Influence of Forest Trees on the Distribution of Mineral Weathering-Associated Bacterial Communities of the Scleroderma citrinum Mycorrhizosphere

In acidic forest soils, availability of inorganic nutrients is a tree-growth-limiting factor. A hypothesis to explain sustainable forest development proposes that tree roots select soil microbes involved in central biogeochemical processes, such as mineral weathering, that may contribute to nutrient mobilization and tree nutrition. Here we showed, by combining soil analyses with cultivation-dependent analyses of the culturable bacterial communities associated with the widespread mycorrhizal fungus Scleroderma citrinum, a significant enrichment of bacterial isolates with efficient mineral weathering potentials around the oak and beech mycorrhizal roots compared to bulk soil. Such a difference did not exist in the rhizosphere of Norway spruce. The mineral weathering ability of the bacterial isolates was assessed using a microplaque assay that measures the pH and the amount of iron released from biotite. Using this microplate assay, we demonstrated that the bacterial isolates harboring the most efficient mineral weathering potential belonged to the Burkholderia genus. Notably, previous work revealed that oak and beech harbored very similar pHs in the 5- to 10-cm horizon in both rhizosphere and bulk soil environments. In the spruce rhizosphere, in contrast, the pH was significantly lower than that in bulk soil. Because the production of protons is one of the main mechanisms responsible for mineral weathering, our results suggest that certain tree species have developed indirect strategies for mineral weathering in nutrient-poor soils, which lie in the selection of bacterial communities with efficient mineral weathering potentials.The mobilization of nutrients via the biotic and abiotic weathering of soil minerals is crucial to satisfying plant nutritional needs (2, 17), especially in acidic forest soils, which are mainly nonfertilized and nutrient poor. Besides the physicochemical weathering reactions, evidence is presently accumulating which indicates that certain soil bacterial strains increase mineral weathering and improve tree nutrition (5, 9, 32, 39-41).By way of their root exudates, plants alter the structure and activity of microbial communities (6, 25, 51) and selectively favor certain ones that are potentially beneficial to them (15, 16, 21, 45, 46). A hypothesis for sustainable forest development proposes that tree roots select from the soil efficient mineral weathering bacterial communities that may contribute to nutrient mobilization and tree growth (20). In this manner, recent studies (10, 46) have revealed that the oak-Scleroderma citrinum ectomycorrhizal symbiosis selects bacterial communities that are more efficient in mineral weathering than those of the surrounding soil, suggesting that the mycorrhizal symbiosis has an indirect effect on plant nutrition through its selective pressure on the functional diversity of the mycorrhizosphere bacterial communities.Distinct impacts of the tree species on the soil bacterial community structure have been previously reported (23, 38), suggesting that the composition and activity of soil bacterial communities depend on tree physiology and notably on its impact on the soil physicochemical properties and nutrient cycling (24, 26, 37). However, no study has ever addressed the question of the impact of tree species on the structure of forest soil bacterial communities involved in mineral weathering. This question regarding the impact of tree species on the functional diversity of the bacterial communities remains a major issue in forestry, especially in the context of today''s climate change, which will give rise to a shift in the spatial distribution of forest tree species.To appreciate the effect of tree species on mycorrhizosphere bacterial communities, we focused on a single but ubiquitous mycorrhizal fungus, S. citrinum, which forms mycorrhizae with different tree species. Since no functional genes have been identified to date, a cultivation-dependent analysis was developed in this study. A total of 155 bacterial isolates were randomly chosen among a collection of 400 bacterial isolates from the soil-Scleroderma citrinum mycorrhiza interface (ectomycorrhizosphere), the extramatrical mycelium (hyphosphere), and the surrounding soil (bulk soil) in 28-year-old stands of oak (Quercus sessiliflora Smith), beech (Fagus sylvatica L.), and Norway spruce (Picea abies Karst.). The mineral weathering potential of each bacterial isolate was evaluated by way of an in vitro microplate assay, putting in interaction a calibrated bacterial suspension and the biotite, a mineral widespread in soils (46). The bacterial isolates were genotypically characterized by amplifying and sequencing a portion of the 16S rRNA gene. Their mineral weathering efficiencies and the functional structure of the bacterial communities were compared with the physicochemical characteristics of the surrounding soil.     

Inter- and Intraspecific Variations of Bacterial Communities Associated with Marine Sponges from San Juan Island,Washington

This study attempted to assess whether conspecific or congeneric sponges around San Juan Island, Washington, harbor specific bacterial communities. We used a combination of culture-independent DNA fingerprinting techniques (terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis [DGGE]) and culture-dependent approaches. The results indicated that the bacterial communities in the water column consisted of more diverse bacterial ribotypes than and were drastically different from those associated with the sponges. High levels of similarity in sponge-associated bacterial communities were found only in Myxilla incrustans and Haliclona rufescens, while the bacterial communities in Halichondria panicea varied substantially among sites. Certain terminal restriction fragments or DGGE bands were consistently obtained for different individuals of M. incrustans and H. rufescens collected from different sites, suggesting that there are stable or even specific associations of certain bacteria in these two sponges. However, no specific bacterial associations were found for H. panicea or for any one sponge genus. Sequencing of nine DGGE bands resulted in recovery of seven sequences that best matched the sequences of uncultured Proteobacteria. Three of these sequences fell into the sponge-specific sequence clusters previously suggested. An uncultured alphaproteobacterium and a culturable Bacillus sp. were found exclusively in all M. incrustans sponges, while an uncultured gammaproteobacterium was unique to H. rufescens. In contrast, the cultivation approach indicated that sponges contained a large proportion of Firmicutes, especially Bacillus, and revealed large variations in the culturable bacterial communities associated with congeneric and conspecific sponges. This study revealed sponge species-specific but not genus- or site-specific associations between sponges and bacterial communities and emphasized the importance of using a combination of techniques for studying microbial communities.Marine sponges (phylum Porifera) harbor a remarkable array of microorganisms, including bacteria (51, 54), unicellular algae (50), cyanobacteria (45, 48), dinoflagellates (14), zoochlorellae (58), and members of the domain Archaea (33). Of these microorganisms, bacteria are the most dominant group of microbial associates in sponges and can account for up to 40 to 50% of a sponge''s biomass (17). The density of bacteria can be up to 10⁸ to 10¹⁰ bacteria per g (wet weight) of sponge (18). The great abundance of bacteria in sponges caused workers to coin the term “bacteriosponges” (35) and has attracted much research interest in the role and specificity of the sponge-bacterium association.There are generally two pathways by which sponges may acquire their bacterial associates. The first pathway is filter feeding and selective retention of bacteria (44). Bacteria in the surrounding seawater can be captured by sponges when sponges filter the food particles out of the water column. The bacteria that resist digestion by sponge choanocytes and archaeocytes can survive and live inside the sponges. Sponge-associated bacterial communities acquired via this pathway are therefore heavily influenced by the type of bacteria in the water column. The second pathway is vertical transmission of bacterial associates from adult sponges to their progeny (10, 40, 49). Bacteria acquired through this pathway may exhibit specificity for genera or species of sponges due to coevolution (for a review, see reference 44).Sponge-bacterium symbioses are often mutualistic; while bacteria may benefit from the favorable nutritional conditions in sponges (18, 44), some bacterial associates may help their hosts eliminate metabolic waste (4), stabilize the sponge skeleton (36), and defend against pathogens, predators, or competitors via the production of bioactive secondary metabolites (5, 21, 39, 47). In addition, some cyanobacterial symbionts are a source of nutrients for their hosts because of their photosynthetic and nitrogen-fixing abilities (3, 57).There is a large body of knowledge concerning the possible roles of bacterial associates in sponges, but whether sponges harbor specific bacterial communities deserves more detailed study. Many previous investigations demonstrated that associated bacterial communities in certain species of sponges were highly similar and consistently different from the bacterial communities in the ambient environment (6, 22, 46). For instance, Hentschel et al. (16) showed that there were uniform microbial communities in the marine sponges Aplysina aerophoba, Rhopaloeides odorabile, and Theonella swinhoei from different geographic regions that were distinct from those in the water column or in sediments. In addition, Friedrich et al. (12) demonstrated that the composition of sponge-associated bacterial communities was resistant to environmental perturbations resulting from transplantation to different habitats. These studies suggest that there is a stable, specific, and perhaps mutualistic relationship between the two types of organisms (44). However, some bacterium-sponge symbioses do not appear to be consistent. For instance, Wichels et al. (56) demonstrated that the bacterial communities associated with the North Sea sponge Halichondria panicea varied substantially over time. Qian et al. (34) and Lee et al. (25) showed that the congeneric Callyspongia and Mycale sponges from different biogeographic regions had different bacterial associates. Although the discrepancies may be attributed to the methods employed in different studies, whether consistent sponge-bacterium associations occur in different species or genera of sponges remains unclear. Furthermore, most of the previous studies focused on only one sponge species or involved a few sponges from geographically separated regions. So far, there has been no large-scale study comparing the bacterial communities associated with sponges of different genera and species.In this study, we compared the bacterial communities associated with marine sponges around San Juan Island, Washington, in order to investigate the specificity of congeneric and conspecific sponge-associated bacterial communities. Most previous studies have relied on only one method to assess the bacterial communities, limiting the resolution of community assessments. In addition, different studies have used different approaches to address the same question, creating uncertainties and making generalizations difficult. Here, we employed both culture-independent and -dependent approaches to compensate for the limitations of different methods and to obtain a more reliable assessment of the associated bacterial communities. We used two DNA fingerprinting techniques, terminal restriction fragment length polymorphism (TRFLP) (27) and denaturing gradient gel electrophoresis (DGGE) (11). TRFLP is an effective, sensitive, high-throughput technique that differentiates bacterial community structures, while DGGE allows subsequent identification of bacteria of interest by excision and sequencing of specific bands. Both techniques have been successfully and widely used for characterization of bacterial communities in marine samples (13, 26, 28, 43, 56). In addition, bacteria associated with sponges were isolated using cultivation methods and identified by comparative analysis of 16S rRNA gene sequences. Phylogenetic affiliations of the isolates were determined and compared for different samples in order to study the specificity of the sponge-associated culturable bacterial communities.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Phylogenetic and Multivariate Analyses To Determine the Effects of Different Tillage and Residue Management Practices on Soil Bacterial Communities

