Phosphate-solubilising microorganisms for improved crop productivity: a critical assessment.

Phosphate-solubilising microorganisms (PSM) are often reported to have positive effects on crop productivity through enhanced phosphorus (P) nutrition. Our aim was to evaluate the validity of this concept. Most studies that report 'positive effects' of PSM on plant growth have been conducted under controlled conditions, whereas field experiments more frequently fail to demonstrate a positive response. Many studies have indicated that the mechanisms seen in vitro do not translate into improved crop P nutrition in complex soil-plant systems. Furthermore, associated mechanisms are often not rigorously assessed. We suggest that PSM do not mobilise sufficient P to change the crops' nutritional environment under field conditions. The current concept, in which PSM solubilise P 'for the plant' should thus be revised. Although PSM have the capacity to solubilise P to meet their own needs, it is the turnover of the microbial biomass that subsequently provides P to plants over a longer time. Therefore, the existing concept of PSM function is unlikely to deliver a reliable strategy for increasing crop P nutrition. A further mechanistic understanding is needed to determine how P mobilisation by PSM as a component of the whole soil community can be manipulated to become more effective for plant P nutrition.

Different Effects of Soil Fertilization on Bacterial Community Composition in the Penicillium canescens Hyphosphere and in Bulk Soil.

This study investigated the effects of long-term soil fertilization on the composition and potential for phosphorus (P) and nitrogen (N) cycling of bacterial communities associated with hyphae of the P-solubilizing fungus Penicillium canescens Using a baiting approach, hyphosphere bacterial communities were recovered from three soils that had received long-term amendment in the field with mineral or mineral plus organic fertilizers. P. canescens hyphae recruited bacterial communities with a decreased diversity and an increased abundance of Proteobacteria relative to what was observed in soil communities. As core bacterial taxa, Delftia and Pseudomonas spp. were present in all hyphosphere samples irrespective of soil fertilization. However, the type of fertilization showed significant impacts on the diversity, composition, and distinctive taxa/operational taxonomic units (OTUs) of hyphosphere communities. The soil factors P (Olsen method), exchangeable Mg, exchangeable K, and pH were important for shaping soil and hyphosphere bacterial community compositions. An increased relative abundance of organic P metabolism genes was found in hyphosphere communities from soil that had not received P fertilizers, which could indicate P limitation near the fungal hyphae. Additionally, P. canescens hyphae recruited bacterial communities with a higher abundance of N fixation genes than found in soil communities, which might imply a role of hyphosphere communities for fungal N nutrition. Furthermore, the relative abundances of denitrification genes were greater in several hyphosphere communities, indicating an at least partly anoxic microenvironment with a high carbon-to-N ratio around the hyphae. In conclusion, soil fertilization legacy shapes P. canescens hyphosphere microbiomes and their functional potential related to P and N cycling.IMPORTANCE P-solubilizing Penicillium strains are introduced as biofertilizers to agricultural soils to improve plant P nutrition. Currently, little is known about the ecology of these biofertilizers, including their interactions with other soil microorganisms. This study shows that communities dominated by Betaproteobacteria and Gammaproteobacteria colonize P. canescens hyphae in soil and that the compositions of these communities depend on the soil conditions. The potential of these communities for N and organic P cycling is generally higher than that of soil communities. The high potential for organic P metabolism might complement the ability of the fungus to solubilize inorganic P, and it points to the hyphosphere as a hot spot for P metabolism. Furthermore, the high potential for N fixation could indicate that P. canescens recruits bacteria that are able to improve its N nutrition. Hence, this community study identifies functional groups relevant for the future optimization of next-generation biofertilizer consortia for applications in soil.

The biofilm matrix polysaccharides cellulose and alginate both protect Pseudomonas putida mt-2 against reactive oxygen species generated under matric stress and copper exposure.

