Plant growth-promoting microbes improve stormwater retention of a newly-built vertical greenery system.

On-site decentralized urban stormwater management has gained significant momentum in urban planning. Recently, vegetated roofs have been recommended as a viable decentralized stormwater management system and nature-based solution to meet the challenge of urban floods. However, as another type of unconventional green infrastructure, vertical greenery systems (VGS), also known as vegetated facades, have received much less research attention. Even though some researchers suggest that stormwater management by VGS is comparable to that of vegetated roofs, empirical evidence to substantiate this claim is limited. In this study, we conducted rain simulations on newly-built vegetation containers with water storage compartments. These vegetation containers were designed to be incorporated into a VGS specifically for stormwater management. We tested variables that could influence water retention efficiency and evapotranspiration of the containers under field conditions, i.e., inoculation of plant growth-promoting microbes (PGPMs) (Rhizophagus irregularis and Bacillus amyloliquefaciens), different substrate types (sandy loam and reed-based substrate), simulated rain quantity, natural precipitation, substrate moisture, and air temperature. The inoculation of PGPMs significantly reduced runoff quantity from the vegetation containers. Meanwhile, the well-ventilated sandy-loam substrate significantly reduced the remaining water in the water storage compartments over 1-week periods between rain simulation events, achieving high water-use efficiency. The selected microbes were established successfully in the containers and promoted the growth of 2 out of 5 plant species. R. irregularis colonization responded to substrate type and host plant species, while B. amyloliquefaciens population density in the substrate did not respond to these factors. Environmental conditions, such as antecedent substrate moisture, air temperature, and natural precipitation also influenced the efficiency of stormwater retention and/or evapotranspiration. In conclusion, this study provides instructive and practical insights to reduce urban flood risk by using VGS.

Ericoid plant species and Pinus sylvestris shape fungal communities in their roots and surrounding soil.

Root-colonizing fungi can form mycorrhizal or endophytic associations with plant roots, the type of association depending on the host. We investigated the differences and similarities of the fungal communities of three boreal ericoid plants and one coniferous tree, and identified the community structure of fungi utilizing photosynthates from the plants studied. The fungal communities of roots and soils of Vaccinium myrtillus, Vaccinium vitis-idaea, Calluna vulgaris and Pinus sylvestris were studied in an 18-month-long experiment where the plants were grown individually in natural substrate. Photosynthates utilizing fungi were detected with DNA stable-isotope probing using 13 CO2 (13 C-DNA-SIP). The results indicated that the plants studied provide different ecological niches preferred by different fungal species. Those fungi which dominated the community in washed roots had also the highest 13 C-uptake. In addition, a common root endophyte without confirmed mycorrhizal status also obtained 13 C from all the plants, indicating close plant-association of this fungal species. We detect several fungal species inhabiting the roots of both ericoid mycorrhizal and ectomycorrhizal plants. Our results highlight that the ecological role of co-occurrence of fungi with different life styles (e.g. mycorrhizal or endophytic) in plant root systems should be further investigated.

Ericoid Roots and Mycospheres Govern Plant-Specific Bacterial Communities in Boreal Forest Humus.

In this study, the bacterial populations of roots and mycospheres of the boreal pine forest ericoid plants, heather (Calluna vulgaris), bilberry (Vaccinium myrtillus), and lingonberry (Vaccinium vitis-idaea), were studied by qPCR and next-generation sequencing (NGS). All bacterial communities of mycosphere soils differed from soils uncolonized by mycorrhizal mycelia. Colonization by mycorrhizal hyphae increased the total number of bacterial 16S ribosomal DNA (rDNA) gene copies in the humus but decreased the number of different bacterial operational taxonomic units (OTUs). Nevertheless, ericoid roots and mycospheres supported numerous OTUs not present in uncolonized humus. Bacterial communities in bilberry mycospheres were surprisingly similar to those in pine mycospheres but not to bacterial communities in heather and lingonberry mycospheres. In contrast, bacterial communities of ericoid roots were more similar to each other than to those of pine roots. In all sample types, the relative abundances of bacterial sequences belonging to Alphaproteobacteria and Acidobacteria were higher than the sequences belonging to other classes. Soil samples contained more Actinobacteria, Deltaproteobacteria, Opitutae, and Planctomycetia, whereas Armatimonadia, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia were more common to roots. All mycosphere soils and roots harbored bacteria unique to that particular habitat. Our study suggests that the habitation by ericoid plants increases the overall bacterial diversity of boreal forest soils.

Improving runoff quality in vertical greenery systems: Substrate type outweighed the effect of plant growth promoting microbes.