Bacterial communities are important not only in the cycling of organic compounds but also in maintaining ecosystems. Specific bacterial groups can be affected as a result of changes in environmental conditions caused by human activities, such as agricultural practices. The aim of this study was to analyze the effects of different forms of tillage and residue management on soil bacterial communities by using phylogenetic and multivariate analyses. Treatments involving zero tillage (ZT) and conventional tillage (CT) with their respective combinations of residue management, i.e., removed residue (−R) and kept residue (+R), and maize/wheat rotation, were selected from a long-term field trial started in 1991. Analysis of bacterial diversity showed that soils under zero tillage and crop residue retention (ZT/+R) had the highest levels of diversity and richness. Multivariate analysis showed that beneficial bacterial groups such as fluorescent Pseudomonas spp. and Burkholderiales were favored by residue retention (ZT/+R and CT/+R) and negatively affected by residue removal (ZT/−R). Zero-tillage treatments (ZT/+R and ZT/−R) had a positive effect on the Rhizobiales group, with its main representatives related to Methylosinus spp. known as methane-oxidizing bacteria. It can be concluded that practices that include reduced tillage and crop residue retention can be adopted as safer agricultural practices to preserve and improve the diversity of soil bacterial communities.Agricultural sustainability is linked to soil management and efficient use of natural and economic resources (25, 53). Sustainable handling of resources can be obtained by applying conservation agricultural practices, i.e., reduced tillage, crop residue retention, and crop rotation (26). Reduced tillage and crop residue retention have been proposed, as they facilitate water infiltration, reduce erosion, improve soil structure, increase soil organic matter and carbon content, and moderate soil temperatures (13, 16, 30, 33, 56). Compared with conventional tillage and crop residue removal, these practices can also decrease production costs by reducing the use of heavy machinery, fuels, water, and fertilizers (19, 23). The positive effect of these practices seems to be correlated with the improvement of soil structure and a higher availability of organic substrates for microorganisms (3, 30). Improved soil structure allows better soil aeration and diffusion of water and nutrients through the soil profile, while the retention of crop residues enhances microbial activity and the soil microbial biomass content (12, 28). These improvements in soil quality can also increase soil microbial diversity, thus protecting crops against pests and diseases through competition for soil nutrients (8).Until now, most research has focused on microbial communities affected by agricultural practices, i.e., tillage and residue management, by using indicators such as plate counting and microbial biomass or by analyzing denaturing gradient gel bacterial banding patterns (21, 22, 37). Salles et al. (46) reported the use of canonical correspondence analysis on denaturing gradient gel electrophoresis banding pattern data to understand the effect of crop and land history on Burkholderia communities. However, few studies have applied phylogenetic and multivariate analyses to understand the effect of soil management practices, i.e., tillage and residue management, on microbial communities.It is necessary to interpret the changes in microbial communities as a function of contextual environmental parameters to analyze the effect of anthropogenic activities on microbial communities (42). Once modifications in microbial communities are interpreted as a function of contextual environments, it becomes possible to determine the kind of organisms that dominate such environments and to establish whether specific practices could lead to changes in beneficial or nonbeneficial microorganisms for agro-ecosystems. Changes in microbial communities can then be related to food production, soil quality, and greenhouse gas emissions (19, 20, 36).Govaerts et al. (19, 20, 21, 22) had previously characterized the soils used in this study. They showed that soils under zero tillage (ZT) and crop residue retention (+R) have better soil quality, crop yields, and catabolic diversity and a higher diversity of microflora groups than do soils under conventional tillage (CT) with or without crop residue retention (−R). The aim of this study was to complement the results of Govaerts et al. (19, 20, 21, 22) by using phylogenetic approaches and the additive main effect and multiplicative interactions (AMMI) model (18, 60) to analyze the effect of the above treatments on soil bacterial communities.     

Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur

Marine bacteria play a central role in the degradation of dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) and acrylic acid, DMS being critical to cloud formation and thereby cooling effects on the climate. High concentrations of DMSP and DMS have been reported in scleractinian coral tissues although, to date, there have been no investigations into the influence of these organic sulfur compounds on coral-associated bacteria. Two coral species, Montipora aequituberculata and Acropora millepora, were sampled and their bacterial communities were characterized by both culture-dependent and molecular techniques. Four genera, Roseobacter, Spongiobacter, Vibrio, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of clones retrieved from coral mucus and tissue 16S rRNA gene clone libraries. Clones affiliated with Roseobacter sp. constituted 28% of the M. aequituberculata tissue libraries, while 59% of the clones from the A. millepora libraries were affiliated with sequences related to the Spongiobacter genus. Vibrio spp. were commonly isolated from DMS and acrylic acid enrichments and were also present in 16S rRNA gene libraries from coral mucus, suggesting that under “normal” environmental conditions, they are a natural component of coral-associated communities. Genes homologous to dddD, and dddL, previously implicated in DMSP degradation, were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize this sulfur compound when present in coral tissues. Our results demonstrate that DMSP, DMS, and acrylic acid potentially act as nutrient sources for coral-associated bacteria and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals, with important consequences for the health of both corals and coral reef ecosystems.Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound implicated in the formation of clouds via its cleavage product dimethyl sulfide (DMS) and therefore has the potential to exert major cooling effects on climate (9, 38). The production of DMSP is mainly restricted to a few classes of marine macro- and microalgae (27, 68), with the main producers being phytoplankton species belonging to prymnesiophyte and dinoflagellate taxa (28, 62, 67). Recently, significant concentrations of DMSP and DMS have been recorded in association with animals that harbor symbiotic algae such as scleractinian corals and giant clams (7, 8, 68), raising questions about the role of coral reefs in sulfur cycling. The densities of symbiotic dinoflagellates (genus Symbiodinium, commonly known as zooxanthellae) in coral tissues are similar to those recorded for dinoflagellates in phytoplankton blooms (11, 68). Since dinoflagellates are among the most significant producers of DMSP and high intracellular concentrations of DMSP have been found in both cultured zooxanthellae (26) and scleractinian corals (6-8, 25), these observations suggest that endosymbiotic zooxanthellae have an integral role in sulfur cycling in oligotrophic reef waters.Most of the DMSP produced by planktonic dinoflagellates is exuded into the surrounding water, where it is degraded by bacteria via two possible pathways: the first one converts a large fraction (ca. 75%) of dissolved DMSP to methylmercaptopropionate, which is subsequently incorporated into the biomass of microbial cells (22, 27, 66). The second pathway transforms the remaining part of the dissolved DMSP to equimolar concentrations of DMS and acrylic acid (43, 66, 72). This metabolic pathway for DMSP degradation has been identified in the alphaproteobacterial species Sulfitobacter sp. and the enzyme involved (DMSP-dependent DMS lyase [DddL]) characterized (10). Another pathway for DMS formation (without production of acrylate) has been described for Marinomonas sp. and the gene responsible, dddD, identified. In addition, the protein DddR has been directly implicated in the regulation of the gene encoding DddD (66). The DMS produced by these enzymes are then released into the surrounding water (27). Prior to the 1980s, diffusion of supersaturated DMS from the oceans to the atmosphere was thought to be the major removal pathway of this compound from the oceans (35, 72). More recently, however, it has been estimated that between 50 and 80% of the DMS produced by DMSP-degrading bacteria is degraded directly by other types of bacteria (58, 59), although the populations and metabolic pathways involved in the degradation of DMS are still poorly understood.Coral-associated bacterial communities are known to be diverse and highly abundant (12, 30, 48, 49, 52). These dynamic communities exploit a number of habitats associated with corals, including mucus on coral surfaces (48), intracellular niches within coral tissues (3, 16, 45, 47, 52), spaces within coral skeletons (15, 51), and seawater surrounding corals (16, 61). Each of these habitats is believed to harbor different bacterial populations (4, 52). Despite high bacterial diversity, corals have been reported to harbor species-specific microbial communities for beneficial effects; however, their role in coral health is poorly understood (47-50). In coral reef environments, bacteria are dependent upon organic compounds produced by photoautotrophic organisms such as endosymbiotic zooxanthellae (48); therefore, photosynthates translocated to coral tissues and mucus may determine microbial communities closely associated with corals (48, 52). The high levels of DMSP and DMS produced by corals, coupled with the dependence of DMSP and DMS conversion on processes typically involving bacteria, suggest that corals are likely to harbor bacterial species involved in the cycling of these compounds. To investigate the potential of the organosulfur compound DMSP and its breakdown products, DMS and acrylic acid, to drive coral-associated microbial communities, we used these compounds as sole carbon sources to isolate bacteria from two coral species (Montipora aequituberculata and Acropora millepora) and then directly compared these microbial communities with coral-associated microbiota identified using culture-independent analyses. Genes implicated in the metabolism of DMSP were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize organic sulfur compounds present in coral tissues.     

Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO2

Elevated atmospheric CO₂ can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritus-based ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO₂ would result in changes in the size and composition of microbial communities colonizing leaves in a woodland stream. Three tree species, Populus tremuloides, Salix alba, and Acer saccharum, were grown under ambient (360 ppm) or elevated (720 ppm) CO₂, and their leaves were incubated in a woodland stream. Elevated-CO₂ treatment resulted in significant increases in the phenolic and tannin contents and C/N ratios of leaves. Microbial effects, which occurred only for P. tremuloides leaves, included decreased fungal biomass and decreased bacterial counts. Analysis of fungal and bacterial communities on P. tremuloides leaves via terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing revealed that fungal community composition was mostly unchanged by the elevated-CO₂ treatment, whereas bacterial communities showed a significant shift in composition and a significant increase in diversity. Specific changes in bacterial communities included increased numbers of alphaproteobacterial and cytophaga-flavobacter-bacteroides (CFB) group sequences and decreased numbers of betaproteobacterial and firmicutes sequences, as well as a pronounced decrease in overall Gram-positive bacterial sequences.The concentration of atmospheric CO₂ has been increasing for the last 150 years, from 270 ppm prior to the industrial revolution (49) to the current level of approximately 388 ppm (http://www.mlo.noaa.gov), and is projected to exceed 700 ppm by the end of the century (57). This ongoing increase in atmospheric CO₂ is believed to be due to the extensive use of fossil fuels and changes in land use patterns (5). Elevated atmospheric CO₂ has global climate implications due to its role in the greenhouse effect (39), and it has also been shown to have direct biological effects. Specifically, elevated CO₂ can increase the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco) (13), resulting in increased carbon fixation by C3 plants (49). This increased carbon fixation can result in increased above- and below-ground plant biomass (21, 47, 63, 72), as well as altered foliar chemical composition (31, 46, 58, 70).Elevated atmospheric CO₂ is unlikely to have direct impacts on forested headwater streams, as they are primarily heterotrophic systems (2) in which CO₂ is typically supersaturated (41). However, changes in leaf chemistry may have an impact, as forested headwater streams are detritus-based ecosystems that derive up to 99% of their carbon inputs from terrestrial organic matter (71), which is mainly leaf litter (29). Microbes play a key role in the entry of this allochthonous organic material into stream food webs. Fungi and bacteria colonize leaf litter after its deposition in a stream and begin decomposition of the leaf material (34). The resulting growth of microbial assemblages associated with leaf litter provides a critical food resource for detritus-feeding invertebrate consumers (6, 18, 23, 44), which through their feeding activities help facilitate the further transformation and breakdown of plant litter and the flow of carbon and nutrients to higher-trophic-level organisms, including fish. Prior research has demonstrated that aquatic invertebrates show a clear preference to eat leaves that have been extensively colonized, or “conditioned,” by microbes (4, 18, 65). This is likely due to the fact that microbial colonization significantly increases the nutrient content of detritus, as microbes can incorporate soluble nutrients from stream water (e.g., nitrogen) into the microbial biomass (64, 66). In addition, microbes convert indigestible leaf components (e.g., lignin and cellulose) into microbial biomass, which invertebrates can digest more efficiently (6). Therefore, fungi and bacteria are significant contributors to the transfer of carbon and nutrients from terrestrial to aquatic ecosystems.Microbial decomposition of leaves in streams is influenced by the chemical composition of the leaf material. This has been illustrated by a number of studies comparing decomposition of leaves from different tree species (for a review, see reference 62). These studies have demonstrated that leaves from species, such as oaks and conifers, that are relatively high in polyphenolic compounds, including lignin and tannins, tend to decompose more slowly than leaves from species with lower concentrations of these compounds, such as alder (62). The leaf carbon-to-nitrogen (C/N) ratio also impacts decomposition rates; leaf litter with a high C/N ratio tends to decompose more slowly than litter with a low C/N ratio (62). These trends are relevant to atmospheric CO₂ concentrations because elevated atmospheric CO₂ has been shown to increase the concentrations of phenolic compounds (lignin and tannins), as well as the C/N ratio of leaves of C3 plants (31, 46, 58, 70). Therefore, it is reasonable to hypothesize that growth of trees under elevated CO₂ could have negative impacts on microbial colonization and decomposition of leaves. Rier et al. (58) tested this hypothesis with one tree species, Populus tremuloides (quaking aspen), and found that leaves produced under elevated CO₂ decomposed more slowly in streams and supported less fungal and bacterial biomass than leaves produced under ambient conditions (58).In addition to impacting microbial community size, it is reasonable to hypothesize that changes in leaf chemistry caused by growth of trees under elevated CO₂ could impact microbial community composition. Several studies have demonstrated that the compositions of microbial communities colonizing leaves in streams can differ based on tree species (36, 45). No study we are aware of has examined the effects of tree growth under elevated atmospheric CO₂ on the compositions of microbial communities colonizing leaf litter in streams; however, such changes in microbial community composition could be highly relevant to stream food webs. For example, different groups of fungi and bacteria differ in their abilities to degrade various components of leaf litter (1, 67), so the species compositions of microbial communities could potentially impact rates of decomposition and production of microbial biomass (26). This in turn could impact the transfer of carbon and energy to higher-trophic-level organisms. In addition, different groups of fungi and bacteria differ in chemical composition (9, 32), and thus, they may differ in their nutritional values to aquatic invertebrates.In the current study, we tested the hypothesis that changes in leaf chemistry caused by elevated CO₂ would result in changes in the biomass and composition of detrital microbial communities by growing three tree species under ambient or elevated CO₂, collecting leaves after abscission, incubating the leaves in a woodland stream, and determining the biomass and composition of the microbial communities colonizing the leaves.     