In natural environments most bacteria live in biofilms embedded in complex matrices of extracellular polymeric substances (EPS). This lifestyle is known to increase protection against environmental stress. Pseudomonas putida mt-2 harbours genes for the production of at least four different EPS polysaccharides, including alginate and cellulose. Little is known about the functional properties of cellulose, while alginate attenuates the accumulation of reactive oxygen species (ROS) caused by matric stress. By using mutants that are deficient in either alginate or cellulose production we show that even cellulose attenuates the accumulation of matric stress-induced ROS for cells in biofilms. Further, both cellulose and alginate attenuate ROS generated through exposure to copper. Interestingly, the two EPS polysaccharides protect cells in both liquid culture and in biofilms against ROS caused by matric stress, indicating that cellulose and alginate do not need to be produced as an integral part of the biofilm lifestyle to provide tolerance towards environmental stressors.

Cupriavidus pinatubonensis AEO106 deals with copper-induced oxidative stress before engaging in biodegradation of the herbicide 4-chloro-2-methylphenoxyacetic acid.

BACKGROUND: Microbial degradation of phenoxy acid (PA) herbicides in agricultural soils is important to minimize herbicide leaching to groundwater reservoirs. Degradation may, however, be hampered by exposure of the degrader bacteria to toxic metals as copper (Cu) in the soil environment. Exposure to Cu leads to accumulation of intracellular reactive oxygen species (ROS) in some bacteria, but it is not known how Cu-derived ROS and an ensuing oxidative stress affect the degradation of PA herbicides. Based on the previously proposed paradigm that bacteria deal with environmental stress before they engage in biodegradation, we studied how the degradation of the PA herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) by the model PA degrader Cupriavidus pinatubonensis AEO106 was affected by Cu exposure. RESULTS: Exposure of C. pinatubonensis in batch culture to sublethal concentrations of Cu increased accumulation of ROS measured by the oxidant sensing probe 2,7-dichlorodihydrofluorescein diacetate and flow cytometry, and resulted in upregulation of a gene encoding a protein belong to the Ohr/OsmC protein family. The ohr/osmC gene was also highly induced by H2O2 exposure suggesting that it is involved in the oxidative stress response in C. pinatubonensis. The increased ROS accumulation and increased expression of the oxidative stress defense coincided with a delay in the catabolic performance, since both expression of the catabolic tfdA gene and MCPA mineralization were delayed compared to unexposed control cells. CONCLUSIONS: The current study suggests that Cu-induced ROS accumulation in C. pinatubonensis activates a stress response involving the product of the ohr/osmC gene. Further, the stress response is launched before induction of the catabolic tfdA gene and mineralization occurs.

Adhesion to sand and ability to mineralise low pesticide concentrations are required for efficient bioaugmentation of flow-through sand filters.

Pesticide-polluted drinking water may be remediated by inoculating waterworks sand filters with specific degrading bacteria. However, degradation efficiency is often hampered by the poor adhesion behaviour of the introduced bacteria. The phenoxy acid herbicide 4-chloro-2-methyl-phenoxy-acetic acid (MCPA) is a widespread groundwater contaminant. The aim of this study was to investigate whether specific surface characteristics of MCPA-degrading bacteria could be linked to their degrading capabilities in sand filters. Four MCPA degraders with different taxonomic affiliations and original habitats (Sphingomonas sp. PM2, Sphingomonas sp. ERG5, Burkholderia sp. TFD34, Cupriavidus sp. TFD38) were characterised with regard to their motility, cell surface hydrophobicity, biofilm formation, adhesion behaviour and ability to mineralise MCPA. Strains PM2 and ERG5 were non-motile and hydrophobic, whilst strains TFD34 and TFD38 were motile and less hydrophobic. All the strains except ERG5 showed low biofilm formation on polystyrene, although it was significantly higher on glass. PM2 was the most efficient MCPA degrader as it displayed no lag phase and reached >50 % mineralisation at all concentrations (0.0016-25 mg L-1). PM2 adhered significantly better to sand than the other strains. No link was found between motility, biofilm formation and the ability to adhere to sand. PM2 completely removed MCPA for 14 days when inoculated in sand columns with a constant inlet of 1 mg L-1 MCPA. These results demonstrate that besides the ability to degrade the contaminant, surface hydrophobicity and adherence abilities are significant parameters controlling sustained degradation in flow-through sand columns and must be considered when selecting bacteria for bioaugmentation.

Lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms.

