antagonists were hierarchically evaluated. In contrast to the highly specific 16S rRNA fingerprints of bacterial communities in soil and cultivated medicinal plants, internal transcribed spacer profiles of fungal communities were less discriminative and mainly characterised by potential pathogens. Therefore, we focused on in vitro bacterial antagonists against pathogenic fungi. Based on the antifungal potential and genomic diversity, 45 unique strains were selected and characterised in detail. Bacillus/Paenibacillus were most frequently identified from agricultural soil, but antagonists from the surrounding desert soil mainly belonged to Streptomyces. All strains produced antibiotics against the nematode Meloidogyne incognita, and one-third showed additional activity against the bacterial pathogen Ralstonia solanacearum. Altogether, 13 broad-spectrum antagonists with antibacterial, antifungal and nematicidal activity were found. They belong to seven different bacterial species of the genera Bacillus and Streptomyces. These Gram-positive, spore-forming bacteria are promising drought-resistant BCAs and a potential source for antibiotics. Their rhizosphere competence was shown by fluorescence in situ hybridisation combined with laser scanning microscopy.

Stenotrophomonas rhizophila DSM14405T is of high biotechnological interest as plant growth stimulator, especially for salinated conditions. The objective of this study was to determine the effect of plant species (cotton, tomato, and sweet pepper) on colonisation and plant growth promotion of this beneficial bacterium in gnotobiotic systems and in non-sterile soil. All plant structures (leaves, stems, and roots) were densely colonised by DSM14405T reaching up to 109 cells g−1 fresh weight; under gnotobiotic conditions the abundances were 4–5 orders of magnitude higher than in non-sterile soil. Under non-sterile conditions and ambient humidity, tomato shoots were more densely colonised than shoots of sweet pepper and cotton. S. rhizophila DSM14405T was shown to grow endophytically and colonise the vicinity of root hairs of tomato. Plant growth promotion was particularly apparent in tomato. In general, the impact of plant species on colonisation and plant growth promotion was more pronounced in soil than under gnotobiotic conditions and likely due to the control of diseases and deleterious microorganisms. S. rhizophila DSM14405T was shown to control diseases in sweet pepper and in cotton. Molecular profiling via single strand conformation polymorphism of internal transcribed spacers and 16S rRNA genes (PCR-single strand conformational polymorphism (SSCP)) revealed that S. rhizophila DSM14405T strongly affected fungal, but not bacterial communities in the rhizosphere of tomato and sweet pepper. Major SSCP bands related to uncultured fungi and Candida subhashii, disappeared in tomato rhizosphere after Stenotrophomonas treatment. This suggests an indirect, species-specific plant growth promotion effect of S. rhizophila via the elimination of deleterious rhizosphere organisms.

From the rhizosphere of two salt tolerant plant species, Hordeum secalinum and Plantago winteri growing in a naturally salt meadow, 100 strains were isolation on enrichment media for various plant growth-promoting (PGP) functions (ACC deaminase activity, auxin synthesis, calcium phosphate mobilization and nitrogen fixation). Based on the taxonomic affiliation of the isolated bacteria and their enrichment medium 22 isolates were selected to test their growth promotion effect on the crop barley (Hordeum vulgare) under salt stress in pot experiment. In parallel the isolates were characterized in pure culture for their plant growth-promoting activities. Surprisingly the best promotors did not display a promising set of PGP activities. Isolates with multiple PGP-activities in pure culture like Microbacterium natoriense strain E38 and Pseudomonas brassicacearum strain E8 did not promote plant growth. The most effective isolate was strain E108 identified as Curtobacterium flaccumfaciens, which increased barley growth up to 300%. In pure culture strain E108 showed only two out of six plant growth promoting activities and would have been neglected. Our results highlight that screening based on pure culture assays may not be suitable for recognition of best plant growth promotion candidates and could preclude the detection of both new PGPR and new plant promotion mechanisms.

