The Feather Moss Hylocomium splendens Affects the Transcriptional Profile of a Symbiotic Cyanobacterium in Relation to Acquisition and Turnover of Key Nutrients.

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss-cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.

Nitrogen fixation and nifH gene diversity in cyanobacteria living on feather mosses in a subalpine forest of Mt. Fuji.

In the boreal forests, feather mosses such as Hylocomium splendens and Pleurozium schreberi are colonized by cyanobacteria, which provide large amounts of nitrogen to forest ecosystems through nitrogen fixation. Although these feather mosses are also ubiquitous in subalpine forests of East Asia, little is known regarding their associated cyanobacteria and their ability to fix nitrogen. In this study, we investigated (1) whether cyanobacteria co-exist and fix nitrogen in the two species of feather mosses that cover the ground surface in a subalpine forest of Mt. Fuji, (2) whether cyanobacteria belonging to a common cluster with boreal forests are found in feather mosses in Mt. Fuji, and (3) whether moss-associated nitrogen fixation rates differed among moss growing substrates, canopy openness, and moss nitrogen concentrations in the same forest area. Our results showed that cyanobacteria colonized feather mosses in the subalpine forests of Mt. Fuji and acetylene reduction rates as an index of nitrogen fixation tended to be higher in H. splendens than in P. schreberi. Based on analysis of the nifH gene, 43 bacterial operational taxonomic units (OTUs) were identified, 28 of which represented cyanobacteria. Among the five clusters of cyanobacteria classified based on their nifH gene and identified in northern Europe, four (Nostoc cluster I, Nostoc cluster II, Stigonema cluster, and nifH2 cluster) were also found at Mt. Fuji. The acetylene reduction rate differed depending on the moss growing substrate and the total nitrogen concentration of moss shoots, and a strong negative correlation was observed with the total nitrogen concentration.

Chemical Stimulation of Heterocyte Differentiation by the Feather Moss Hylocomium splendens: a Potential New Step in Plant-Cyanobacteria Symbioses.

Cyanobacteria associated with mosses play a key role in the nitrogen (N) cycle in unpolluted ecosystems. Mosses have been found to release molecules that induce morphophysiological changes in epiphytic cyanobionts. Nevertheless, the extent of moss influence on these microorganisms remains unknown. To evaluate how mosses or their metabolites influence N2 fixation rates by cyanobacteria, we assessed the nitrogenase activity, heterocyte frequency and biomass of a cyanobacterial strain isolated from the feather moss Hylocomium splendens and a non-symbiotic strain when they were either growing by themselves, together with H. splendens or exposed to H. splendens water, acetone, ethanol, or isopropanol extracts. The same cyanobacterial strains were added to another moss (Taxiphyllum barbieri) and a liverwort (Monosolenium tenerum) to assess if these bryophytes affect N2 fixation differently. Although no significant increases in nitrogenase activity by the cyanobacteria were observed when in contact with H. splendens shoots, both the symbiotic and non-symbiotic cyanobacteria increased nitrogenase activity as well as heterocyte frequency significantly upon exposure to H. splendens ethanol extracts. Contact with T. barbieri shoots, on the other hand, did lead to increases in nitrogenase activity, indicating low host-specificity to cyanobacterial activity. These findings suggest that H. splendens produces heterocyte-differentiating factors (HDFs) that are capable of stimulating cyanobacterial N2 fixation regardless of symbiotic competency. Based on previous knowledge about the chemical ecology and dynamics of moss-cyanobacteria interactions, we speculate that HDF expression by the host takes place in a hypothetical new step occurring after plant colonization and the repression of hormogonia.

Dominance of coniferous and broadleaved trees drives bacterial associations with boreal feather mosses.

The composition of ecologically important moss-associated bacterial communities seems to be mainly driven by host species but may also be shaped by environmental conditions related with tree dominance. The moss phyllosphere has been studied in coniferous forests while broadleaf forests remain understudied. To determine if host species or environmental conditions defined by tree dominance drives the bacterial diversity in the moss phyllosphere, we used 16S rRNA gene amplicon sequencing to quantify changes in bacterial communities as a function of host species (Pleurozium schreberi and Ptilium crista-castrensis) and forest type (coniferous black spruce versus deciduous broadleaf trembling aspen) in eastern Canada. The overall composition of moss phyllosphere was defined by the interaction of both factors, though most of the bacterial phyla were determined by a strong effect of forest type. Bacterial α-diversity was highest in spruce forests, while there was greater turnover (ß-diversity) and higher γ-diversity in aspen forests. Unexpectedly, Cyanobacteria were much more relatively abundant in aspen than in spruce forests, with the cyanobacteria family Nostocaceae differing the most between forest types. Our results advance the understanding of moss-associated microbial communities among coniferous and broadleaf deciduous forests, which are important with the increasing changes in tree dominance in the boreal system.