Assessing Susceptibility to Age-related Macular Degeneration with Proteomic and Genomic Biomarkers

Age-related macular degeneration (AMD) is a progressive disease and major cause of severe visual loss. Toward the discovery of tools for early identification of AMD susceptibility, we evaluated the combined predictive capability of proteomic and genomic AMD biomarkers. We quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. CEP adducts are uniquely generated from oxidation of docosahexaenoate-containing lipids that are abundant in the retina. Mean CEP adduct and autoantibody levels were found to be elevated in AMD plasma by ∼60 and ∼30%, respectively. The odds ratio for both CEP markers elevated was 3-fold greater or more in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high temperature requirement factor A1 (HTRA1), complement factor H, and complement C3, and the risk of AMD was predicted based on genotype alone or in combination with the CEP markers. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2–3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. The results compellingly demonstrate higher mean CEP marker levels in AMD plasma over a broad age range. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ∼76% accuracy and in combination with genomic markers provide up to ∼80% discrimination accuracy. Plasma CEP marker levels were altered slightly by several demographic and health factors that warrant further study. We conclude that CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for assessing susceptibility to this blinding, multifactorial disease.Age-related macular degeneration (AMD)¹ is the most common cause of legal blindness in the elderly in developed countries (1). It is a complex, progressive disease involving multiple genetic and environmental factors that can result in severe visual loss. Early risk factors include the macular deposition of debris (drusen) on Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE). Later stages of “dry” AMD involve the degeneration of photoreceptor and RPE cells resulting in geographic atrophy. In “wet” AMD, abnormal blood vessels grow from the choriocapillaris through Bruch membrane (choroidal neovascularization (CNV)). CNV occurs in 10–15% of AMD cases yet accounts for over 80% of debilitating visual loss in AMD. Anti-vascular endothelial growth factor treatments can effectively inhibit the progression of CNV (1), and antioxidant vitamins and zinc can slow dry AMD progression for select individuals (2). However, there are no universally effective therapies for the prevention of dry AMD or the progression from dry to wet AMD nor are there therapies to repair retinal damage in advanced AMD. The prevalence of advanced AMD in the United States is projected to increase by 50% to ∼3 million by the year 2020 largely because of the rapidly growing elderly population (3). Accordingly early identification of AMD susceptibility and implementation of preventive measures are important therapeutic strategies (1).The molecular mechanisms causing AMD remain unknown, although inflammatory processes have been implicated by the identification of AMD susceptibility genes encoding complement factors (4–10) and the presence of complement proteins in drusen (11–13). Oxidative stress has long been associated with AMD pathology as shown by the finding that smoking significantly increases the risk of AMD (14) and that antioxidant vitamins can selectively slow AMD progression (2). A direct molecular link between oxidative damage and AMD was established by the finding that carboxyethylpyrrole (CEP), an oxidative protein modification generated from docosahexaenoate (DHA)-containing phospholipids, was elevated in Bruch membrane and drusen from AMD patients (11). Subsequently CEP adducts as well as CEP autoantibodies were found to be elevated in plasma from AMD donors (15), and CEP adducts were found to stimulate neovascularization in vivo, suggesting a role in the induction of CNV (16). From such observations, oxidative protein modifications were hypothesized to serve as catalysts of AMD pathology (11, 15, 17). In support of this hypothesis, mice immunized with CEP-adducted mouse albumin develop a dry AMD-like phenotype that includes sub-RPE deposits resembling drusen and RPE lesions mimicking geographic atrophy (18).Although identified AMD susceptibility genes account for over half of AMD cases (19), many individuals carrying AMD risk genotypes may never develop the disease. Likewise only a fraction of those diagnosed with early AMD progress to advanced stage disease with severe visual loss (2). Toward the discovery of better methods to predict susceptibility to advanced AMD, we quantified CEP adducts and autoantibodies in over 1400 plasma donors and also genotyped many of these donors for AMD risk polymorphisms in complement factor H (CFH) (4–7), complement C3 (9, 10), age-related maculopathy susceptibility 2 (ARMS2; also known as LOC387715) (19–22), and high temperature requirement factor A1 (HTRA1) (23, 24). The results demonstrate that combined CEP proteomic and genomic biomarker measurements are more effective in assessing AMD risk than either method alone.     

Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods

Ciliates are an important component of aquatic ecosystems, acting as predators of bacteria and protozoa and providing nutrition for organisms at higher trophic levels. Understanding of the diversity and ecological role of ciliates in stream biofilms is limited, however. Ciliate diversity in biofilm samples from four streams subject to different impacts by human activity was assessed using microscopy and terminal restriction fragment length polymorphism (T-RFLP) analysis of 18S rRNA sequences. Analysis of 3′ and 5′ terminal fragments yielded very similar estimates of ciliate diversity. The diversity detected using microscopy was consistently lower than that suggested by T-RFLP analysis, indicating the existence of genetic diversity not apparent by morphological examination. Microscopy and T-RFLP analyses revealed similar relative trends in diversity between different streams, with the lowest level of biofilm-associated ciliate diversity found in samples from the least-impacted stream and the highest diversity in samples from moderately to highly impacted streams. Multivariate analysis provided evidence of significantly different ciliate communities in biofilm samples from different streams and seasons, particularly between a highly degraded urban stream and less impacted streams. Microscopy and T-RFLP data both suggested the existence of widely distributed, resilient biofilm-associated ciliates as well as ciliate taxa restricted to sites with particular environmental conditions, with cosmopolitan taxa being more abundant than those with restricted distributions. Differences between ciliate assemblages were associated with water quality characteristics typical of urban stream degradation and may be related to factors such as nutrient availability and macroinvertebrate communities. Microscopic and molecular techniques were considered to be useful complementary approaches for investigation of biofilm ciliate communities.Heterotrophic microeukaryotes such as ciliates are thought to be of considerable importance in aquatic ecosystems, as they are major predators of bacteria and constitute a nutritional resource for other protozoa, invertebrates, and probably fish larvae (9, 22, 36, 52, 62, 63, 71). In addition, protozoan bacterivory contributes to enhanced decomposition of leaf detritus—a vital nutrient resource in streams—by increasing turnover of bacterial populations through predation (57). It is not well understood, however, how ciliate diversity and community structure in streams are affected by changing environmental conditions, or how ciliate communities affect other stream biota and processes. The effects of various physical, chemical, and biological factors on freshwater protozoan communities have been considered by a number of studies, but most of these have focused upon planktonic organisms in lentic habitats (for example, see references 2, 11, and 44). However, the complex microbial communities in biofilms have been recognized as important contributors to critical ecological processes, such as auxotrophic primary production, nitrogen fixation, and nutrient cycling, and may underpin the function of stream food webs (31, 45, 61). The few studies which have investigated benthic habitats in lotic systems have found evidence of the existence of diverse communities of abundant ciliates (3, 20, 56) and shifts in community structure in response to ecophysiological parameters (30, 42, 43). With one exception, however, these investigations were based on aquatic sediments, and the organisms within epilithic biofilms have continued to receive little attention.Most studies of ciliate diversity and ecology have utilized microscopy-based methods of identification (for example, see references 3 and 56), as ciliate cells are relatively large and morphologically diverse. Such methods demand a high level of taxonomic expertise, however, and are difficult and time-consuming—for example, many ciliates are fragile and fast moving, and they often require difficult fixing and staining protocols for reliable identification. Molecular biological tools offer the possibility of more accurate and efficient methods for protozoan study and may provide a useful complement to traditional approaches (12, 18, 28, 65), yet we know of only a few molecular studies of environmental ciliate diversity (18, 20, 37). A series of recent investigations used culture-independent analysis of 18S rRNA gene sequences to reveal the existence of diverse microeukaryote communities in assorted marine, anoxic, and extreme environments (40, 48, 66, 69, 70, 72). Furthermore, a growing body of evidence suggests the existence of significant genetic diversity among various ciliate taxa which has escaped detection by microscopy (14, 18, 23, 34, 60, 64, 78), pointing to the potential for molecular techniques to generate new insights into ciliate diversity and ecology, and suggesting a need for comparison of the effectiveness of these different techniques in environmental samples.Terminal restriction fragment length polymorphism (T-RFLP) analysis provides an efficient, inexpensive, and semiquantitative means for comparing microbial molecular diversity between different samples and has been widely used to investigate bacterial communities, although only a few studies have applied T-RFLP methods to the analysis of microeukaryote diversity (6, 16, 17). In this study, ciliate diversity and community structure were investigated in biofilm samples from streams representing a range of levels of anthropogenic degradation, with the objective of testing the null hypothesis that human impacts have no effect upon this important heterotrophic component of stream ecosystems. To achieve this, ciliate-targeted PCR primers were used in conjunction with T-RFLP and multivariate statistical analyses. Additionally, ciliate diversity measures obtained using molecular techniques were compared with those derived from microscopy-based methods in order to assess the relative effectiveness of these approaches.     

