Henry Müller

Species-specific structural and functional diversity of bacterial communities in lichen symbioses

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 108 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.

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.

Desert farming benefits from microbial potential in arid soils and promotes diversity and plant health.

Background To convert deserts into arable, green landscapes is a global vision, and desert farming is a strong growing area of agriculture world-wide. However, its effect on diversity of soil microbial communities, which are responsible for important ecosystem services like plant health, is still not known. Methodology/Principal Findings We studied the impact of long-term agriculture on desert soil in one of the most prominent examples for organic desert farming in Sekem (Egypt). Using a polyphasic methodological approach to analyse microbial communities in soil as well as associated with cultivated plants, drastic effects caused by 30 years of agriculture were detected. Analysing bacterial fingerprints, we found statistically significant differences between agricultural and native desert soil of about 60%. A pyrosequencing-based analysis of the 16S rRNA gene regions showed higher diversity in agricultural than in desert soil (Shannon diversity indices: 11.21/7.90), and displayed structural differences. The proportion of Firmicutes in field soil was significantly higher (37%) than in the desert (11%). Bacillus and Paenibacillus play the key role: they represented 96% of the antagonists towards phytopathogens, and identical 16S rRNA sequences in the amplicon library and for isolates were detected. The proportion of antagonistic strains was doubled in field in comparison to desert soil (21.6%/12.4%); disease-suppressive bacteria were especially enriched in plant roots. On the opposite, several extremophilic bacterial groups, e.g., Acidimicrobium, Rubellimicrobium and Deinococcus-Thermus, disappeared from soil after agricultural use. The N-fixing Herbaspirillum group only occurred in desert soil. Soil bacterial communities were strongly driven by the a-biotic factors water supply and pH. Conclusions/Significance After long-term farming, a drastic shift in the bacterial communities in desert soil was observed. Bacterial communities in agricultural soil showed a higher diversity and a better ecosystem function for plant health but a loss of extremophilic bacteria. Interestingly, we detected that indigenous desert microorganisms promoted plant health in desert agro-ecosystems.

Quorum-sensing effects in the antagonistic rhizosphere bacterium Serratia plymuthica HRO-C48

The rhizosphere-associated bacterium Serratia plymuthica HRO-C48 is not only able to suppress symptoms caused by soil-borne pathogens but is also able to stimulate growth of plants. Detailed knowledge about the underlying mechanisms and regulation are crucial for the application in biocontrol strategies. To analyse the influence of N-acyl homoserine lactone (AHL)-mediated communication on the biocontrol activity, the AHL-degrading lactonase AiiA was heterologously expressed in the strain, resulting in abolished AHL production. The comparative analysis of the wild type and AHL negative mutants led to the identification of new AHL-regulated phenotypes. In the pathosystem Verticillium dahliae-oilseed rape, the essential role of AHL-mediated signaling for disease suppression was demonstrated. In vitro, the regulatory function of AHLs in the synthesis of the plant growth hormone indole-3-acetic acid is shown for the first time. Additionally, swimming motility was found to be negatively AHL regulated. In contrast, production of extracellular hydrolytic enzymes is shown to be positively AHL-regulated. HRO-C48 emits a broad spectrum of volatile organic compounds that are involved in antifungal activity and, interestingly, whose relative abundances are influenced by quorum sensing (QS). This study shows that QS is crucial for biocontrol activity of S. plymuthica and discusses the impact for the application of the strain as a biocontrol agent.

