Extracellular enzyme activity in the mycorrhizospheres of a boreal fire chronosequence

Saprotrophic microbes are typically credited with producing extracellular enzymes that recycle organic matter, though roots and mycorrhizal fungi also can contribute and may compete with the saprotrophs. We examined extracellular enzyme activity associated with the mycorrhizospheres of arbuscular mycorrhizal, ectomycorrhizal, dual-colonized (arbuscular and ectomycorrhizal), and ericoid mycorrhizal plants in a fire chronosequence in Alaska. Bulk soil and soil from beneath host plants were gathered in July 2004 and assayed for five enzymes that target organic C, P, and N substrates. Compared to bulk soil, activities of the C-targeting enzymes β-1,4-glucosidase and peroxidase were lower in arbuscular mycorrhizospheres and ericoid mycorrhizospheres, respectively. Moreover, extracellular enzyme activity varied among mycorrhizosphere types. Specifically, N-targeting leucine aminopeptidase was highest in arbuscular mycorrhizospheres, followed by ericoid and ectomycorrhizal/dual-colonized mycorrhizospheres; β-1,4-glucosidase had the reverse pattern. In addition, enzymatic stoichiometry suggested that extracellular enzyme producers invested more in C-acquisition than in N-acquisition in recent fire scars compared to mature forests. These data extend previous findings that roots and mycorrhizal fungi compete with saprotrophs by showing that the strength of this effect varies by mycorrhizal host. As a result the community composition of mycorrhizal host plants might mediate enzymatic activity in boreal soils. © 2011 Elsevier GmbH

In Situ Enzyme Activity in the Dissolved and Particulate Fraction of the Fluid from Four Pitcher Plant Species of the Genus Nepenthes

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×106 to 2.2×108 cells ml−1. We measured the activity of three common enzymes in the fluid: acid phosphatases, β-d-glucosidases, and β-d-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant

Soil organic matter and litter chemistry response to experimental N deposition in northern temperate deciduous forest ecosystems

The effects of atmospheric nitrogen (N) deposition on organic matter decomposition vary with the biochemical characteristics of plant litter. At the ecosystem-scale, net effects are difficult to predict because various soil organic matter (SOM) fractions may respond differentially. We investigated the relationship between SOM chemistry and microbial activity in three northern deciduous forest ecosystems that have been subjected to experimental N addition for 2 years. Extractable dissolved organic carbon (DOC), DOC aromaticity, C : N ratio, and functional group distribution, measured by Fourier transform infrared spectra (FTIR), were analyzed for litter and SOM. The largest biochemical changes were found in the sugar maple–basswood (SMBW) and black oak–white oak (BOWO) ecosystems. SMBW litter from the N addition treatment had less aromaticity, higher C : N ratios, and lower saturated carbon, lower carbonyl carbon, and higher carboxylates than controls; BOWO litter showed opposite trends, except for carbonyl and carboxylate contents. Litter from the sugar maple–red oak (SMRO) ecosystem had a lower C : N ratio, but no change in DOC aromaticity. For SOM, the C : N ratio increased with N addition in SMBW and SMRO ecosystems, but decreased in BOWO; N addition did not affect the aromaticity of DOC extracted from mineral soil. All ecosystems showed increases in extractable DOC from both litter and soil in response to N treatment. The biochemical changes are consistent with the divergent microbial responses observed in these systems. Extracellular oxidative enzyme activity has declined in the BOWO and SMRO ecosystems while activity in the SMBW ecosystem, particularly in the litter horizon, has increased. In all systems, enzyme activities associated with the hydrolysis and oxidation of polysaccharides have increased. At the ecosystem scale, the biochemical characteristics of the dominant litter appear to modulate the effects of N deposition on organic matter dynamics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72320/1/j.1365-2486.2005.01001.x.pd

The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse, Tracheoniscus rathkei

The digestive tract of the common woodlouse, Tracheoniscus rathkei Brandt (Isopoda: Oniscoidea), contains digestive enzymes active against α-1,4-glucans, which are the chief storage polysaccharides of vascular plants, algae, fungi, and animals, and β-1,3-glucans, which are present in algae and fungi. Digestive tract extracts also exhibit significant activity toward xylan and carboxymethyl-cellulose but negligible activity toward microcrystalline cellulose, substrates representative of the major structural polysaccharides of vascular plants. Low activity was detected toward pectin, and no activity was detected toward chitin. Activity toward xylan is due in part to microbial enzymes acquired from the leaf litter which was the isopod's normal food. Although ingested microbial xylanases are stable and active in the gut fluid, they do not make a quantitatively significant contribution to the isopod's ability to assimilate the hemicellulosic component of its diet. However, the assimilation of carbon from labeled plant fiber is enhanced in isopods which have acquired a cellulase by ingestion of leaf litter amended with a commercial preparation of the cellulase complex from the fungus, Penicillium funiculosum . This result demonstrates the potential contribution of acquired enzymes to the digestion of plant fiber in terrestrial detritivores. We urge caution, however, in assigning an important digestive function to ingested enzymes on the basis of evidence that only indicates that such enzymes are present in the gut fluid without additional evidence that their presence results in an enhancement of digestive efficiency.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47763/1/442_2004_Article_BF00377057.pd