Diversity of Bacteria in the Marine Sponge Aplysina fulva in Brazilian Coastal Waters

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using “direct” and “indirect” approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups—Pseudomonas and Actinobacteria—revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.The phylum Porifera (sponges) consists of benthic (sessile) organisms that occur primarily in marine environments at different depths (26). Sponges are classified into three groups, namely, the Calcarea (calcareous sponges), Hexactinellida (glass sponges), and Demospongiae (5, 26). The group Demospongiae, also called demosponges, encompasses 95% of the ca. 5,500 living sponge species described thus far (5). As typical filter feeders, demosponges are the prime bacterial filters of the sea. They are capable of pumping thousands of liters of water per day (23), using prokaryotic microorganisms as the main source of food (1, 43, 47). In addition to demosponges feeding on microorganisms, the presence of bacteria in high density in internal sponge layers (mesohyl) indicates that a selective process favoring particular prokaryotes, involving microbe-sponge interactions, is likely to occur (64). Furthermore, the dawn of the interactions between Prokarya and higher organisms may actually lie in the demosponges, whose origin is estimated to date back to 550 million years ago (5, 33).Putative interactions between demosponges and microorganisms, presumably mostly consisting of Bacteria and Archaea, were first demonstrated by transmission electron microscopy (TEM), where high amounts of microorganisms were observed in the mesohyl (1, 14, 16, 64). Hence, these bacterium-rich sponges have been termed “bacteriosponges” (46). While investigating 11 taxonomically different demosponges using TEM, Vacelet and Donadey (64) identified two different sponge types in respect of their association with bacteria. Sponges with thick mesohyl contained abundant, dense, and morphologically diverse microbial communities (i.e., bacteriosponge), while those with a well-developed aquiferous system and low-density mesohyl contained few bacterial cells and typically only single bacterial morphotypes. The two types have recently been called “high-microbial-abundance” (HMA) and “low-microbial-abundance” (LMA) sponges, respectively (23). In HMA sponges, bacterial densities may reach 10⁸ to 10¹⁰ bacterial cells per g (wet weight) of sponge, exceeding seawater concentrations by 2 to 4 orders of magnitude (15, 23). Based on the analysis of 16S rRNA genes, over 15 bacterial phyla have thus far been reported to occur in association with marine sponges (11, 23, 56). Among these are typical sponge-associated bacteria such as members of the Cyanobacteria, Chloroflexi, Proteobacteria, Acidobacteria, Verrucomicrobia, and the candidate phyla “Poribacteria” and TM6 (14, 30, 51, 56, 60, 68).Increasing research interest in the sponge-associated microbiota has emerged in the past few years, mainly due to the in spongium production of an enormous diversity of biologically active secondary metabolites (56). Recent studies suggest that certain bioactive compounds retrieved from marine sponges—such as complex polypeptides and nonribosomal peptides—are likely to be synthesized by the symbiont bacteria (27, 41, 42). Such bioactive secondary metabolites offer great promise for use in biotechnology and medicine (3, 22, 27, 41, 42, 51, 59). In particular, cytotoxic compounds, i.e., antitumoral substances and polyketides, may find application in anticancer therapies (13, 42, 51). Recent investigations revealed the presence of dibromotyrosine-derived metabolites in Aplysina fulva (Pallas, 1766) specimens collected along the Brazilian shore (39). However, a putative role of microbial symbionts in the production of such metabolites, commonly found to display biological activity, remains to be evaluated.Despite the global-scale occurrence of sponges in Earth''s marine ecosystems, the investigation of their associated bacterial communities has thus far been restricted only to certain areas (1, 11, 13, 14, 27, 54, 58, 68). To our knowledge, no studies have been conducted, to date, on sponge-associated microbes in subtropical, South Atlantic open shore waters. In the present study, we assess the diversity and composition of the bacterial community associated with the demosponge A. fulva collected at two different sites at the Brazilian shore. A suite of tools, ranging from plate count estimations and TEM to sponge DNA-based analyses of bacterial 16S rRNA genes, was used. We hypothesized that a distinct bacterial community occurs in A. fulva, which is different from that in the surrounding bulk water, as well as from those in other sponge species.     

Microbial Decomposer Communities Are Mainly Structured by Trophic Status in Circumneutral and Alkaline Streams

In streams, the release of nitrogen and phosphorus is reported to affect microbial communities and the ecological processes they govern. Moreover, the type of inorganic nitrogen (NO₃, NO₂, or NH₄) may differently impact microbial communities. We aimed to identify the environmental factors that structure aquatic microbial communities and drive leaf litter decomposition along a gradient of eutrophication. We selected five circumneutral (Portuguese) and five alkaline (French) streams differing in nutrient concentrations to monitor mass loss of alder leaves, bacterial and fungal diversity by PCR-denaturing gradient gel electrophoresis, fungal biomass and reproduction, and bacterial biomass during 11 weeks of leaf immersion. The concentrations of inorganic nutrients in the stream water ranged from 5 to 300 μg liter⁻¹ soluble reactive phosphorus, 0.30 to 5.50 mg liter⁻¹ NO₃-N, 2 to 103 μg liter⁻¹ NO₂-N, and <4 to 7,100 μg liter⁻¹ NH₄-N. Species richness was maximum in moderately anthropized (eutrophic) streams but decreased in the most anthropized (hypertrophic) streams. Different species assemblages were found in subsets of streams with different trophic statuses. In both geographic areas, the limiting nutrient, either nitrate or phosphate, stimulated the microbial activity in streams of intermediate trophic status. In the hypertrophic streams, fungal biomass and reproduction were significantly lower, and bacterial biomass dramatically decreased at the site with the highest ammonium concentration. The limiting nutrients that defined the trophic status were the main factor structuring fungal and bacterial communities, whatever the geographic area. A very high ammonium concentration in stream water most probably has negative impacts on microbial decomposer communities.There is evidence that increases in nitrate and phosphate concentrations stimulate microbial respiration and fungal and bacterial activity (biomass buildup, sporulation, and/or productivity) on plant litter, leading to faster leaf decomposition in freshwaters (16, 17, 26, 34). However, fungal demands of nitrate and phosphate are reported to be fulfilled at relatively low levels (1, 12), and further increases in these nutrients in the stream water do not necessarily result in enhanced fungal activity. Besides, the form in which inorganic nutrients are present in streams, their biological availability, and even their toxicity have different ecological consequences. In densely anthropized hypertrophic streams, high levels of nitrate and phosphate were associated with decreased fungal biomass and leaf breakdown, most probably because of the high concentrations of ammonium and ammonia (2). On the other hand, the positive effects of nutrients on biomass and productivity of leaf-associated fungi can be offset by other factors, such as low oxygen concentration and sedimentation, leading to retarded decomposition (26, 33, 34).Changes in inorganic nutrient concentrations in the stream water were reported to alter the structure of fungal communities on plant litter (16, 36). Nutrient additions to moderate levels increased the diversity of fungal communities in circumneutral soft-water Appalachian mountain streams (18) but not in a Mediterranean alkaline stream (1). Moreover, fungal diversity was lower in circumneutral eutrophic streams than in reference streams (10, 35). Fungal diversity has been assessed mostly through the morphological analysis of produced conidia, not taking into account nonsporulating fungi. This raises the question of whether the differential impacts of eutrophication on fungal diversity could be due partly to difficulties in measuring actual diversity. Besides, the study of bacterial diversity on decomposing leaves has been strongly restricted to a few cultivable bacteria (<1%). Molecular typing, such as denaturing gradient gel electrophoresis (DGGE) of a specific rRNA gene region, has proved useful for assessing diversity in both leaf-associated fungi and bacteria (7, 8, 9, 11, 30).We aimed to identify the environmental factors that drive the ecological processes in freshwaters impacted by eutrophication through examination of leaf litter decomposition and associated microbial communities along a gradient of nutrient enrichment. Specifically, we addressed the following two questions: (i) which are the environmental factors that mainly structure the fungal and bacterial communities and (ii) what are the relationships between concentrations of inorganic nutrients in the stream water, leaf litter decomposition, and the activity of associated microbes? We selected 10 stream sites spanning wide concentration ranges of dissolved inorganic nitrogen (NO₃-N, NO₂-N, NH₄-N, and NH₃-N) and soluble reactive phosphorus (SRP), including 5 in northwestern Portugal with circumneutral pH and 5 in southwestern France with an alkaline pH. With these two groups of stream sites, we assessed the structure of and diversity in both sporulating and nonsporulating fungal communities, using asexual spore morphology and DGGE fingerprints of the ITS2 region, and in bacterial communities, using DGGE fingerprints of the 16S rRNA gene region. Additionally, we examined leaf mass loss and microbial activity on decomposing leaves by determining bacterial and fungal biomass and fungal reproduction.     