Transcriptional profiling reveals conserved and species-specific plant defense responses during the interaction of Physcomitrium patens with Botrytis cinerea.

KEY MESSAGE: Evolutionary conserved defense mechanisms present in extant bryophytes and angiosperms, as well as moss-specific defenses are part of the immune response of Physcomitrium patens. Bryophytes and tracheophytes are descendants of early land plants that evolved adaptation mechanisms to cope with different kinds of terrestrial stresses, including drought, variations in temperature and UV radiation, as well as defense mechanisms against microorganisms present in the air and soil. Although great advances have been made on pathogen perception and subsequent defense activation in angiosperms, limited information is available in bryophytes. In this study, a transcriptomic approach uncovered the molecular mechanisms underlying the defense response of the bryophyte Physcomitrium patens (previously Physcomitrella patens) against the important plant pathogen Botrytis cinerea. A total of 3.072 differentially expressed genes were significantly affected during B. cinerea infection, including genes encoding proteins with known functions in angiosperm immunity and involved in pathogen perception, signaling, transcription, hormonal signaling, metabolic pathways such as shikimate and phenylpropanoid, and proteins with diverse role in defense against biotic stress. Similarly as in other plants, B. cinerea infection leads to downregulation of genes involved in photosynthesis and cell cycle progression. These results highlight the existence of evolutionary conserved defense responses to pathogens throughout the green plant lineage, suggesting that they were probably present in the common ancestors of land plants. Moreover, several genes acquired by horizontal transfer from prokaryotes and fungi, and a high number of P. patens-specific orphan genes were differentially expressed during B. cinerea infection, suggesting that they are important players in the moss immune response.

Environmental patterns of brown moss- and Sphagnum-associated microbial communities.

Northern peatlands typically develop through succession from fens dominated by the moss family Amblystegiaceae to bogs dominated by the moss genus Sphagnum. How the different plants and abiotic environmental conditions provided in Amblystegiaceae and Sphagnum peat shape the respective moss associated microbial communities is unknown. Through a large-scale molecular and biogeochemical study spanning Arctic, sub-Arctic and temperate regions we assessed how the endo- and epiphytic microbial communities of natural northern peatland mosses relate to peatland type (Sphagnum and Amblystegiaceae), location, moss taxa and abiotic environmental variables. Microbial diversity and community structure were distinctly different between Amblystegiaceae and Sphagnum peatlands, and within each of these two peatland types moss taxon explained the largest part of microbial community variation. Sphagnum and Amblystegiaceae shared few (< 1% of all operational taxonomic units (OTUs)) but strikingly abundant (up to 65% of relative abundance) OTUs. This core community overlapped by one third with the Sphagnum-specific core-community. Thus, the most abundant microorganisms in Sphagnum that are also found in all the Sphagnum plants studied, are the same OTUs as those few shared with Amblystegiaceae. Finally, we could confirm that these highly abundant OTUs were endophytes in Sphagnum, but epiphytes on Amblystegiaceae. We conclude that moss taxa and abiotic environmental variables associate with particular microbial communities. While moss taxon was the most influential parameter, hydrology, pH and temperature also had significant effects on the microbial communities. A small though highly abundant core community is shared between Sphagnum and Amblystegiaceae.

Time-gated confocal microscopy reveals accumulation of exocyst subunits at the plant-pathogen interface.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites, allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labelled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d, and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labelled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

Methane emission from feather moss stands.

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH4 . However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH4 turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH4 cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH4 emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH4 h-1 gdw-1 . Temperature and water content positively influenced CH4 emission. Methanogenic potentials determined under N2 atmosphere in darkness ranged between 22 and 157 pmol CH4 h-1 gdw-1 . Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH4 was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 104 and 105 copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH4 was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH4 produced was depleted in 13 C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3-62 mM). The δ13 C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH4 by hydrogenotrophic methanogenesis and causing net emission of CH4 under ambient conditions, albeit at low rates.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

An Innate Immunity Pathway in the Moss Physcomitrella patens.