Rhizobiales (Alphaproteobacteria) are well-known beneficial partners in plant-microbe interactions. Less is known about the occurrence and function of Rhizobiales in the lichen symbiosis, although it has previously been shown that Alphaproteobacteria are the dominating group in growing lichen thalli. We have analyzed the taxonomic structure and assigned functions to Rhizobiales within a metagenomic dataset of the lung lichen Lobaria pulmonaria L. One third (32.2%) of the overall bacteria belong to the Rhizobiales, in particular to the families Methylobacteriaceae, Bradyrhizobiaceae, and Rhizobiaceae. About 20% of our metagenomic assignments could not be placed in any of the Rhizobiales lineages, which indicates a yet undescribed bacterial diversity. SEED-based functional analysis focused on Rhizobiales and revealed functions supporting the symbiosis, including auxin and vitamin production, nitrogen fixation and stress protection. We also have used a specifically developed probe to localize Rhizobiales by confocal laser scanning microscopy after fluorescence in situ hybridization (FISH-CLSM). Bacteria preferentially colonized fungal surfaces, but there is clear evidence that members of the Rhizobiales are able to intrude at varying depths into the interhyphal gelatinous matrix of the upper lichen cortical layer and that at least occasionally some bacteria also are capable to colonize the interior of the fungal hyphae. Interestingly, the gradual development of an endosymbiotic bacterial life was found for lichen- as well as for fungal- and plant-associated bacteria. The new tools to study Rhizobiales, FISH microscopy and comparative metagenomics, suggest a similar beneficial role for lichens than for plants and will help to better understand the Rhizobiales-host interaction and their biotechnological potential.

Complex and distinct bacterial communities inhabit marine sponges and are believed to be essential to host survival, but our present-day inability to domesticate sponge symbionts in the laboratory hinders our access to the full metabolic breadth of these microbial consortia. We address bacterial cultivation bias in marine sponges using a procedure that enables direct comparison between cultivated and uncultivated symbiont community structures. Bacterial community profiling of the sympatric keratose species Sarcotragus spinosulus and lrcinia variabilis (Dictyoceratida, Irciniidae) was performed by polymerase chain reaction-denaturing gradient gel electrophoresis and 454-pyrosequecing of 16S rRNA gene fragments. Whereas cultivation-independent methods revealed species-specific bacterial community structures in these hosts, cultivation-dependent methods resulted in equivalent community assemblages from both species. Between 15 and 18 bacterial phyla were found in S. spinosulus and I. variabilis using cultivation-independent methods. However, Alphaproteobacteria and Gammaproteobacteria dominated the cultivation-dependent bacterial community. While cultivation-independent methods revealed about 200 and 220 operational taxonomic units (OTUs, 97% gene similarity) in S. spinosulus and I. variabilis, respectively, only 33 and 39 OTUs were found in these species via culturing. Nevertheless, around 50% of all cultured OTUs escaped detection by cultivation-independent methods, indicating that standard cultivation makes otherwise host-specific bacterial communities similar by selectively enriching for rarer and generalist symbionts. This study sheds new light on the diversity spectrum encompassed by cultivated and uncultivated sponge-associated bacteria. Moreover, it highlights the need to develop alternative culturing technologies to capture the dominant sponge symbiont fraction that currently remains recalcitrant to laboratory manipulation. Portuguese Foundation for Science and Technology (FCT) [PTDC/MAR/101431/2008]; FCT [SFRH/BD/60873/2009]; FCT postdoctoral fellowship [SFRII/BPD/62946/2009]; European Regional Development Fund (ERDF) through the COMPETE (Operational Competitiveness) Programme; FCT under the project [PEst-C/MAR/LA0015/2011]; FCT-funded research project [PTDC/BIA-MIC/3865/2012] info:eu-repo/semantics/publishedVersion

