Plant functional group diversity promotes soil protist diversity

We tested whether effects of plant diversity can propagate through food webs, down to heterotrophic protists not linked directly to plants. To this end we synthesised grassland ecosystems with varying numbers of plant functional groups (FGN) and assessed corresponding changes in testate amoebae communities. The number of plant species was kept constant. When FGN was increased from 1 to 3, species number and total community density of live testate amoebae were enhanced according to a linear and a saturating function, respectively. From FGN 1 to 2, the appearance of new testate amoebae species did not affect the presence of the resident species, whereas, from FGN 2 to 3 about one quarter of the resident testate amoebae species was replaced, without altering the total species number. Overall, density by species increased, while evenness of the testate amoebae community was not affected by FGN; although Trinema lineare, one of the most common species, became more abundant. The observed relationship between plant functional group diversity and testate amoebae diversity could shed new light on the biogeographical distribution patterns of protists.     

Woody species diversity influences productivity and soil nutrient availability in tropical plantations

We investigated the relationship between plant diversity and ecological function (production and nutrient cycling) in tropical tree plantations. Old plantations (65–72 years) of four different species, namely Araucaria cunninghamii, Agathis robusta, Toona ciliata and Flindersia brayleyana, as well as natural secondary forest were examined at Wongabel State Forest, in the wet tropics region of Queensland, Australia. Two young plantations (23 years) of Araucaria cunninghamii and Pinus caribaea were also examined. The close proximity of the older plantations and natural forests meant they had similar edaphic and climatic conditions. All plantations had been established as monocultures, but had been colonised by a range of native woody plants from the nearby rainforest. The extent to which this had occurred varied with the identity of the plantation species (from 2 to 17 species in 0.1 ha blocks). In many cases these additional species had grown up and joined the forest canopy. This study is one of the few to find a negative relationship between overstorey plant diversity and productivity. The conversion of natural forest with highly productive, low-diversity gymnosperm-dominated plantations (young and old Araucaria cunninghamii and Pinus caribaea) was found to be associated with lower soil nutrient availability (approximately five times less phosphorus and 2.5 times less nitrogen) and lower soil pH (mean = 6.28) compared to the other, less productive plantations. The dominant effects of two species, Araucaria cunninghamii and Hodgkinsonia frutescens, indicate that ecosystem functions such as production and nutrient availability are not determined solely by the number of species, but are more likely to be determined by the characteristics of the species present. This suggests that monoculture plantations can be used to successfully restore some functions (e.g. nutrient cycling and production), but that the level to which such functions can be restored will depend upon the species chosen and site conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Phosphorous availability influences the dissolution of apatite by soil fungi

Apatite (Ca₁₀(PO₄)₆(OH,F,Cl)₂) is the primary inorganic source of phosphorus in the biosphere. Soil fungi are known to increase plant‐available phosphorus by promoting dissolution of various phosphate minerals. Yet no apatite dissolution studies exist using fungi as weathering agents, and regulation of fungal weathering activity in response to different levels of phosphorus availability is largely unknown. Fungi were isolated from a grassland soil in northern California. Three pathways of tri‐calcium phosphate (Ca₃(PO₄)₂) (TCP) dissolution in liquid culture were identified among biogeochemically active fungi: (1) acidification (pH 3.3 ± 0.16), (2) moderate acidification (pH 4.9 ± 0.11) and (3) no acidification. Isolates representing pathway 1 and 2 were Zygomycetes in the order of Mucorales. All non‐acidifying isolates in pathway 3 were Ascomycetes and cleared the media by altering TCP into hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and sequestering it within mycelial spheres. One isolate representing each pathway was used in fluorapatite dissolution experiments either with the fungi present or under abiotic conditions using cell‐free liquid media conditioned by fungal growth at different phosphorus and calcium availabilities. Both Mucorales isolates acidify their substrate when growing in the presence of phosphorus. Mucorales exudates were mainly oxalic acid, and conditioned cell‐free media with phosphorus induced fluorapatite dissolution at a rate of 10^?0.9±0.14 and 10^?1.2±0.22 µmol P m^?2 s^?1. The ascomycete isolate on the other hand, induced fluorapatite dissolution at a rate of 10^?1.1±0.05 µmol P m^?2 s^?1 by lowering the pH of the media under phosphorus‐limited conditions, without producing significant amounts of low molecular weight organic acids (LMWOAs). Oxalate strongly etches fluorapatite along channels parallel to [001], forming needle‐like features, while exudates from the ascomycete‐induced surface rounding. We conclude that while LMWOAs are well‐studied weathering agents, these do not appear to be produced by fungi in response to phosphorus‐limiting growth conditions.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Oxidative consumption of nitric oxide by heterotrophic bacteria in soil