Soil carbon loss by experimental warming in a tropical forest

Tropical soils contain one-third of the carbon stored in soils globally1, so destabilization of soil organic matter caused by the warming predicted for tropical regions this century2 could accelerate climate change by releasing additional carbon dioxide (CO2) to the atmosphere3,4,5,6. Theory predicts that warming should cause only modest carbon loss from tropical soils relative to those at higher latitudes5,7, but there have been no warming experiments in tropical forests to test this8. Here we show that in situ experimental warming of a lowland tropical forest soil on Barro Colorado Island, Panama, caused an unexpectedly large increase in soil CO2 emissions. Two years of warming of the whole soil profile by four degrees Celsius increased CO2 emissions by 55 per cent compared to soils at ambient temperature. The additional CO2 originated from heterotrophic rather than autotrophic sources, and equated to a loss of 8.2 ± 4.2 (one standard error) tonnes of carbon per hectare per year from the breakdown of soil organic matter. During this time, we detected no acclimation of respiration rates, no thermal compensation or change in the temperature sensitivity of enzyme activities, and no change in microbial carbon-use efficiency. These results demonstrate that soil carbon in tropical forests is highly sensitive to warming, creating a potentially substantial positive feedback to climate chang

Microbial Community Structure and Oxidative Enzyme Activity in Nitrogen-amended North Temperate Forest Soils

Large regions of temperate forest are subject to elevated atmospheric nitrogen (N) deposition which can affect soil organic matter dynamics by altering mass loss rates, soil respiration, and dissolved organic matter production. At present there is no general model that links these responses to changes in the organization and operation of microbial decomposer communities. Toward that end, we studied the response of litter and soil microbial communities to high levels of N amendment (30 and 80 kg ha −1 yr −1 ) in three types of northern temperate forest: sugar maple/basswood (SMBW), sugar maple/red oak (SMRO), and white oak/black oak (WOBO). We measured the activity of extracellular enzymes (EEA) involved directly in the oxidation of lignin and humus (phenol oxidase, peroxidase), and indirectly, through the production of hydrogen peroxide (glucose oxidase, glyoxal oxidase). Community composition was analyzed by extracting and quantifying phospholipid fatty acids (PLFA) from soils. Litter EEA responses at SMBW sites diverged from those at oak-bearing sites (SMRO, BOWO), but the changes were not statistically significant. For soil, EEA responses were consistent across forests types: phenol oxidase and peroxidase activities declined as a function of N dose (33–73% and 5–41%, respectively, depending on forest type); glucose oxidase and glyoxal oxidase activities increased (200–400% and 150–300%, respectively, depending on forest type). Principal component analysis (PCA) ordinated forest types and treatment responses along two axes; factor 1 (44% of variance) was associated with phenol oxidase and peroxidase activities, factor 2 (31%) with glucose oxidase. Microbial biomass did not respond to N treatment, but nine of the 23 PLFA that formed >1 mol% of total biomass showed statistically significant treatment responses. PCA ordinated forest types and treatment responses along three axes (36%, 26%, 12% of variance). EEA factors 1 and 2 correlated negatively with PLFA factor 1 ( r = −0.20 and −0.35, respectively, n = 108) and positively with PLFA factor 3 ( r = +0.36 and +0.20, respectively, n = 108). In general, EEA responses were more strongly tied to changes in bacterial PLFA than to changes in fungal PLFA. Collectively, our data suggests that N inhibition of oxidative activity involves more than the repression of ligninase expression by white-rot basidiomycetes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48115/1/248_2003_Article_9001.pd

Fungi Unearthed: Transcripts Encoding Lignocellulolytic and Chitinolytic Enzymes in Forest Soil