Comparison of Effects of Compost Amendment and of Single-Strain Inoculation on Root Bacterial Communities of Young Cucumber Seedlings

Compost amendment and inoculations with specific microorganisms are fundamentally different soil treatment methods, commonly used in agriculture for the improvement of plant growth and health. Although distinct, both methods affect the rhizosphere and the plant roots. In the present study we used a 16S rRNA gene approach to achieve an overview of early consequences of these treatments on the assemblage of plant root bacterial communities. For this purpose, cucumber seedlings were grown, under controlled conditions, in perlite potting mix amended with biosolid compost or straw compost, or inoculated with Streptomyces spp. A uniform trend of response of root bacterial communities for all treatments was observed. Root bacterial density, measured as bacterial targets per plant tef gene by real-time PCR, was reduced in 31 to 67%. In addition, increased taxonomic diversity accompanied shifts in composition (α-diversity). The magnitude of change in these parameters relative to the perlite control varied between the different treatments but not in relation to the treatment method (compost amendments versus inoculations). Similarity between the compositions of root and of potting mix bacterial communities (β-diversity) was relatively unchanged. The abundance of Oxalobacteraceae was >50% of the total root bacterial community in the untreated perlite. Root domination by this group subsided >10-fold (straw compost) to >600-fold (Streptomyces sp. strain S1) after treatment. Thus, loss of dominance appears to be the major phenomenon underlining the response trend of the root bacterial communities.Environmental concern over conventional agricultural fertilization and disease control measures has led to increased interest in finding environmentally friendly alternatives. The most explored ones include compost amendments (18, 36) and the application of different microbial preparations (11, 19, 37). These are widely distinct applications. The first approach adds to the amended medium not only a rich and diverse consortium of biological agents but also organic matter and nutrients. It was confirmed that the efficacy of such treatments involves the response of the soil, the plant, and the rhizosphere microbial communities (19, 56). The activities of rhizosphere microorganisms alter the rhizosphere and thus affect plant health and root growth and development (24). Therefore, one of the main objectives of compost amendment or of inoculation with specific microbial strains is manipulation of the plant rhizosphere conditions, particularly via manipulation of the microbial community composition (32).The response of rhizosphere bacterial communities to different anthropogenic and other disturbances has been discussed in terms of resilience (3, 32). Generally, the introduction of new microorganisms produces only restricted spatial and temporal effects on the soil, rhizosphere, and root microbial communities (4, 29, 35). Thus, the plant growth-promoting effect of such treatments may be related to microbial events occurring during the early stages of plant development. Such early effects were pointed out for inoculants of different bacterial species (14, 42) and for compost amendment (15, 21, 50).Consequences of compost amendment or of single species inoculation often include shifts in the plant roots hormonal balance or a plant systemic response, namely, induced systemic resistance (8, 38, 52). Thus, direct or indirect activities of the introduced microorganisms may result in similar modifications of the root habitat. If so, bacterial assemblages of treated roots may share qualitative and quantitative characteristics different from those exhibited by untreated roots.The objective of the present study was therefore to describe and compare responses of bacterial communities of young plant roots to the application of compost or bacterial inoculants. This was performed in a simple model comprised of cucumber seedlings grown in potting mixes amended with compost or inoculated with Streptomyces spp. isolated from the two different composts.     

The Carnivorous Pale Pitcher Plant Harbors Diverse,Distinct, and Time-Dependent Bacterial Communities

The ability of American carnivorous pitcher plants (Sarracenia) to digest insect prey is facilitated by microbial associations. Knowledge of the details surrounding this interaction has been limited by our capability to characterize bacterial diversity in this system. To describe microbial diversity within and between pitchers of one species, Sarracenia alata, and to explore how these communities change over time as pitchers accumulate and digest insect prey, we collected and analyzed environmental sequence tag (454 pyrosequencing) and genomic fingerprint (automated ribosomal intergenic spacer analysis and terminal restriction fragment length polymorphism) data. Microbial richness associated with pitcher plant fluid is high; more than 1,000 unique phylogroups were identified across at least seven phyla and 50 families. We documented an increase in bacterial diversity and abundance with time and observed repeated changes in bacterial community composition. Pitchers from different plants harbored significantly more similar bacterial communities at a given time point than communities coming from the same genetic host over time. The microbial communities in pitcher plant fluid also differ significantly from those present in the surrounding soil. These findings indicate that the bacteria associated with pitcher plant leaves are far from random assemblages and represent an important step toward understanding this unique plant-microbe interaction.Characterization of the phyllosphere is fundamental to our understanding of the ecology and evolution of plant populations and plant diversity and their interactions with other organisms (46, 64, 66). The carnivorous pitcher plant genus Sarracenia is an obvious system to address basic questions in plant-microbe interaction because each pitcher (a modified leaf) of the plant contains a microcosm composed of larval insects, fungi, algae, rotifers, nematodes, and bacteria that, together, ultimately break down nutrients from insect prey for the plant (1, 10, 20, 28, 37). Each pitcher represents a naturally defined and discrete community with a finite volume and a discrete life span (each leaf lasts only one season). Several investigations have explored species interactions within Sarracenia pitchers (13, 20, 34, 54), and competition, predation and dispersal frequency appear to be important drivers of community composition in the system (1, 20, 43, 44). Studies involving community patterns on a larger scale within pitchers, however, are few, and the processes that produce these patterns remain unknown (33).     

Pyrosequencing-Based Assessment of Soil pH as a Predictor of Soil Bacterial Community Structure at the Continental Scale

Soils harbor enormously diverse bacterial populations, and soil bacterial communities can vary greatly in composition across space. However, our understanding of the specific changes in soil bacterial community structure that occur across larger spatial scales is limited because most previous work has focused on either surveying a relatively small number of soils in detail or analyzing a larger number of soils with techniques that provide little detail about the phylogenetic structure of the bacterial communities. Here we used a bar-coded pyrosequencing technique to characterize bacterial communities in 88 soils from across North and South America, obtaining an average of 1,501 sequences per soil. We found that overall bacterial community composition, as measured by pairwise UniFrac distances, was significantly correlated with differences in soil pH (r = 0.79), largely driven by changes in the relative abundances of Acidobacteria, Actinobacteria, and Bacteroidetes across the range of soil pHs. In addition, soil pH explains a significant portion of the variability associated with observed changes in the phylogenetic structure within each dominant lineage. The overall phylogenetic diversity of the bacterial communities was also correlated with soil pH (R² = 0.50), with peak diversity in soils with near-neutral pHs. Together, these results suggest that the structure of soil bacterial communities is predictable, to some degree, across larger spatial scales, and the effect of soil pH on bacterial community composition is evident at even relatively coarse levels of taxonomic resolution.The biogeographical patterns exhibited by microbial communities have been examined in a wide range of environments, and studies focusing on microbial biogeography continue to be published at a rapid pace. We know that microbial community diversity and composition can vary considerably across space, and this variation is theorized to be linked to changes in a number of biotic or abiotic factors (22, 36, 41). There are numerous overarching reasons for this interest in understanding microbial biogeography. For example, comparing microbial patterns to those commonly observed in plant and animal taxa is of intense theoretical interest (22, 25). From a more practical standpoint, studies of microbial biogeography can often provide key insights into the physiologies, environmental tolerances, and ecological strategies of microbial taxa, particularly those difficult-to-culture taxa that often dominate in natural environments. However, perhaps the most important rationale for studying microbial biogeography is the most basic one: microbes are diverse, ubiquitous, and abundant, yet their biogeographical patterns and the factors driving these spatial patterns often remain poorly understood.No single biogeographical pattern is shared by all microorganisms, just as there is no single biogeographical pattern followed by all “macrobial” (i.e., plant and animal) communities (31). The specific biogeographical patterns exhibited by microorganisms are variable and highly dependent on a number of factors, including the taxonomic group in question (29), the degree of phylogenetic resolution at which the communities are examined (e.g., Pseudomonas) (7), and the spatial scale of the study (40). However, some common patterns emerge if we specifically examine the biogeography of soil microorganisms. In particular, the structure and diversity of soil bacterial communities have been found to be closely related to soil environmental characteristics (5, 37, 47), and soil pH is often correlated with the observed biogeographical patterns (19, 24). However, due to the paucity of detailed and comprehensive studies of soil bacterial biogeography, particularly across larger spatial scales, our understanding of soil microbial biogeography remains incomplete.Previous studies of soil bacterial biogeography have focused on either surveying a few soils in detail or surveying a larger number of soils by techniques that offer less detailed phylogenetic information. For example, a few recent studies used pyrosequencing or Sanger sequencing-based techniques to deeply survey the diversity and composition of the bacterial communities within a single soil or a few soils (1, 14, 20, 39, 42). Such studies are valuable in that they provide our best assessments of overall bacterial diversity and community structure and the relative abundances of specific bacterial taxa within soils. However, because such studies often examine only a limited number of soils, they do not allow for robust assessment of biogeographical patterns and the factors that may drive these patterns. Other studies have examined bacterial communities across a larger number of soils, using more limited techniques, such as fingerprinting methods that offer little specific phylogenetic information on bacterial community structure or techniques that describe communities at very coarse levels of taxonomic resolution (18, 19). A comprehensive assessment of the biogeographical patterns exhibited by soil bacterial communities requires both depth (individual communities surveyed at a reasonable level of phylogenetic detail) and breadth (examining a sufficiently large number of samples to assess spatial patterns). With the recent development of the bar-coded pyrosequencing technique (23), we need not sacrifice depth for breadth, or vice versa. This was demonstrated in several recent studies (2, 12, 17, 28) that used bar-coded pyrosequencing to simultaneously analyze relatively large numbers of individual samples, surveying the bacterial community in each sample to an extent that would be difficult (or prohibitively expensive) using standard cloning and Sanger sequencing techniques.Here we apply the bar-coded pyrosequencing technique to examine the structure and diversity of bacterial communities in 88 soils collected from across North and South America. This work expands on a previous fingerprinting-based survey of bacterial communities across a similar set of soils (19), using the pyrosequencing technique to extend the analyses and to answer the following questions. Which taxa are most abundant in soil? How does the phylogenetic structure of bacterial communities vary across the continental scale? Which environmental factors best predict bacterial community structure and diversity? Are some soil bacterial phyla more diverse than others?     