Pseudomonads produce several lipopeptide biosurfactants that have antimicrobial properties but that also facilitate surface motility and influence biofilm formation. Detailed studies addressing the significance of lipopeptides for biofilm formation and architecture are rare. Hence, the present study sets out to determine the specific role of the lipopeptide viscosin in Pseudomonas fluorescens SBW25 biofilm formation, architecture and dispersal, and to relate viscA gene expression to viscosin production and effect. Initially, we compared biofilm formation of SBW25 and the viscosin-deficient mutant strain SBW25ΔviscA in static microtitre assays. These experiments demonstrated that viscosin had little influence on the amount of biofilm formed by SBW25 during the early stages of biofilm development. Later, however, SBW25 formed significantly less biofilm than SBW25ΔviscA. The indication that viscosin is involved in biofilm dispersal was confirmed by chemical complementation of the mutant biofilm. Furthermore, a fluorescent bioreporter showed that viscA expression was induced in biofilms 4 h prior to dispersal. Subsequent detailed studies of biofilms formed in flow cells for up to 5 days revealed that SBW25 and SBW25ΔviscA developed comparable biofilms dominated by well-defined, mushroom-shaped structures. Carbon starvation was required to obtain biofilm dispersal in this system. Dispersal of SBW25 biofilms was significantly greater than of SBW25ΔviscA biofilms after 3 h and, importantly, carbon starvation strongly induced viscA expression, in particular for cells that were apparently leaving the biofilm. Thus, the present study points to a role for viscosin-facilitated motility in dispersal of SBW25 biofilms.

Pseudomonas putida mt-2 tolerates reactive oxygen species generated during matric stress by inducing a major oxidative defense response.

BACKGROUND: Soil bacteria typically thrive in water-limited habitats that cause an inherent matric stress to the cognate cells. Matric stress gives rise to accumulation of intracellular reactive oxygen species (ROS), which in turn may induce oxidative stress, and even promote mutagenesis. However, little is known about the impact of ROS induced by water limitation on bacteria performing important processes as pollutant biodegradation in the environment. We have rigorously examined the physiological consequences of the rise of intracellular ROS caused by matric stress for the toluene- and xylene-degrading soil bacterium Pseudomonas putida mt-2. METHODS: For the current experiments, controlled matric potential stress was delivered to P. putida cells by addition of polyethylene glycol to liquid cultures, and ROS formation in individual cells monitored by a specific dye. The physiological response to ROS was then quantified by both RT-qPCR of RNA transcripts from genes accredited as proxies of oxidative stress and the SOS response along with cognate transcriptional GFP fusions to the promoters of the same genes. RESULTS: Extensive matric stress at -1.5 MPa clearly increased intracellular accumulation of ROS. The expression of the two major oxidative defense genes katA and ahpC, as well as the hydroperoxide resistance gene osmC, was induced under matric stress. Different induction profiles of the reporters were related to the severity of the stress. To determine if matric stress lead to induction of the SOS-response, we constructed a DNA damage-inducible bioreporter based on the LexA-controlled phage promoter PPP3901. According to bioreporter analysis, this gene was expressed during extensive matric stress. Despite this DNA-damage mediated gene induction, we observed no increase in the mutation frequency as monitored by emergence of rifampicin-resistant colonies. CONCLUSIONS: Under conditions of extensive matric stress, we observed a direct link between matric stress, ROS formation, induction of ROS-detoxifying functions and (partial) activation of the SOS system. However, such a stress-response regime did not translate into a general DNA mutagenesis status. Taken together, the data suggest that P. putida mt-2 can cope with this archetypal environmental stress while preserving genome stability, a quality that strengthens the status of this bacterium for biotechnological purposes.

The biosurfactant viscosin transiently stimulates n-hexadecane mineralization by a bacterial consortium.