Abstract: Uptake rate constants for nitric oxide were measured in a neutral calcic cambisol (KBE) and an acidic luvisol (PBE). The NO uptake was higher under oxic than under anoxic incubation conditions by a factor of about three. Gassing the soils with air containing 10 ppmv NO resulted in the accumulation of nitrate which accounted for 57–94% of the NO consumed. Aerobic heterotrophic bacteria were isolated on glucose-yeast extract medium from soil dilutions corresponding to a most probable number of 10⁸–10⁹ bacteria per gram dry weight soil. One of the isolates (strain PS88, a Pseudomonas sp.) exhibited NO consumption activity that was much higher under oxic than anoxic incubation conditions. When sterile KBE amended with strain PS88 was gassed with air containing 10 ppmv NO, 88% of the consumed NO was recovered as nitrate and nitrite. A screening of various bacteria obtained from culture collections showed a widespread ability for consumption of low NO concentrations. Our results indicate that NO consumption in soil is not only possible by reductive denitrification, but also by oxidation due to aerobic heterotrophic bacteria such as strain PS88.     

Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria from soil

Two heterotrophic bacteria that oxidized thiosulfate to tetrathionate were isolated from soil. The enzyme system in one of the isolates (C-3) was constitutive, but in the other isolate (A-50) it was induced by thiosulfate or tetrathionate. The apparent K(m) for oxygen for thiosulfate oxidation by A-50 was about 223 mum, but, for lactate oxidation by A-50 or thiosulfate oxidation by C-3, the apparent K(m) for oxygen was below 2 mm. The oxidation of thiosulfate by A-50 was first order with respect to oxygen from 230 mum. The rate of oxidation was greatest at pH 6.3 to 6.8 and at about 10 mm thiosulfate, and it was strongly inhibited by several metal-binding reagents. Extracts of induced A-50 reduced ferricyanide, endogenous cytochrome c, and mammalian cytochrome c in the presence of thiosulfate. A-50, once induced to oxidize thiosulfate, also reduced tetrathionate to thiosulfate in the presence of an electron donor such as lactate. The optimal pH for this reaction was at 8.5 to 9.5, and the reaction was first order with respect to tetrathionate. There was no correlation between the formation of the thiosulfate-oxidizing enzyme of A-50 and the incorporation of thiosulfate-sulfur into cell sulfur. Thiosulfate did not affect the growth rate or yield of A-50.     

Phylogenetic diversity and activity of aerobic heterotrophic bacteria from a hypersaline oil-polluted microbial mat

The diversity and function of aerobic heterotrophic bacteria (AHB) in cyanobacterial mats have been largely overlooked. We used culture-dependent and molecular techniques to explore the species diversity, degradative capacities and functional guilds of AHB in the photic layer (2mm) of an oil-polluted microbial mat from Saudi Arabia. Enrichment isolation was carried out at different salinities (5% and 12%) and temperatures (28 and 45 degrees C) and on various substrates (acetate, glycolate, Spirulina extract and crude oils). Counts of most probable number showed a numerical abundance of AHB in the range of 1.15-8.13x10(6) cellsg(-1) and suggested the presence of halotolerant and thermotolerant populations. Most of the 16S rRNA sequences of the obtained clones and isolates were phylogenetically affiliated to the groups Gammaproteobacteria, Bacteriodetes and Alphaproteobacteria. Groups like Deltaproteobacteria, Verrucomicrobia, Planctomycetes, Spirochaetes, Acidobacteria and Deinococcus-Thermus were only detected by cloning. The strains isolated on acetate and glycolate belonged to the genera Marinobacter, Halomonas, Roseobacter and Rhodobacter whereas the strains enriched on crude oil belonged to Marinobacter and Alcanivorax. Members of the Bacteriodetes group were only enriched on Spirulina extract indicating their specialization in the degradation of cyanobacterial dead cells. The substrate spectra of representative strains showed the ability of all AHB to metabolize cyanobacterial photosynthetic and fermentation products. However, the unique in situ conditions of the mat apparently favored the enrichment of versatile strains that grew on both the cyanobacterial exudates and the hydrocarbons. We conclude that AHB in cyanobacterial mats represent a diverse community that plays an important role in carbon-cycling within microbial mats.     