Community Genomic and Proteomic Analyses of Chemoautotrophic Iron-Oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) Bacteria in Acid Mine Drainage Biofilms

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.To understand how microorganisms contribute to biogeochemical cycling, it is necessary to determine the roles of uncultivated as well as cultivated groups and to establish how these roles vary during ecological succession and when environmental conditions change. Shotgun genomic sequencing (metagenomics) has opened new opportunities for culture-independent studies of microbial communities. Examples include investigations of acid mine drainage (AMD) biofilm communities (4, 43, 75), symbiosis in a marine worm involving sulfur-oxidizing and sulfate-reducing bacteria (85), and enhanced biological phosphorous removal by sludge communities (32). From these genomic data sets, it has been possible to reconstruct aspects of the metabolism of individual organisms (32) and coexisting community members (29, 75) and to identify which organisms contribute community-essential functions (75). An interesting question relates to how differences in metabolic potential between organisms from the same lineage allow them to occupy distinct niches. Identification of potentially adaptive traits in closely related organisms is also important from an evolutionary perspective.Genomic data do not reveal how organisms alter their metabolisms in response to the presence of other organisms or environmental conditions. Proteomics methods for analysis of metabolic responses of isolates (16, 17, 42, 80, 81) have been extended to analyze the functioning of the dominant members of natural consortia (56, 69), with strain-level resolution (43, 82). In these studies, peptides are separated by liquid chromatography (LC) and identified by tandem mass spectrometry (MS-MS) through reference to appropriate genomic databases. Proteomic analysis is possible even if the genome sequences are not identical to those of the organisms present (24); however, missing sequence information reduces the resolution of such proteogenomic studies.Due to dominance by a few organism types, chemoautotrophic microbial AMD biofilms from Richmond Mine, Iron Mountain, CA, are tractable model systems used to develop cultivation-independent metagenomic and proteogenomic methods for analysis of community structure, function, and ecology (13). Acidophilic Leptospirillum bacteria dominate this AMD system (15), other AMD systems (54), and bioleaching systems used for recovery of metals (19, 53, 86). These bacteria play pivotal roles in sulfide mineral dissolution because they are iron oxidizers (53, 75), and ferric iron drives sulfide oxidation, leading to formation of metal-rich sulfuric acid solutions. According to a recent microscopy-based study (83), Leptospirillum group II are the first colonists in AMD biofilm communities whereas Leptospirillum group III generally appear later, sometimes partitioned within biofilm interiors. Because only Leptospirillum group III appear to be able to fix nitrogen, they may be keystone species in AMD ecosystems (75). This observation enabled the isolation of one representative, “Leptospirillum ferrodiazotrophum” (76). In prior work, we reported near-complete genome sequences of two Leptospirillum group II types (43, 65), but detailed functional annotations and metabolic analyses have not been published. Genomic data have been used to explore the metabolism of Leptospirillum bacteria in one biofilm community (56), but proteomic and genomic analyses of the same biofilm community have not been performed.Here, we report a near-complete genomic sequence for Leptospirillum group III, derived from a biofilm obtained from the UBA site within the Richmond Mine, Iron Mountain, CA; a detailed functional annotation of the genomes of Leptospirillum groups II and III; and a genomic and proteomic comparison of them. In addition, we report the sequence of an extrachromosomal plasmid associated with these organisms. This study represents the first comprehensive genomics-based analysis of the metabolism of bacteria in the Nitrospirae phylum and the first environmental community proteogenomic study where the genomic and proteomic data were derived from the same sample. We compared the proteomic profiles of three different biofilm communities to evaluate the importance of shared and unique genes and pathways in environmental adaptation.     

Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers

Researchers agree that climate change factors such as rising atmospheric [CO₂] and warming will likely interact to modify ecosystem properties and processes. However, the response of the microbial communities that regulate ecosystem processes is less predictable. We measured the direct and interactive effects of climatic change on soil fungal and bacterial communities (abundance and composition) in a multifactor climate change experiment that exposed a constructed old-field ecosystem to different atmospheric CO₂ concentration (ambient, +300 ppm), temperature (ambient, +3°C), and precipitation (wet and dry) might interact to alter soil bacterial and fungal abundance and community structure in an old-field ecosystem. We found that (i) fungal abundance increased in warmed treatments; (ii) bacterial abundance increased in warmed plots with elevated atmospheric [CO₂] but decreased in warmed plots under ambient atmospheric [CO₂]; (iii) the phylogenetic distribution of bacterial and fungal clones and their relative abundance varied among treatments, as indicated by changes in 16S rRNA and 28S rRNA genes; (iv) changes in precipitation altered the relative abundance of Proteobacteria and Acidobacteria, where Acidobacteria decreased with a concomitant increase in the Proteobacteria in wet relative to dry treatments; and (v) changes in precipitation altered fungal community composition, primarily through lineage specific changes within a recently discovered group known as soil clone group I. Taken together, our results indicate that climate change drivers and their interactions may cause changes in bacterial and fungal overall abundance; however, changes in precipitation tended to have a much greater effect on the community composition. These results illustrate the potential for complex community changes in terrestrial ecosystems under climate change scenarios that alter multiple factors simultaneously.Soil microbial communities are responsible for the cycling of carbon (C) and nutrients in ecosystems, and their activities are regulated by biotic and abiotic factors such as the quantity and quality of litter inputs, temperature, and moisture. Atmospheric and climatic changes will impact both abiotic and biotic drivers in ecosystems and the response of ecosystems to these changes. Feedbacks from ecosystem to the atmosphere may also be regulated by soil microbial communities (3). Although microbial communities regulate important ecosystem processes, it is often unclear how the abundance and composition of microbial communities correlate with climatic perturbations and interact to effect ecosystem processes. As such, much of the ecosystem climate change research conducted to date has focused on macroscale responses to climatic change such as changes in plant growth (43, 44), plant community composition (2, 37), and coarse scale soil processes (14, 18, 21, 26), many of which may also indirectly interact to effect microbial processes. Studies that have addressed the role of microbial communities and processes have most often targeted gross parameters, such as microbial biomass, enzymatic activity, or basic microbial community profiles in response to single climate change factors (22, 28, 29, 33, 61, 63).Climate change factors such as atmospheric CO₂ concentrations, warming, and altered precipitation regimes can potentially have both direct and indirect impacts on soil microbial communities. However, the direction and magnitude of these responses is uncertain. For example, the response of soil microbial communities to changes in atmospheric CO₂ concentrations can be positive or negative, and consistent overall trends between sites and studies have not been observed (1, 28, 34-36). Further, depending on what limits ecosystem productivity, precipitation and soil moisture changes may increase or decrease the ratio of bacteria and fungi, as well as shift their community composition (8, 50, 58). Increasing temperatures can increase in microbial activity, processing, and turnover, causing the microbial community to shift in favor of representatives adapted to higher temperatures and faster growth rates (7, 46, 60, 64, 65). Atmospheric and climatic changes are happening in concert with one another so that ecosystems are experiencing higher levels of atmospheric CO₂, warming, and changes in precipitation regimes simultaneously. Although the many single factor climate change studies described above have enabled a better understanding of how microbial communities may respond to any one factor, understanding how multiple climate change factors interact with each other to influence microbial community responses is poorly understood. For example, elevated atmospheric [CO₂] and precipitation changes might increase soil moisture in an ecosystem, but this increase may be counteracted by warming (10). Similarly, warming may increase microbial activity in an ecosystem, but this increase may be eliminated if changes in precipitation lead to a drier soil condition or reduced litter quantity, quality, and turnover. Such interactive effects of climate factors in a multifactorial context have been less commonly studied even in plant communities (45), and detailed studies are rarer still in soil microbial communities (25). Clearly, understanding how microbial communities will respond to these atmospheric and climate change drivers is important to make accurate predications of how ecosystems may respond to future climate scenarios.To address how multiple climate change drivers will interact to shape soil microbial communities, we took advantage of a multifactor climatic change experiment that manipulated atmospheric CO₂ (+300 ppm, ambient), warming (+3°C, ambient) and precipitation (wet and dry) in a constructed old-field ecosystem that had been ongoing for 3.5 years at the time of sampling. Previous work on this project has demonstrated direct and interactive effects of the treatments on plant community composition and biomass (15, 30), soil respiration (56), microbial activity (30), nitrogen fixation (21), and soil carbon stocks (20). These results led us to investigations of how the soil bacterial and fungal communities, important regulators of some of these processes, were responding using culture-independent molecular approaches. Our research addresses two overarching questions. (i) Do climatic change factors and their interactions alter bacterial and fungal abundance and diversity? (ii) Do climatic change factors and their interactions alter bacterial or fungal community composition?     