Pseudomonas produces powerful lipopeptide biosurfactants including viscosin, massetolide A, putisolvin, and amphisin, but their ability to stimulate alkane mineralization and their utility for bioremediation have received limited attention. The four Pseudomonas lipopeptides yielded emulsification indices on hexadecane of 20-31% at 90 mg/l, which is comparable to values for the synthetic surfactant Tween 80. Viscosin was the optimal emulsifier and significantly stimulated n-hexadecane mineralization by diesel-degrading bacterial consortia but exclusively during the first 2 days of batch culture experiments. Growth of the consortia, as determined by OD600 measurements and quantification of the alkB marker gene for alkane degradation, was arrested after the first day of the experiment. In contrast, the control consortia continued to grow and reached higher OD600 values and higher alkB copy numbers during the next days. Due to the short-lived stimulation of n-hexadecane mineralization, the stability of viscosin was analyzed, and it was observed that added viscosin was degraded by the bacterial consortium during the first 2 days. Hence, viscosin has a potential as stimulator of alkane degradation, but its utility in bioremediation may be limited by its rapid degradation and growth-inhibiting properties.

Removal of a complex VOC mixture by potted plants-effects on soil microorganisms.

Microorganisms in the soil of potted plants are important for removal of volatile organic compounds (VOCs) from indoor air, but little is known about the subject. The aim of this study was therefore to obtain a better understanding of the effect of VOCs on the microbial community in potted plants. Hedera helix was exposed to gasoline vapors under dynamic chamber conditions for 21 days and three main parameters were investigated. These were (1) removal of the target compounds heptane, 3-methylhexane, benzene, toluene, ethylbenzene, m,p-xylene, and naphthalene from the gasoline mixture; (2) toluene mineralization; and (3) bacterial abundance and bacterial community structure. H. helix was able to reduce the concentration of the target compounds in the continuously emitted gasoline by 25-32%, except for naphthalene, which was too low in concentration. The soil microcosm of gasoline exposed plants had for an initial 66 h increased toluene mineralization rate compared to the soil microcosm in the soil of plants exposed to clean air. Bacterial abundance was decreased in response to gasoline exposure while bacterial community structure was changed. The change in bacterial community structure was, however, different between the two experiments indicating that several taxonomic units can degrade gasoline components. Especially the genera Rhodanobacter and Pseudonorcardia significantly increased in abundance in response to gasoline vapors. Bauldia, Devosia, and Bradyrhizobium, on the other hand, decreased.

Selection for Cu-tolerant bacterial communities with altered composition, but unaltered richness, via long-term Cu exposure.

Toxic metal pollution affects the composition and metal tolerance of soil bacterial communities. However, there is virtually no knowledge concerning the responses of members of specific bacterial taxa (e.g., phyla or classes) to metal toxicity, and contradictory results have been obtained regarding the impact of metals on operational taxonomic unit (OTU) richness. We used tag-coded pyrosequencing of the 16S rRNA gene to elucidate the impacts of copper (Cu) on bacterial community composition and diversity within a well-described Cu gradient (20 to 3,537 µg g(-1)) stemming from industrial contamination with CuSO(4) more than 85 years ago. DNA sequence information was linked to analysis of pollution-induced community tolerance (PICT) to Cu, as determined by the [(3)H]leucine incorporation technique, and to chemical characterization of the soil. PICT was significantly correlated to bioavailable Cu, as determined by the results seen with a Cu-specific bioluminescent biosensor strain, demonstrating a specific community response to Cu. The relative abundances of members of several phyla or candidate phyla, including the Proteobacteria, Bacteroidetes, Verrumicrobia, Chloroflexi, WS3, and Planctomycetes, decreased with increasing bioavailable Cu, while members of the dominant phylum, the Actinobacteria, showed no response and members of the Acidobacteria showed a marked increase in abundance. Interestingly, changes in the relative abundances of classes frequently deviated from the responses of the phyla to which they belong. Despite the apparent Cu impacts on Cu resistance and community structure, bioavailable Cu levels did not show any correlation to bacterial OTU richness (97% similarity level). Our report highlights several bacterial taxa responding to Cu and thereby provides new guidelines for future studies aiming to explore the bacterial domain for members of metal-responding taxa.

Cellulose amendment promotes P solubilization by Penicillium aculeatum in non-sterilized soil.