Microbial Diversity Inside Pumpkins: Microhabitat-Specific Communities Display a High Antagonistic Potential Against Phytopathogens

Recent and substantial yield losses of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) are primarily caused by the ascomycetous fungus Didymella bryoniae but bacterial pathogens are frequently involved as well. The diversity of endophytic microbial communities from seeds (spermosphere), roots (endorhiza), flowers (anthosphere), and fruits (carposphere) of three different pumpkin cultivars was studied to develop a biocontrol strategy. A multiphasic approach combining molecular, microscopic, and cultivation techniques was applied to select a consortium of endophytes for biocontrol. Specific community structures for Pseudomonas and Bacillus, two important plant-associated genera, were found for each microenvironment by fingerprinting of 16S ribosomal RNA genes. All microenvironments were dominated by bacteria; fungi were less abundant. Of the 2,320 microbial isolates analyzed in dual culture assays, 165 (7%) were tested positively for in vitro antagonism against D. bryoniae. Out of these, 43 isolates inhibited the growth of bacterial pumpkin pathogens (Pectobacterium carotovorum, Pseudomonas viridiflava, Xanthomonas cucurbitae); here only bacteria were selected. Microenvironment-specific antagonists were found, and the spermosphere and anthosphere were revealed as underexplored reservoirs for antagonists. In the latter, a potential role of pollen grains as bacterial vectors between flowers was recognized. Six broad spectrum antagonists selected according to their activity, genotypic diversity, and occurrence were evaluated under greenhouse conditions. Disease severity on pumpkins of D. bryoniae was significantly reduced by Pseudomonas chlororaphis treatment and by a combined treatment of strains (Lysobacter gummosus, P. chlororaphis, Paenibacillus polymyxa, and Serratia plymuthica). This result provides a promising prospect to biologically control pumpkin diseases.

Fungal diversity in the rhizosphere of endemic plant species of Tenerife (Canary Islands): relationship to vegetation zones and environmental factors

Knowledge about fungal diversity scaling relationships relative to that of plants is important to understand ecosystem functioning. Tenerife Island, a natural laboratory to study terrestrial biodiversity, is represented by six different vegetation zones characterized by specific abiotic conditions and plant communities with a high proportion of endemic plants. Little is known about the biodiversity of associated fungi. To understand the relationship between plant and fungal communities, we analysed soil/rhizosphere fungi from all vegetation zones. From 12 sampling points dispersed on the whole island, molecular analysis of fungal communities was determined by single-strand conformation polymorphism (SSCP) analysis using universal and specific primers for Trichoderma. The highly diverse fungal communities were mainly characterized by ectomycorrhiza-forming Basidiomycota and a high proportion of yet-unidentified species. Besides, Trichoderma-specific SSCP resulted in low diversity of mainly cosmopolitan species, for example Hypocrea lixii/T. harzianum. The dominance of T. harzianum was confirmed by cultivation. All Trichoderma isolates show an extraordinarily high antagonistic potential towards different groups of plant pathogens, supporting the hypothesis of extensive colonization by highly competitive Trichoderma species from the continent. In contrast, biodiversity patterns of the whole fungal and plant communities follow the same ecological rules. Furthermore, a high statistical correlation between fungal communities and the main environmental factors, temperature and precipitation, was found.

Functional and Structural Microbial Diversity in Organic and Conventional Viticulture: Organic Farming Benefits Natural Biocontrol Agents

ABSTRACT Statistically significant differences in the structure and function of above-ground grapevine-associated microorganisms from organically and conventionally managed vineyards were found. Aureobasidium pullulans, a copper-detoxifying fungus and biocontrol agent, plays a key role in explaining these differences. The black fungus was strongly enriched in the communities of organically managed plants and yielded a higher indigenous antiphytopathogenic potential.