Lettuce belongs to the most commonly raw eaten food worldwide and its microbiome plays an important role for both human and plant health. Yet, little is known about the impact of potentially occurring pathogens and beneficial inoculants of the indigenous microorganisms associated with lettuce. To address this question we studied the impact of the phytopathogenic fungus Rhizoctonia solani and the biological control agent Bacillus amyloliquefaciens FZB42 on the indigenous rhizosphere and phyllosphere community of greenhouse-grown lettuce at two plant stages. The rhizosphere and phyllosphere gammaproteobacterial microbiomes of lettuce plants showed clear differences in their overall and core microbiome composition as well as in corresponding diversity indices. The rhizosphere was dominated by Xanthomonadaceae (48%) and Pseudomonadaceae (37%) with Rhodanobacter, Pseudoxanthomonas, Dokdonella, Luteimonas, Steroidobacter, Thermomonas as core inhabitants, while the dominating taxa associated to phyllosphere were Pseudomonadaceae (54%), Moraxellaceae (16%) and Enterobacteriaceae (25%) with Alkanindiges, Pantoea and a group of Enterobacteriaceae unclassified at genus level. The preferential occurrence of enterics in the phyllosphere was the most significant difference between both habitats. Additional enhancement of enterics on the phyllosphere was observed in bottom rot diseased lettuce plants, while Acinetobacter and Alkanindiges were identified as indicators of healthy plants. Interestingly, the microbial diversity was enhanced by treatment with both the pathogen, and the co-inoculated biological control agent. The highest impact and bacterial diversity was found by Rhizoctonia inoculation, but FZB42 lowered the impact of Rhizoctonia on the microbiome. This study shows that the indigenous microbiome shifts as a consequence to pathogen attack but FZB42 can compensate these effects, which supports their role as biocontrol agent and suggests a novel mode of action.

No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.

The correlation between the taxonomic composition of Alphaproteobacteria, Burkholderia and nitrogen fixers associated with the lichen Lobaria pulmonaria and the geographical distribution of the host was studied across four sites in Europe. Results proved that the diversity of Alphaproteobacteria is affected by geography, while those of Burkholderia and nitrogen fixers were mostly driven by local habitat. This difference indicates a higher stability of the association between Alphaproteobacteria and the lichen host.

Knowledge about Sphagnum-associated microbial communities, their structure and their origin is important to understand and maintain climate-relevant Sphagnum-dominated bog ecosystems. We studied bacterial communities of two cosmopolitan Sphagnum species, which are well adapted to different abiotic parameters (Sphagnum magellanicum, which are strongly acidic and ombrotrophic, and Sphagnum fallax, which are weakly acidic and mesotrophic), in three Alpine bogs in Austria by a multifaceted approach. Great differences between bacterial fingerprints of both Sphagna were found independently from the site. This remarkable specificity was confirmed by a cloning and a deep sequencing approach. Besides the common Alphaproteobacteria, we found a discriminative spectrum of bacteria; although Gammaproteobacteria dominated S. magellanicum, S. fallax was mainly colonised by Verrucomicrobia and Planctomycetes. Using this information for fluorescent in situ hybridisation analyses, corresponding colonisation patterns for Alphaproteobacteria and Planctomycetes were detected. Bacterial colonies were found in high abundances inside the dead big hyalocytes, but they were always connected with the living chlorocytes. Using multivariate statistical analysis, the abiotic factors nutrient richness and pH were identified to modulate the composition of Sphagnum-specific bacterial communities. Interestingly, we found that the immense bacterial diversity was transferred via the sporophyte to the gametophyte, which can explain the high specificity of Sphagnum-associated bacteria over long distances. In contrast to higher plants, which acquire their bacteria mainly from the environment, mosses as the phylogenetically oldest land plants maintain their bacterial diversity within the whole lifecycle.