Successful application of microbial biofertilizers, such as phosphorus (P) solubilizing fungi to agroecosystems, is constrained from the lack of knowledge about their ecology; for example in terms of how they respond to an external input of carbon (C) to get established in the soil. In two soil incubation experiments we examined the performance of the P solubilizing fungus Penicillium aculeatum in non-sterile and semi-sterile (γ-irradiated) soil with different C and P sources. Results from the first experiment with C sources showed that starch and cellulose generally improved P solubilization by P. aculeatum measured as water extractable P (Pwep), though only significantly in non-sterile soil. This coincided with an increased population density of P. aculeatum measured with a hygromycin B resistant strain of this fungus. Soil respiration used to measure soil microbial activity was overall much higher in treatments with C compounds than without C in both non-sterile and semi-sterile soil. However, soil respiration was highest with cellulose in semi-sterile soil, especially in combination with P. aculeatum. Hence, for the second experiment with P sources (tricalcium phosphate (TCP) and sewage sludge ash) cellulose was used as a C source for P. aculeatum growth in all treatments. Main results showed that P. aculeatum in combination with cellulose soil amendment increased soil Pwep independent of soil sterilization and P source treatments. Soil resin P (Pres) and microbial P (Pmic), which represents stocks of potentially plant available P, were also affected from P. aculeatum inoculation. Increased soil Pres from TCP and sewage sludge ash was observed with P. aculeatum independent of soil type. On the other hand soil Pmic was higher after P. aculeatum inoculation only in semi-sterile soil. Population density of P. aculeatum measured with qPCR was maintained or increased in non-sterile and semi-sterile soil, respectively, compared to the original inoculum load of P. aculeatum. In conclusion, our results underline the importance of C source addition for P. aculeatum if used as a biofertilizer. For this, cellulose seems to be a promising option promoting P. aculeatum growth and P solubilization also in non-sterilized soil.

The cytokinin-producing plant beneficial bacterium Pseudomonas fluorescens G20-18 primes tomato (Solanum lycopersicum) for enhanced drought stress responses.

Plant growth-promoting rhizobacteria (PGPR) are known for exerting beneficial effects on plant growth and tolerance to plant pathogens. However, their specific role in mediating protection against abiotic stress remains underexplored. The aim of this study was to characterise the ability of the cytokinin-producing beneficial bacterium Pseudomonas fluorescens G20-18 to enhance tomato growth and boost tolerance to drought stress. Tomato seedlings were root inoculated and their growth and physiological and molecular responses assessed under well-watered conditions and also in response to progressive drought stress and a subsequent recovery period. Root inoculation with G20-18 had a significant positive impact on tomato growth. Furthermore, G20-18 inoculated and drought-stressed plants showed higher leaf chlorophyll and abscisic acid (ABA) content and stomatal closure than non-inoculated controls. Root inoculation also increased the activity of different carbohydrate metabolism enzymes, which are important for root and leaf growth and development in drought stressed plants. A significant increase in the activity of different antioxidant enzymes and total antioxidant capacity correlated with elevated levels of relevant secondary metabolites, such as phenolics, anthocyanins and flavonoids. RNA sequencing revealed distinct qualitative and quantitative differences in gene regulation in response to G20-18. Notably, the number of genes differentially regulated in response to G20-18 was approximately sevenfold higher during drought stress, indicating that root inoculation with the bacteria primed the plants for a much stronger transcriptionally regulated systemic drought stress response. The regulated genes are related to phenylalanine metabolism and other key processes linked to plant growth, development and drought stress resilience. A role of the ability of G20-18 to produce the plant hormone cytokinin for interaction with tomato was established by the cytokinin-deficient biosynthesis mutants CNT1 and CNT2. In comparison with G20-18, the inoculation of plants with CNT1 resulted in a reduced number of differentially regulated genes. The relative change was most prominent under well-watered conditions with a 85 % reduction, corresponding to 462 genes. However, under drought conditions the absolute number of differentially regulated genes was reduced by even 2219 in response to the CNT1 mutant. The relevance of the ability of G20-18 to produce cytokinins for interaction with plants was also evident from differences in growth and specific cell and ecophysiological parameters in response to CNT1 and CNT2. These findings provide novel insights about G20-18's ability to improve drought stress responses and the role of interkingdom signalling by bacterial-derived cytokinins, and contribute to enhance the robustness of the practical application of these microorganisms to improve crop resilience in agricultural production.

Succession of the wheat seed-associated microbiome as affected by soil fertility level and introduction of Penicillium and Bacillus inoculants in the field.