BACKGROUND: Fungi are the main organisms responsible for the degradation of biopolymers such as lignin, cellulose, hemicellulose, and chitin in forest ecosystems. Soil surveys largely target fungal diversity, paying less attention to fungal activity. METHODOLOGY/PRINCIPAL FINDINGS: Here we have focused on the organic horizon of a hardwood forest dominated by sugar maple that spreads widely across Eastern North America. The sampling site included three plots receiving normal atmospheric nitrogen deposition and three that received an extra 3 g nitrogen m(2) y(1) in form of sodium nitrate pellets since 1994, which led to increased accumulation of organic matter in the soil. Our aim was to assess, in samples taken from all six plots, transcript-level expression of fungal genes encoding lignocellulolytic and chitinolytic enzymes. For this we collected RNA from the forest soil, reverse-transcribed it, and amplified cDNAs of interest, using both published primer pairs as well as 23 newly developed ones. We thus detected transcript-level expression of 234 genes putatively encoding 26 different groups of fungal enzymes, notably major ligninolytic and diverse aromatic-oxidizing enzymes, various cellulose- and hemicellulose-degrading glycoside hydrolases and carbohydrate esterases, enzymes involved in chitin breakdown, N-acetylglucosamine metabolism, and cell wall degradation. Among the genes identified, 125 are homologous to known ascomycete genes and 105 to basidiomycete genes. Transcripts corresponding to all 26 enzyme groups were detected in both control and nitrogen-supplemented plots. CONCLUSIONS/SIGNIFICANCE: Many of these enzyme groups are known to be important in soil turnover processes, but the contribution of some is probably underestimated. Our data highlight the importance of ascomycetes, as well as basidiomycetes, in important biogeochemical cycles. In the nitrogen-supplemented plots, we have detected no transcript-level gap likely to explain the observed increased carbon storage, which is more likely due to community changes and perhaps transcriptional and/or post-transcriptional down-regulation of relevant genes

Comparative metagenomic, phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients

Terrestrial ecosystems are receiving elevated inputs of nitrogen (N) from anthropogenic sources and understanding how these increases in N availability affect soil microbial communities is critical for predicting the associated effects on belowground ecosystems. We used a suite of approaches to analyze the structure and functional characteristics of soil microbial communities from replicated plots in two long-term N fertilization experiments located in contrasting systems. Pyrosequencing-based analyses of 16S rRNA genes revealed no significant effects of N fertilization on bacterial diversity, but significant effects on community composition at both sites; copiotrophic taxa (including members of the Proteobacteria and Bacteroidetes phyla) typically increased in relative abundance in the high N plots, with oligotrophic taxa (mainly Acidobacteria) exhibiting the opposite pattern. Consistent with the phylogenetic shifts under N fertilization, shotgun metagenomic sequencing revealed increases in the relative abundances of genes associated with DNA/RNA replication, electron transport and protein metabolism, increases that could be resolved even with the shallow shotgun metagenomic sequencing conducted here (average of 75 000 reads per sample). We also observed shifts in the catabolic capabilities of the communities across the N gradients that were significantly correlated with the phylogenetic and metagenomic responses, indicating possible linkages between the structure and functioning of soil microbial communities. Overall, our results suggest that N fertilization may, directly or indirectly, induce a shift in the predominant microbial life-history strategies, favoring a more active, copiotrophic microbial community, a pattern that parallels the often observed replacement of K-selected with r-selected plant species with elevated N

Simulated Atmospheric N Deposition Alters Fungal Community Composition and Suppresses Ligninolytic Gene Expression in a Northern Hardwood Forest

High levels of atmospheric nitrogen (N) deposition may result in greater terrestrial carbon (C) storage. In a northern hardwood ecosystem, exposure to over a decade of simulated N deposition increased C storage in soil by slowing litter decay rates, rather than increasing detrital inputs. To understand the mechanisms underlying this response, we focused on the saprotrophic fungal community residing in the forest floor and employed molecular genetic approaches to determine if the slower decomposition rates resulted from down-regulation of the transcription of key lignocellulolytic genes, by a change in fungal community composition, or by a combination of the two mechanisms. Our results indicate that across four Acer-dominated forest stands spanning a 500-km transect, community-scale expression of the cellulolytic gene cbhI under elevated N deposition did not differ significantly from that under ambient levels of N deposition. In contrast, expression of the ligninolytic gene lcc was significantly down-regulated by a factor of 2–4 fold relative to its expression under ambient N deposition. Fungal community composition was examined at the most southerly of the four sites, in which consistently lower levels of cbhI and lcc gene expression were observed over a two-year period. We recovered 19 basidiomycete and 28 ascomycete rDNA 28S operational taxonomic units; Athelia, Sistotrema, Ceratobasidium and Ceratosebacina taxa dominated the basidiomycete assemblage, and Leotiomycetes dominated the ascomycetes. Simulated N deposition increased the proportion of basidiomycete sequences recovered from forest floor, whereas the proportion of ascomycetes in the community was significantly lower under elevated N deposition. Our results suggest that chronic atmospheric N deposition may lower decomposition rates through a combination of reduced expression of ligninolytic genes such as lcc, and compositional changes in the fungal community