High diversity in DNA of soil bacteria

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plant diversity effects on soil heterotrophic activity in experimental grassland ecosystems

The loss of plant species from terrestrial ecosystems may cause changes in soil decomposer communities and in decomposition of organic material with potential further consequences for other ecosystem processes. This was tested in experimental communities of 1, 2, 4, 8, 32 plant species and of 1, 2 or 3 functional groups (grasses, legumes and non-leguminous forbs). As plant species richness was reduced from the highest species richness to monocultures, mean aboveground plant biomass decreased by 150%, but microbial biomass (measured by substrate induced respiration) decreased by only 15% (P = 0.05). Irrespective of plant species richness, the absence of legumes (across diversity levels) caused microbial biomass to decrease by 15% (P = 0.02). No effect of plant species richness or composition was detected on the microbial metabolic quotient (qCO₂) and no plant species richness effect was found on feeding activity of the mesofauna (assessed with a bait-lamina-test). Decomposition of cellulose and birchwood sticks was also not affected by plant species richness, but when legumes were absent, cellulose samples were decomposed more slowly (16% in 1996, 27% in 1997, P = 0.006). A significant decrease in earthworm population density of 63% and in total earthworm biomass by 84% was the single most prominent response to the reduction of plant species richness, largely due to a 50% reduction in biomass of the dominant `anecic' earthworms. Voles (Arvicola terrestris L.) also had a clear preference for high-diversity plots. Soil moisture during the growing season was unaffected by plant species richness or the number of functional groups present. In contrast, soil temperature was 2 K higher in monocultures compared with the most diverse mixtures on a bright day at peak season. We conclude that the lower abundance and activity of decomposers with reduced plant species richness was related to altered substrate quantity, a signal which is not reflected in rates of decomposition of standard test material. The presence of nitrogen fixers seemed to be the most important component of the plant diversity manipulation for soil heterotrophs. The reduction in plant biomass due to the simulated loss of plant species had more pronounced effects on voles and earthworms than on microbes, suggesting that higher trophic levels are more strongly affected than lower trophic levels.     

Amplified functional DNA restriction analysis to determine catechol 2,3-dioxygenase gene diversity in soil bacteria

To determine phylogenetic diversity of a functional gene from strain collections or environmental DNA amplifications, new and fast methods are required. Catechol 2,3-dioxygenase (C23O) subfamily I.2.A genes, known to be of crucial importance for aromatic degradation, were used as a model to adapt the amplified ribosomal DNA restriction analysis to functional genes. Sequence data of C23O genes from 13 reference strains, representing the main branches of the C23O family I.2.A phylogeny, were used for simulation of theoretical restriction patterns. Among other restriction enzymes, Sau3A1 theoretically produce characteristic profiles from each subfamily I.2.A member and their similarities reassembled the main divergent branches of C23O gene phylogeny. This enzyme was used to perform an amplified functional DNA restriction analysis (AFDRA) on C23O genes of reference strains and 19 isolates. Cluster analyses of the restriction fragment profiles obtained from isolates showed patterns with distinct similarities to the reference strain profiles, allowing to distinguish four different groups. Sequences of PCR fragments from isolates were in close agreement with the phylogenetic correlations predicted with the AFDRA approach. AFDRA thus provided a quick assessment of C23O diversity in a strain collection and insights of its gene phylogeny affiliation among known family members. It cannot only be easily applied to a vast number of isolates but also to define the predominant polymorphism of a functional gene present in environmental DNA extracts. This approach may be useful to differentiate functional genes also for many other gene families.     