During germination, the seed releases nutrient-rich exudates into the spermosphere, thereby fostering competition between resident microorganisms. However, insight into the composition and temporal dynamics of seed-associated bacterial communities under field conditions is currently lacking. This field study determined the temporal changes from 11 to 31 days after sowing in the composition of seed-associated bacterial communities of winter wheat as affected by long-term soil fertilization history, and by introduction of the plant growth-promoting microbial inoculants Penicillium bilaiae and Bacillus simplex. The temporal dynamics were the most important factor affecting the composition of the seed-associated communities. An increase in the relative abundance of genes involved in organic nitrogen metabolism (ureC and gdhA), and in ammonium oxidation (amoA), suggested increased mineralization of plant-derived nitrogen compounds over time. Dynamics of the phosphorus cycling genes ppt, ppx and cphy indicated inorganic phosphorus and polyphosphate cycling, as well as phytate hydrolysis by the seed-associated bacteria early after germination. Later, an increase in genes for utilization of organic phosphorus sources (phoD, phoX and phnK) indicated phosphorus limitation. The results indicate that community temporal dynamics are partly driven by changed availability of major nutrients, and reveal no functional consequences of the added inoculants during seed germination.

Low concentration of copper inhibits colonization of soil by the arbuscular mycorrhizal fungus Glomus intraradices and changes the microbial community structure.

Common agricultural practices result in accumulation of copper in agricultural soils worldwide. The effect of bioavailable copper ([Cu](bio)) on colonization of soil by the AM fungus Glomus intraradices and other soil microorganisms was investigated in microcosms containing copper-amended soil. To avoid indirect effects through the plant, copper was only added to root-free microcosm compartments. [Cu](bio) was measured using a Pseudomonas fluorescens biosensor strain. In the range of 0-1.5 µg g(-1) [Cu](bio), a log-log linear relationship between added copper and [Cu](bio) was found. Microbial colonization of the root-free compartment was evaluated by whole-cell fatty acid analysis (WCFA) and amplified rDNA restriction analysis (ARDRA). The WCFA analysis showed that the AM fungus soil colonization was severely inhibited by Cu with a 50% reduction of mycorrhizal growth at 0.26 µg g(-1) [Cu](bio). The growth of other main microbial groups was not significantly affected by copper. However, ARDRA analysis showed a very strong effect of copper on the bacterial community composition probably caused by an increased proportion of Cu-resistant bacteria. Our results suggest that problems with plant yield may arise when converting slightly copper-contaminated soils to land uses such as low-input and sustainable agriculture that are dependent on AM fungal symbiosis.

Vogesella mureinivorans sp. nov., a peptidoglycan-degrading bacterium from lake water.

A novel, non-pigmented, rod-shaped, Gram-negative strain was isolated from mesotrophic lake water in Zealand, Denmark. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium, designated strain 389(T), indicated that the strain belonged to the genus Vogesella and formed a monophyletic group with Vogesella perlucida DS-28(T) (99.1 % nucleotide similarity); it was less related to Vogesella indigofera ATCC 19706(T) (96.9 % similarity) and Vogesella lacus LMG 24504(T) (96.8 % similarity). Hybridization of DNA from strain 389(T) and V. perlucida demonstrated a reassociation of 50.6 ± 9.6 %. The DNA G+C content of strain 389(T) was 61.2 mol%. The fatty acid profile of the strain differed from those of the other strains representing the genus Vogesella by a high content of C16:1ω7c and/or iso-C15:0 2-OH (71.6 %) and a lower content of C16: 0. Strain 389(T) was capable of degrading peptidoglycan and had chitinase and lysozyme activities, possibly associated with the degradation of peptidoglycan, and had capacity for degradation of several other polymer compounds. Based on phenotypic and genotypic characteristics, strain 389(T) represents a novel species, for which we propose the name Vogesella mureinivorans sp. nov. The type strain is 389(T) (=DSM 21247(T) =LMG 25302(T)).

Cu exposure under field conditions coselects for antibiotic resistance as determined by a novel cultivation-independent bacterial community tolerance assay.