MAP kinase (MPK) cascades in Arabidopsis thaliana and other vascular plants are activated by developmental cues, abiotic stress, and pathogen infection. Much less is known of MPK functions in nonvascular land plants such as the moss Physcomitrella patens Here, we provide evidence for a signaling pathway in P. patens required for immunity triggered by pathogen associated molecular patterns (PAMPs). This pathway induces rapid growth inhibition, a novel fluorescence burst, cell wall depositions, and accumulation of defense-related transcripts. Two P. patens MPKs (MPK4a and MPK4b) are phosphorylated and activated in response to PAMPs. This activation in response to the fungal PAMP chitin requires a chitin receptor and one or more MAP kinase kinase kinases and MAP kinase kinases. Knockout lines of MPK4a appear wild type but have increased susceptibility to the pathogenic fungi Botrytis cinerea and Alternaria brassisicola Both PAMPs and osmotic stress activate some of the same MPKs in Arabidopsis. In contrast, abscisic acid treatment or osmotic stress of P. patens does not activate MPK4a or any other MPK, but activates at least one SnRK2 kinase. Signaling via MPK4a may therefore be specific to immunity, and the moss relies on other pathways to respond to osmotic stress.

Seasonal variation in nifH abundance and expression of cyanobacterial communities associated with boreal feather mosses.

Dinitrogen (N2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, 'Stigonema cluster' made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82-94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N2-fixation being greatly explained by variations in nifH expression of the 'Stigonema cluster'. These results suggest that Stigonema is potentially the most influential N2-fixer in symbiosis with boreal forest feather mosses.

Dependence on epiphytic bacteria for freezing protection in an Antarctic moss, Bryum argenteum.

Mosses are the dominant flora of Antarctica, but their mechanisms of survival in the face of extreme low temperatures are poorly understood. A variety of Bryum argenteum from 77° S was previously shown to have strong ice-pitting activity, a sign of the presence of ice-binding proteins (IBPs) that mitigate freezing damage. Here, using samples that had been stored at -25(o) C for 10 years, it is shown that much if not all of the activity is due to bacterial ice-binding proteins secreted on the leaves of the moss. Sequencing of the leaf metagenome revealed the presence of hundreds of genes from a variety of bacteria (mostly Actinobacteria and Bacteroidetes) that encode a domain (DUF3494) that is associated with ice binding. The frequency of occurrence of this domain is one to two orders of magnitude higher than it is in representative mesophilic bacterial metagenomes. Genes encoding 42 bacterial IBPs with N-terminal secretion signals were assembled. There appears to be a commensal relationship in which the moss provides sustenance to the bacteria in return for freezing protection.

Paenibacillus physcomitrellae sp. nov., isolated from the moss Physcomitrella patens.

A Gram-stain-positive, facultatively anaerobic and rod-shaped bacterium, designated strain XBT, was isolated from Physcomitrella patens growing in Beijing, China. The isolate was identified as a member of the genus Paenibacillus based on phenotypic characteristics and phylogenetic inferences. The novel strain was spore-forming, motile, catalase-negative and weakly oxidase-positive. Optimal growth of strain XBT occurred at 28°C and pH 7.0-7.5. The major polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unidentified components, including one phospholipid, two aminophospholipids, three glycolipids, one aminolipid and one lipid. The predominant isoprenoid quinone was MK-7. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major fatty acid components (>5 %) were anteiso-C15 : 0 (51.2 %), anteiso-C17 : 0 (20.6 %), iso-C16 : 0 (8.3 %) and C16 : 0 (6.7 %). The G+C content of the genomic DNA was 53.3 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain XBT fell within the evolutionary distances encompassed by the genus Paenibacillus; its closest phylogenetic neighbour was Paenibacillus yonginensis DCY84T (96.6 %). Based on phenotypic, chemotaxonomic and phylogenetic properties, strain XBT is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus physcomitrellae sp. nov., is proposed. The type strain is XBT ( = CGMCC 1.15044T = DSM 29851T).

Systemic acquired resistance in moss: further evidence for conserved defense mechanisms in plants.

Vascular plants possess multiple mechanisms for defending themselves against pathogens. One well-characterized defense mechanism is systemic acquired resistance (SAR). In SAR, a plant detects the presence of a pathogen and transmits a signal throughout the plant, inducing changes in the expression of various pathogenesis-related (PR) genes. Once SAR is established, the plant is capable of mounting rapid responses to subsequent pathogen attacks. SAR has been characterized in numerous angiosperm and gymnosperm species; however, despite several pieces of evidence suggesting SAR may also exist in non-vascular plants6-8, its presence in non-vascular plants has not been conclusively demonstrated, in part due to the lack of an appropriate culture system. Here, we describe and use a novel culture system to demonstrate that the moss species Amblystegium serpens does initiate a SAR-like reaction upon inoculation with Pythium irregulare, a common soil-borne oomycete. Infection of A. serpens gametophores by P. irregulare is characterized by localized cytoplasmic shrinkage within 34 h and chlorosis and necrosis within 7 d of inoculation. Within 24 h of a primary inoculation (induction), moss gametophores grown in culture became highly resistant to infection following subsequent inoculation (challenge) by the same pathogen. This increased resistance was a response to the pathogen itself and not to physical wounding. Treatment with ß-1,3 glucan, a structural component of oomycete cell walls, was equally effective at triggering SAR. Our results demonstrate, for the first time, that this important defense mechanism exists in a non-vascular plant, and, together with previous studies, suggest that SAR arose prior to the divergence of vascular and non-vascular plants. In addition, this novel moss - pathogen culture system will be valuable for future characterization of the mechanism of SAR in moss, which is necessary for a better understanding of the evolutionary history of SAR in plants.