Influence of Species Specificity and Other Factors on Bacteria Associated with the Coral Stylophora pistillata in Taiwan

Species of bacteria associated with Stylophora pistillata were determined by analyses of 16S ribosomal genes. Coral samples were taken from two distinct sites at Kenting, in the far south of Taiwan; three coral colonies at each site were tagged and sampled in the winter and summer of 2007. Six hundred 16S rRNA gene clones were selected and sequenced for diversity analysis and community comparison. LIBSHUFF and nonparametric multiple dimensional scaling analyses showed variations in the composition of the coral-associated bacteria in the different samples, suggesting that seasonal and geographic factors and variations in individual coral colonies were all vital drivers of the structure of the S. pistillata-associated bacterial community. To examine the association between species specificity and environmental impacts on the structure of the coral-associated bacterial community, we conducted an integrated, comparative analysis of 44 coral-associated bacterial data sets, including the present study''s data. The clustering analysis suggests that the influence of spatial and temporal factors on the coral-associated bacteria population structure is considerable; nonetheless, the effect of species specificity is still detectable in some coral species, especially those from the Caribbean Sea.Microbes are abundant in the ocean and thrive around corals. In earlier investigations over the past decades, microbes have been detected in coral mucus (8), in coral tissues (5), and in the surrounding reef waters (25). Although we understand little about the real interactions between these coral-associated microbes and the coral itself, or their mutual roles, much indirect evidence suggests that these microbes may play an important role in the coral holobiont, with respect to coral nutrition, health, and disease (14, 18, 30).It is now known that most microbes are uncultivable by present laboratory methods (1, 28, 10). To understand more about coral-associated microbial communities, to identify the diversity of microbes associated with particular corals, and to assess whether these microbes are indeed species specific or represent only opportunistic interactions with the coral animal, some relatively comprehensive studies have been carried out in recent years based on culture-independent molecular techniques, e.g., construction of 16S rRNA gene clone libraries or denaturing gradient gel electrophoresis (3, 6, 9, 11, 13, 16, 17, 19, 20, 21, 23, 30, 31, 33). Consequently, coral-associated microbes are now known to be not only highly diverse and dynamic but also substantially coral specific.Recently, the specificity of association between coral and bacterial species has been a topic of much discussion. Earlier reports suggested that similar microbial communities were specifically associated with identical coral species, regardless of whether they were isolated from distinct geographic regions or at different times (3, 9, 20, 21); however, environmental factors have also recently been found to significantly influence the specificity of bacteria-coral associations (2, 17). Such inconsistency might reasonably be expected in light of the complexity of interaction in the coral holobiont, which includes coral, algae, fungi, bacteria, archaea, and other biotic and environmental factors (30). Nonetheless, more-detailed studies are needed for a better comprehension of species-specific bacterial associations.In this study, we selected Stylophora pistillata, a widely distributed coral in western Pacific reefs (26), to study the diversity and composition of the coral-associated bacteria and the effects of spatial and temporal differences on such population structures. We also examined the species specificity of such coral-bacteria associations by comparing our data with another 44 coral-associated bacterial data sets. This biodiversity analysis shows the presence of a large variation in the composition of S. pistillata-associated bacterial communities, suggesting that specificity between S. pistillata and associated bacteria is significantly influenced by geographic and seasonal factors. Furthermore, a comparison with 10 previous studies of coral-associated microbes showed that spatial and temporal factors play a role in affecting the population structure of coral-associated bacteria, though distinct species-specific bacterial profiles are detectable in some corals of the Caribbean Sea.     

Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases

The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S₅₀s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 μg/ml and 0.29 μg/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley α-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (∼0.05 μg in 50 mg of leaves) were far lower than the S₅₀ determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.Bacterial plant diseases are a source of great losses in the annual yields of most crops (5). The agrochemical methods and conventional breeding commonly used to control these bacterially induced diseases have many drawbacks. Indiscriminate use of agrochemicals has a negative impact on human, as well as animal, health and contributes to environmental pollution. Conventional plant-breeding strategies have limited scope due to the paucity of genes with these traits in the usable gene pools and their time-consuming nature. Consequently, genetic engineering and transformation technology offer better tools to test the efficacies of genes for crop improvement and to provide a better understanding of their mechanisms. One advance is the possibility of creating transgenic plants that overexpress recombinant DNA or novel genes with resistance to pathogens (36). In particular, strengthening the biological defenses of a crop by the production of antibacterial proteins with other origins (not from plants) offers a novel strategy to increase the resistance of crops to diseases (35, 39, 41). These antimicrobial peptides (AMPs) include such peptides as cecropins (2, 15, 20, 23-24, 27, 31, 42, 50), magainins (1, 9, 14, 29, 47), sarcotoxin IA (35, 40), and tachyplesin I (3). The genes encoding these small AMPs in plants have been used in practice to enhance their resistance to bacterial and fungal pathogens (8, 22, 40). The expression of AMPs in vivo (mostly cecropins and a synthetic analog of cecropin and magainin) with either specific or broad-spectrum disease resistance in tobacco (14, 24, 27), potato (17, 42), rice (46), banana (9), and hybrid poplar (32) have been reported. The transgenic plants showed considerably greater resistance to certain pathogens than the wild types (4, 13, 24, 27, 42, 46, 50). However, detailed studies of transgenic tomatoes expressing natural cecropin have not yet been reported.The tomato (Solanum lycopersicum) is one of the most commonly consumed vegetables worldwide. The annual yield of tomatoes, however, is severely affected by two common bacterial diseases, bacterial wilt and bacterial spot, which are caused by infection with the Gram-negative bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, respectively. Currently available pesticides are ineffective against R. solanacearum, and thus bacterial wilt is a serious problem.Cecropins, one of the natural lytic peptides found in the giant silk moth, Hyalophora cecropia (25), are synthesized in lipid bodies as proteins consisting of 31 to 39 amino acid residues. They adopt an α-helical structure on interaction with bacterial membranes, resulting in the formation of ion channels (12). At low concentrations (0.1 μM to 5 μM), cecropins exhibit lytic antibacterial activity against a number of Gram-negative and some Gram-positive bacteria, but not against eukaryotic cells (11, 26, 33), thus making them potentially powerful tools for engineering bacterial resistance in crops. Moreover, cecropin B (CB) shows the strongest activity against Gram-negative bacteria within the cecropin family and therefore has been considered an excellent candidate for transformation into plants to improve their resistance against bacterial diseases.The introduction of genes encoding cecropins and their analogs into tobacco has been reported to have contradictory results regarding resistance against pathogens (20). However, subsequent investigations of these tobacco plants showed that the expression of CB in the plants did not result in accumulation of detectable levels of CB, presumably due to degradation of the peptide by host peptidases (20, 34). Therefore, protection of CB from cellular degradation is considered to be vital for the exploitation of its antibacterial activity in transgenic plants. The secretory sequences of several genes are helpful, because they cooperate with the desired genes to enhance extracellular secretion (24, 40, 46). In the present study, a natural CB gene was successfully transferred into tomatoes. The transgenic plants showed significant resistance to the tomato diseases bacterial wilt and bacterial spot, as well as with a chemically synthesized CB peptide.     

In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.