Environmental reservoirs of antibiotic resistance are important to human health, and recent evidence indicates that terrestrial resistance reservoirs have expanded during the antibiotic era. Our aim was to study the impact of Cu pollution as a selective driver for the spread of antibiotic resistance in soil. Bacteria were extracted from a well-characterized soil site solely contaminated with CuSO4 more than 80 years ago and from a corresponding control soil. Pollution-induced bacterial community tolerance (PICT) to Cu and a panel of antibiotics was determined by a novel cultivation-independent approach based on [³H]bromodeoxyuridine (BrdU) incorporation into DNA and by resistance profiling of soil bacterial isolates on solid media. High Cu exposure selected for Cu-tolerant bacterial communities but also coselected for increased community-level tolerance to tetracycline and vancomycin. Cu-resistant isolates showed significantly higher incidence of resistance to five out of seven tested antibiotics (tetracycline, olaquindox, nalidixic acid, chloramphenicol, and ampicillin) than Cu-sensitive isolates. Our BrdU-PICT data demonstrate for the first time that soil Cu exposure coselects for resistance to clinically important antibiotics (e.g., vancomycin) at the bacterial community-level. Our study further indicates that Cu exposure provides a strong selection pressure for the expansion of the soil bacterial resistome.

Identification of Root-Associated Bacteria That Influence Plant Physiology, Increase Seed Germination, or Promote Growth of the Christmas Tree Species Abies nordmanniana.

Abies nordmanniana is used for Christmas tree production but poor seed germination and slow growth represent challenges for the growers. We addressed the plant growth promoting potential of root-associated bacteria isolated from A. nordmanniana. Laboratory screenings of a bacterial strain collection yielded several Bacillus and Paenibacillus strains that improved seed germination and produced indole-3-acetic acid. The impact of three of these strains on seed germination, plant growth and growth-related physiological parameters was then determined in greenhouse and field trials after seed inoculation, and their persistence was assessed by 16S rRNA gene-targeted bacterial community analysis. Two strains showed distinct and significant effects. Bacillus sp. s50 enhanced seed germination in the greenhouse but did not promote shoot or root growth. In accordance, this strain did not increase the level of soluble hexoses needed for plant growth but increased the level of storage carbohydrates. Moreover, strain s50 increased glutathione reductase and glutathione-S-transferase activities in the plant, which may indicate induction of systemic resistance during the early phase of plant development, as the strain showed poor persistence in the root samples (rhizosphere soil plus root tissue). Paenibacillus sp. s37 increased plant root growth, especially by inducing secondary root formation, under in greenhouse conditions, where it showed high persistence in the root samples. Under these conditions, it further it increased the level of soluble carbohydrates in shoots, and the levels of starch and non-structural carbohydrates in roots, stem and shoots. Moreover, it increased the chlorophyll level in the field trial. These findings indicate that this strain improves plant growth and vigor through effects on photosynthesis and plant carbohydrate reservoirs. The current results show that the two strains s37 and s50 could be considered for growth promotion programs of A. nordmanniana in greenhouse nurseries, and even under field conditions.

Under the Christmas Tree: Belowground Bacterial Associations With Abies nordmanniana Across Production Systems and Plant Development.

Abies nordmanniana is an economically important tree crop widely used for Christmas tree production. After initial growth in nurseries, seedlings are transplanted to the field. Rhizosphere bacterial communities generally impact the growth and health of the host plant. However, the dynamics of these communities during A. nordmanniana growth in nurseries, and during transplanting, has not previously been addressed. By a 16S rRNA gene amplicon sequencing approach, we characterized the composition and dynamics of bacterial communities in the rhizosphere during early plant growth in field and greenhouse nurseries and for plants transplanted from the greenhouse to the field. Moreover, the N-cycling potential of rhizosphere bacteria across plant age was addressed in both nurseries. Overall, a rhizosphere core microbiome of A. nordmanniana, comprising 19.9% of the taxa at genus level, was maintained across plant age, nursery production systems, and even during the transplantation of plants from the greenhouse to the field. The core microbiome included the bacterial genera Bradyrhizobium, Burkholderia, Flavobacterium, Pseudomonas, Rhizobium, Rhodanobacter, and Sphingomonas, which harbor several N-fixing and plant growth-promoting taxa. Nevertheless, both plant age and production system caused significant changes in the rhizosphere bacterial communities. Concerning community composition, the relative abundance of Rhizobiales (genera Rhizobium, Bradyrhizobium, and Devosia) was higher in the rhizosphere of field-grown A. nordmanniana, whereas the relative abundance of Enterobacteriales and Pseudomonadales (genus Pseudomonas) was higher in the greenhouse. Analysis of community dynamics across plant age showed that in the field nursery, the most abundant bacterial orders showed more dynamic changes in their relative abundance in the rhizosphere than in the bulk soil. In the greenhouse, age-dependent dynamics even occurred but affected different taxa than for the field-grown plants. The N-cycling potential of rhizosphere bacterial communities showed an increase of the relative abundance of genes involved in nitrogen fixation and denitrification by plant age. Similarly, the relative abundance of reported nitrogen-fixing or denitrifying bacteria increased by plant age. However, different community structures seemed to lead to an increased potential for nitrogen fixation and denitrification in the field versus greenhouse nurseries.