High diversity in DNA of soil bacteria.

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbiological characterization of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria

Seven soil samples and seven groundwater samples from a site contaminated with fuel-oil were investigated using several chemical and microbiological techniques. In soil samples, 500 to 7,500 mg/kg of total hydrocarbons were found. These samples contained no n-alkanes but iso- and branched chain alkanes. No polychlorinated biphenyls could be detected. Microbiological investigations included estimations of total cell counts, viable cell counts on different media, and numbers of methylotrophic, denitrifying, sulphate reducing, anaerobic (with the exception of methanogenic organisms), and hydrocarbon degrading bacteria. Viable and hydrocarbon degrading bacteria were found in all samples. A total of 1,366 pure cultures was characterized morphologically and physiologically and identified by numerical identification using a data base of more than 4,000 reference strains. Groundwater samples were dominated by gram-negative bacteria of the generaPseudomonas, Comamonas, Alcaligenes, andAcinetobacter, which were also found in soil samples. In addition, more grampositive bacteria belonging to the generaArthrobacter, Nocardia, andBacillus could be isolated from soil samples.     

Molecular and functional diversity in soil micro-organisms

Traditional approaches to the study of microbial diversity have relied on laboratory cultivation of isolates from natural environments and identification by classical techniques, including analysis of morphology, physiological characteristics and biochemical properties. These approaches provide information on fine-scale diversity but suffer from bias, resulting from the media and cultivation conditions employed, and from the inability to grow and isolate significant proportions of natural communities in the laboratory. An alternative approach is the amplification of ribosomal RNA and functional genes from nucleic acids extracted directly from environmental samples, with subsequent analysis by `fingerprinting' methods or by sequencing and phylogenetic analysis. This approach avoids the need for laboratory cultivation and has provided major insights into species and functional diversity of bacterial and archaeal populations. This article reviews molecular approaches to the characterisation of prokaryote diversity in natural environments, their more recent application to fungal diversity and the advantages and limitations of molecular analyses.     

An experimental test of the effect of plant functional group diversity on arthropod diversity

Characteristics used to categorize plant species into functional groups for their effects on ecosystem functioning may also be relevant to higher trophic levels. In addition, plant and consumer diversity should be positively related because more diverse plant communities offer a greater variety of resources for the consumers. Thus, the functional group composition and richness of a plant community may affect the composition and diversity of the herbivores and even higher trophic levels associated with that community. We tested this hypothesis by sampling arthropods with a vacuum sampler (34 531 individuals of 494 species) from an experiment in which we manipulated plant functional group richness and composition. Plant manipulations included all combinations of three functional groups (forbs, C₃ graminoids, and C₄ graminoids) removed zero, one, or two at a time from grassland plots at Cedar Creek Natural History Area, MN. Although total arthropod species richness was unrelated to plant functional group richness or composition, the species richness of some arthropod orders was affected by plant functional group composition. Two plant characteristics explained most of the effects of plant functional groups on arthropod species richness. Nutritional quality, a characteristic related to ecosystem functioning, and taxonomic diversity, a characteristic not used to designate plant functional groups, seemed to affect arthropod species richness both directly and indirectly. Thus, plant functional groups designated for their effects on ecosystem processes will only be partially relevant to consumer diversity and abundance.     

Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil

Enrichment and isolation of methyl chloride-utilizing bacteria from various terrestrial environments, including woodland and forest soils, resulted in the identification of seven methyl chloride-utilizing strains belonging to the genus Hyphomicrobium, an Aminobacter strain TW23 and strain WG1, which grouped closely with the genus Mesorhizobium. Methyl chloride enrichment cultures were dominated by Hyphomicrobium species, indicating that these bacteria were most suited to growth under the enrichment and isolation conditions used. However, the application of culture-independent techniques such as DNA-stable isotope probing and the use of a functional gene probe targeting cmuA, which encodes the methyltransferase catalysing the first step in bacterial methyl chloride metabolism, indicated a greater diversity of methyl chloride-utilizing bacteria in the terrestrial environment, compared with the diversity of soil isolates obtained via the enrichment and isolation procedure. It also revealed the presence of as yet uncultured and potentially novel methyl chloride-degrading bacteria in soil.