A novel actinobacterium, designated strain CafT13T, was isolated from the thallus of the reindeer lichen Cladonia arbuscula sampled in the Austrian Alps (Koralpe). The organism was aerobic, with rod- to irregular-shaped cells (often forming dense clusters of cells when grown in liquid medium), Gram-stain-positive, oxidase-negative, catalase-positive and non-motile. It was able to grow at 1 °C and at low to neutral pH, but not above 30 °C or at high pH. The peptidoglycan type was B2β with ornithine as the diagnostic diamino acid. The menaquinones were MK-7 and MK-8. The polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids, three unidentified glycolipids and one unidentified aminolipid. The predominant fatty acids were C18 : 1, C14 : 0 2-OH, C17 : 1ω9c, C16 : 0 and anteiso-C15 : 0. The mean DNA G+C content of strain CafT13T was 69.0±0.17 mol%. 16S rRNA gene sequence analysis showed that strain CafT13T belongs to the family Microbacteriaceae, within the genus Frondihabitans. The mean level of DNA–DNA relatedness between strain CafT13T and the type strain of Frondihabitans australicus was 35.2±5.23 %. The enzyme spectrum of strain CafT13T differentiated it from recognized species of the genus Frondihabitans. Based on molecular, chemotaxonomic and physiological data, strain CafT13T is considered to represent a novel species of the genus Frondihabitans, for which the name Frondihabitans cladoniiphilus sp. nov. is proposed; the type strain is CafT13T ( = DSM 23273T = LMG 25550T).

Environmental proteomics, also referred to as metaproteomics, is an emerging technology to study the structure and function of microbial communities. Here, we applied semi-quantitative label-free proteomics using one-dimensional gel electrophoresis combined with LC-MS/MS and normalized spectral counting together with fluorescence in situ hybridization and confocal laser scanning microscopy to characterize the metaproteome of the lung lichen symbiosis Lobaria pulmonaria. In addition to the myco- and photobiont, L. pulmonaria harbors proteins from a highly diverse prokaryotic community, which is dominated by Proteobacteria and including also Archaea. While fungal proteins are most dominant (75.4% of all assigned spectra), about the same amount of spectra were assigned to prokaryotic proteins (10%) and to the green algal photobiont (9%). While the latter proteins were found to be mainly associated with energy and carbohydrate metabolism, a major proportion of fungal and bacterial proteins appeared to be involved in PTMs and protein turnover and other diverse functions.

Lichens are generally considered as mutualisms between fungi and green algae or cyanobacteria. These partnerships allow light-exposed and long-living joint structures. The unique organization of lichens provides still unexplored environments for microbial communities. To study lichen-associated bacterial communities, we analyze samples, by a polyphasic approach, from three lichen species (Cladonia arbuscula, Lecanora polytropa and Umbilicaria cylindrica) from alpine environments. Our results indicate that bacteria can form highly structured, biofilm-like assemblages on fungal surfaces and reach considerable abundances of up to 10(8) cells per gram fresh weight. Fluorescence in situ hybridization reveals the predominance of Alphaproteobacteria. Microbial fingerprints performed by PCR-single-strand conformation polymorphism analysis using universal and group-specific primers show distinct patterns for each lichen species. Characterization of cultivable strains and presence of functional genes in the total fraction suggest the involvement of associated bacteria in nutrient cycling. Ubiquitous nifH genes, which encode the nitrogenase reductase, show a high diversity and are assigned to Alphaproteobacteria and Firmicutes, for example, Paenibacillus. Cultivable strains mainly belonging to the genera Acinetobacter, Bacillus, Burkholderia, Methylobacterium and Paenibacillus show lytic (chitinolytic, glucanolytic, and proteolytic) activities, hormone production (indole-3-acetic acid) as well as phosphate mobilization and antagonistic activity toward other microorganisms. The traditional concept of lichens has to be expanded to consider multiple bacterial partners.

The genus Stenotrophomonas comprises at least eight species. These bacteria are found throughout the environment, particularly in close association with plants. Strains of the most predominant species, Stenotrophomonas maltophilia, have an extraordinary range of activities that include beneficial effects for plant growth and health, the breakdown of natural and man-made pollutants that are central to bioremediation and phytoremediation strategies and the production of biomolecules of economic value, as well as detrimental effects, such as multidrug resistance, in human pathogenic strains. Here, we discuss the versatility of the bacteria in the genus Stenotrophomonas and the insight that comparative genomic analysis of clinical and endophytic isolates of S. maltophilia has brought to our understanding of the adaptation of this genus to various niches.