Due to limited urban green spaces and catchments, researchers are exploring the capacity of vertical greenery systems (VGSs) in stormwater management as complementary strategies. While the literature acknowledges the significant impacts of vegetated roof substrates on stormwater, comparing the stormwater management capacities of organic and non-organic substrates for VGSs remains largely unexplored. It is thus essential to gather empirical evidence to enhance the stormwater management capacity of VGSs. Here, we report on the impact of installation factors (substrate type and plant growth-promoting microbe (PGPM) inoculation) and environmental factors (simulated rainwater quantity and substrate moisture) of an innovative VGS on the concentrations and total loads of 15 elements (N, P, Al, V, Cr, Fe, Mn, Co, Ni, Cu, Zn, As, Se, Cd, and Pb) in the runoff. Results showed that substrate type was the most influential factor: concentrations and total loads were significantly higher from a reed-based substrate with high organic matter than from a sandy loam substrate. Substrate type also had profound interactive effects with other factors. For instance, PGPM inoculation significantly reduced the total loads of As, Cr, N, Ni, and Se, regardless of substrate type, and reduced the total loads of Cd, Co, Cu, Fe, Mn, and Pb in the reed-based substrate only. In addition, PGPM inoculation primarily reduced total loads, yet had little effect on concentrations. Substrate type also interacted with simulated rainwater quantity and substrate moisture: for example, in the reed-based substrate, a higher simulated rainwater quantity reduced concentrations but increased total loads, while concentrations and total loads remained constantly low from the sandy loam substrate under various simulated rainwater quantities. High antecedent substrate moisture increased both concentrations and total loads for most of the elements. We conclude that leaching from VGSs can be contained via substrate selection, maintenance of substrate moisture, and beneficial PGPM inoculation.

L-Amino acid oxidase of the fungus Hebeloma cylindrosporum displays substrate preference towards glutamate.

Catabolism of amino acids is a central process in cellular nitrogen turnover, but only a few of the mechanisms involved have been described from basidiomycete fungi. This study identified one such mechanism, the l-amino acid oxidase (Lao1) enzyme of Hebeloma cylindrosporum, by 2D gel separation and MS. We determined genomic DNA sequences of lao1 and part of its upstream gene, a putative pyruvate decarboxylase (pdc2), and cloned the cDNA of lao1. The two genes were also identified and annotated from the genome of Laccaria bicolor. The lao1 and pdc2 gene structures were conserved between the two fungi. The intergenic region of L. bicolor possessed putative duplications not detected in H. cylindrosporum. Lao1 sequences possessed dinucleotide-binding motifs typical for flavoproteins. Lao1 was less than 23 % identical to Lao sequences described previously. Recombinant Lao1 of H. cylindrosporum was expressed in Escherichia coli, purified and refolded with SDS to gain catalytic activity. The enzyme possessed broad substrate specificity: 37 l-amino acids or derivatives served as effective substrates. The highest activities were recorded with l-glutamate, but positively charged and aromatic amino acids were also accepted. Michaelis constants for six amino acids varied from 0.5 to 6.7 mM. We have thus characterized a novel type of Lao-enzyme and its gene from the basidiomycete fungus H. cylindrosporum.

Effect of weather conditions, substrate pH, biochar amendment and plant species on two plant growth-promoting microbes on vegetated roofs and facades.

Background: Vegetated building envelopes (VBEs), such as vegetated roofs and facades, are becoming more frequent in urban planning nowadays. However, harsh growing conditions restrain the application of VBEs. Plant growth-promoting microbes (PGPMs) might help ease the stresses, but first, it is necessary to investigate how to ensure their survival and growth under VBE conditions. Methods: We conducted three experiments to test the impact of various factors on the microbial populations of inoculated PGPMs in VBEs, a mycorrhizal fungus Rhizophagus irregularis and a bacterium Bacillus amyloliquefaciens. The first experiment was conducted by inoculating the two PGPMs separately in Sedum roof plots, and the microbial populations associated with Poa alpina was monitored for two consecutive years under local weather conditions. The second experiment was conducted in a laboratory testing the effect of substrate pH (substrates collected from balcony gardens) on R. irregularis population associated with Trifolium repens and Viola tricolor. The third experiment was conducted on a meadow roof testing the effect of biochar amendment on R. irregularis population associated with Thymus serpyllum and Fragaria vesca. Results: In the first experiment, Bacillus was found to associate with P. alpina, but Rhizophagus wasn't. Yet, the fungus induced high Bacillus population density in the Rhizophagus treated plots in the first year. In the second experiment, Rhizophagus abundance in T. repens was higher in the neutral substrate (6-6.5), while V. tricolor was more colonized in acidic substrate (5-5.5), suggesting an important interactive effect of substrate pH and plant species on Rhizophagus abundance. The third experiment suggested a negligible impact of biochar amendment on Rhizophagus abundance for both host plants. Conclusion: Three experiments demonstrate that PGPM inoculation on VBEs is feasible, and various factors and interactions affect the PGPM populations. This paper provides reference and inspiration for other VBE research involving substrate microbial manipulation.