A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana) using molecular method and cultivated isolates.

Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed.

Protein secretome of moss plants (Physcomitrella patens) with emphasis on changes induced by a fungal elicitor.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

Isolation and biological activities of an endophytic Mortierella alpina strain from the Antarctic moss Schistidium antarctici.

The Antarctic endophytic fungus (strain ITA1-CCMA 952) was isolated from the moss Schistidium antarctici found in Admiralty Bay, King George Island, Antarctica. Strain ITA1-CCMA 952 was assigned to the specie Mortierella alpina by phylogenetic analysis based on 18S rRNA gene sequences. This strain produces high levels of polyunsaturated fatty acids (PUFAs), including y-(gamma) linolenic acid and arachidonic acid, which when combined represents 48.3% of the total fatty acid content. Fungal extracts demonstrated strong antioxidant activity with the EC50 value of 48.7 µg mL(-1) and also a strong antibacterial activity, mainly against the following bacteria: Escherichia coli, with a MIC of 26.9 µg mL(-1) and Pseudomonas aeruginosa and Enterococcus faecalis, both with a MIC of 107 µg mL(-1). A GC-MS analysis of the chloroform fraction obtained from the crude extract revealed the presence of potential antimicrobials (Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) and Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)) as the major compounds. Therefore, the M. alpina strain ITA1-CCMA 952 is a promising fungus for the biotechnological production of antibiotics, antioxidant substances and PUFAs. This study highlights the need for more research in extreme environments, such as Antarctica.

A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana) using molecular method and cultivated isolates

Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed.

Boreal feather mosses secrete chemical signals to gain nitrogen.

The mechanistic basis of feather moss-cyanobacteria associations, a main driver of nitrogen (N) input into boreal forests, remains unknown. Here, we studied colonization by Nostoc sp. on two feather mosses that form these associations (Pleurozium schreberi and Hylocomium splendens) and two acrocarpous mosses that do not (Dicranum polysetum and Polytrichum commune). We also determined how N availability and moss reproductive stage affects colonization, and measured N transfer from cyanobacteria to mosses. The ability of mosses to induce differentiation of cyanobacterial hormogonia, and of hormogonia to then colonize mosses and re-establish a functional symbiosis was determined through microcosm experiments, microscopy and acetylene reduction assays. Nitrogen transfer between cyanobacteria and Pleurozium schreberi was monitored by secondary ion mass spectrometry (SIMS). All mosses induced hormogonia differentiation but only feather mosses were subsequently colonized. Colonization on Pleurozium schreberi was enhanced during the moss reproductive phase but impaired by elevated N. Transfer of N from cyanobacteria to their host moss was observed. Our results reveal that feather mosses likely secrete species-specific chemo-attractants when N-limited, which guide cyanobacteria towards them and from which they gain N. We conclude that this signalling is regulated by N demands of mosses, and serves as a control of N input into boreal forests.

The cyanobacterial role in the resistance of feather mosses to decomposition--toward a new hypothesis.

Cyanobacteria-plant symbioses play an important role in many ecosystems due to the fixation of atmospheric nitrogen (N) by the cyanobacterial symbiont. The ubiquitous feather moss Pleurozium schreberi (Brid.) Mitt. is colonized by cyanobacteria in boreal systems with low N deposition. Here, cyanobacteria fix substantial amounts of N2 and represent a potential N source. The feather moss appears to be resistant to decomposition, which could be partly a result of toxins produced by cyanobacteria. To assess how cyanobacteria modulated the toxicity of moss, we measured inhibition of bacterial growth. Moss with varying numbers of cyanobacteria was added to soil bacteria to test the inhibition of their growth using the thymidine incorporation technique. Moss could universally inhibit bacterial growth, but moss toxicity did not increase with N2 fixation rates (numbers of cyanobacteria). Instead, we see evidence for a negative relationship between moss toxicity to bacteria and N2 fixation, which could be related to the ecological mechanisms that govern the cyanobacteria-moss relationship. We conclude that cyanobacteria associated with moss do not contribute to the resistance to decomposition of moss, and from our results emerges the question as to what type of relationship the moss and cyanobacteria share.