Root-microbe systems: the effect and mode of interaction of Stress Protecting Agent (SPA) Stenotrophomonas rhizophila DSM14405T

Stenotrophomonas rhizophila has great potential for applications in biotechnology and biological control due to its ability to both promote plant growth and protect roots against biotic and a-biotic stresses, yet little is known about the mode of interactions in the root-environment system. We studied mechanisms associated with osmotic stress using transcriptomic and microscopic approaches. In response to salt or root extracts, the transcriptome of S. rhizophila DSM14405T changed drastically. We found a notably similar response for several functional gene groups responsible for general stress protection, energy production, and cell motility. However, unique changes in the transcriptome were also observed: the negative regulation of flagella-coding genes together with the up-regulation of the genes responsible for biofilm formation and alginate biosynthesis were identified as a single mechanism of S. rhizophila DSM14405T against salt shock. However, production and excretion of glucosylglycerol (GG) were found as a remarkable mechanism for the stress protection of this Stenotrophomonas strain. For S. rhizophila treated with root exudates, the shift from the planktonic lifestyle to a sessile one was measured as expressed in the down-regulation of flagellar-driven motility. These findings fit well with the observed positive regulation of host colonization genes and microscopic images that show different colonization patterns of oilseed rape roots. Spermidine, described as a plant growth regulator, was also newly identified as a protector against stress. Overall, we identified mechanisms of Stenotrophomonas to protect roots against osmotic stress in the environment. In addition to both the changes in life style and energy metabolism, phytohormons, and osmoprotectants were also found to play a key role in stress protection.

Enhancement of biogas production by addition of hemicellulolytic bacteria immobilised on activated zeolite.

Biogas from agricultural biomass and residues is a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process in single- and two-stage reactors. Therefore, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required amongst others in order to optimise the fermentation efficiency and to increase methane yields. Here we show a new strategy for the enhancement of biogas production from hemicellulose-rich substrates. Hemicellulolytic populations from a common biogas fermenter consortium were successively enriched in batch-cultures using a synthetic medium containing xylan powder as single carbon source under anaerobic mesophilic conditions. Enriched hemicellulolytic bacteria were immobilised on trace metal activated zeolite to ensure a stable storage and easy application. Xylanase activity increased continuously during subsequent enrichment cycles by up to 162%. In batch-culture experiments we were able to observe an increase of methane by 53% compared to controls without additionally introduced microorganisms immobilised on zeolite. Specific enrichment of hemicellulolytic bacteria during the process was confirmed by using single strand conformation polymorphism (SSCP) analysis based on amplification of the eubacterial 16S rDNA fragments. Using sequence analysis conspicuous bands from SSCP patterns could be identified as belonging to the groups Bacteroides sp., Azospira oryzae (Dechlorosoma sp.) as well as to a wide spectrum of diverse species within the order of Clostridiales (Firmicutes).

The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions.

Sphagnum‐dominated bogs represent a unique yet widely distributed type of terrestrial ecosystem and strongly contribute to global biosphere functioning. Sphagnum is colonized by highly diverse microbial communities, but less is known about their function. We identified a high functional diversity within the Sphagnum microbiome applying an Illumina‐based metagenomic approach followed by de novo assembly and MG‐RAST annotation. An interenvironmental comparison revealed that the Sphagnum microbiome harbours specific genetic features that distinguish it significantly from microbiomes of higher plants and peat soils. The differential traits especially support ecosystem functioning by a symbiotic lifestyle under poikilohydric and ombrotrophic conditions. To realise a plasticity–stability balance, we found abundant subsystems responsible to cope with oxidative and drought stresses, to exchange (mobile) genetic elements, and genes that encode for resistance to detrimental environmental factors, repair and self‐controlling mechanisms. Multiple microbe–microbe and plant–microbe interactions were also found to play a crucial role as indicated by diverse genes necessary for biofilm formation, interaction via quorum sensing and nutrient exchange. A high proportion of genes involved in nitrogen cycle and recycling of organic material supported the role of bacteria for nutrient supply. 16S rDNA analysis indicated a higher structural diversity than that which had been previously detected using PCR‐dependent techniques. Altogether, the diverse Sphagnum microbiome has the ability to support the life of the host plant and the entire ecosystem under changing environmental conditions. Beyond this, the moss microbiome presents a promising bio‐resource for environmental biotechnology – with respect to novel enzymes or stress‐protecting bacteria.