Effects of Intra- and Interspecific Plant Density on Rhizosphere Bacterial Communities.

There have been very few studies on the effects of plant competition on the rhizosphere bacterial community. To investigate the impacts of intra- and interspecific plant competition, we analyzed the responses of rhizosphere bacterial communities to plant density as determined by 16S rRNA gene targeted sequencing. We included five weedy plant species growing in field soil in monocultures and mixed cultures at three densities in a greenhouse experiment. The rhizosphere bacterial community of each species changed more with density in a mixture of all five plant species than in monocultures, so intra- and interspecific plant competition had different effects on the bacterial community. For the dominant plant competitor, Centaurea cyanus, neither intra- nor interspecific competition had major effects on the composition of its rhizosphere bacterial communities. In contrast, the bacterial communities of the weakest competitor, Trifolium repens, were affected differently by intra- and interspecific competition. During increasing intraspecific competition T. repens maintained a highly specialized bacterial community dominated by Rhizobium; while during interspecific competition, the relative abundance of Rhizobium declined while other nitrogen fixing and potentially plant growth promoting taxa became more abundant. Contrary to previous observations made for soil microbial communities, the bacterial rhizosphere community of the weakest competitor did not become more similar to that of the dominant species. Thus, the process of competition, as well as the plant species themselves, determined the rhizosphere bacterial community. Our results emphasize the role of plant-plant interactions for rhizosphere bacterial communities. These effects may feedback to affect plant-plant interactions, and this is an important hypothesis for future research.

Bacteriophages drive strain diversification in a marine Flavobacterium: implications for phage resistance and physiological properties.

Genetic, structural and physiological differences between strains of the marine bacterium Cellulophaga baltica MM#3 (Flavobacteriaceae) developing in response to the activity of two virulent bacteriophages, Phi S(M) and Phi S(T), was investigated during 3 weeks incubation in chemostat cultures. A distinct strain succession towards increased phage resistance and a diversification of the metabolic properties was observed. During the incubation the bacterial population diversified from a single strain, which was sensitive to 24 tested Cellulophaga phages, into a multistrain and multiresistant population, where the dominant strains had lost susceptibility to up to 22 of the tested phages. By the end of the experiment the cultures reached a quasi steady state dominated by Phi S(T)-resistant and Phi S(M) + Phi S(T)-resistant strains coexisting with small populations of phage-sensitive strains sustaining both phages at densities of > 10(6) plaque forming units (pfu) ml(-1). Loss of susceptibility to phage infection was associated with a reduction in the strains' ability to metabolize various carbon sources as demonstrated by BIOLOG assays. This suggested a cost of resistance in terms of reduced physiological capacity. However, there was no direct correlation between the degree of resistance and the loss of metabolic properties, suggesting either the occurrence of compensatory mutations in successful strains or that the cost of resistance in some strains was associated with properties not resolved by the BIOLOG assay. The study represents the first direct demonstration of phage-driven generation of functional diversity within a marine bacterial host population with significant implications for both phage susceptibility and physiological properties. We propose, therefore, that phage-mediated selection for resistant strains contributes significantly to the extensive microdiversity observed within specific bacterial species in marine environments.