Inputs of Terrestrial Dissolved Organic Matter Enhance Bacterial Production and Methylmercury Formation in Oxic Coastal Water.

Methylmercury (MeHg) is a potent neurotoxin commonly found in aquatic environments and primarily formed by microbial methylation of inorganic divalent mercury (Hg(II)) under anoxic conditions. Recent evidence, however, points to the production of MeHg also in oxic pelagic waters, but the magnitude and the drivers for this process remain unclear. Here, we performed a controlled experiment testing the hypothesis that inputs of terrestrial dissolved organic matter (tDOM) to coastal waters enhance MeHg formation via increased bacterial activity. Natural brackish seawater from a coastal area of the Baltic Sea was exposed to environmentally relevant levels of Hg(II) and additions of tDOM according to climate change scenarios. MeHg formation was observed to be coupled to elevated bacterial production rates, which, in turn, was linked to input levels of tDOM. The increased MeHg formation was, however, not coupled to any specific change in bacterial taxonomic composition nor to an increased abundance of known Hg(II) methylation genes. Instead, we found that the abundance of genes for the overall bacterial carbon metabolism was higher under increased tDOM additions. The findings of this study may have important ecological implications in a changing global climate by pointing to the risk of increased exposure of MeHg to pelagic biota.

The bacterial and fungal community composition in time and space in the nest mounds of the ant Formica exsecta (Hymenoptera: Formicidae).

In a subarctic climate, the seasonal shifts in temperature, precipitation, and plant cover drive the temporal changes in the microbial communities in the topsoil, forcing soil microbes to adapt or decline. Many organisms, such as mound-building ants, survive the cold winter owing to the favorable microclimate in their nest mounds. We have previously shown that the microbial communities in the nest of the ant Formica exsecta are significantly different from those in the surrounding bulk soil. In the current study, we identified taxa, which were consistently present in the nests over a study period of three years. Some taxa were also significantly enriched in the nest samples compared with spatially corresponding reference soils. We show that the bacterial communities in ant nests are temporally stable across years, whereas the fungal communities show greater variation. It seems that the activities of the ants contribute to unique biochemical processes in the secluded nest environment, and create opportunities for symbiotic interactions between the ants and the microbes. Over time, the microbial communities may come to diverge, due to drift and selection, especially given the long lifespan (up to 30 years) of the ant colonies.

Bacterial communities as indicators of environmental pollution by POPs in marine sediments.

Decades of intensive discharge from industrial activities into coastal systems has resulted in the accumulation of a variety of persistent organic pollutants (POPs) in marine waters and sediments, having detrimental impacts on aquatic ecosystems and the resident biota. POPs are among the most hazardous chemicals originating from industrial activities due to their biotoxicity and resistance to environmental degradation. Bacterial communities are known to break down many of these aromatic compounds, and different members of naturally occurring bacterial consortia have been described to work in syntrophic association to thrive in heavily contaminated waters and sediments, making them potential candidates as bioindicators of environmental pollution. In this study environmental, sampling was combined with chemical analysis of pollutants and high-resolution sequencing of bacterial communities using Next Generation Sequencing molecular biology tools. The aim of the present study was to describe the bacterial communities from marine sediments containing high loads of POPs and to identify relevant members of the resident microbial communities that may act as bioindicators of contamination. Marine sediments were collected from a coastal bay area of the Baltic Sea historically influenced by intense industrial activity, including metal smelting, oil processing, and pulp and paper production. Different types of POPs were detected at high concentrations. Fiberbank sediments, resulting from historic paper industry activity, were found to harbour a clearly distinct bacterial community including a number of bacterial taxa capable of cellulolytic and dechlorination activities. Our findings indicate that specific members of the bacterial communities thrive under increasing levels of POPs in marine sediments, and that the abundances of certain taxa correlate with specific POPs (or groups), which could potentially be employed in monitoring, status assessment and environmental management purposes.

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial.

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.

Effect of tree species and mycorrhizal colonization on the archaeal population of boreal forest rhizospheres.

Group 1.1c Crenarchaeota are the predominating archaeal group in acidic boreal forest soils. In this study, we show that the detection frequency of 1.1c crenarchaeotal 16S rRNA genes in the rhizospheres of the boreal forest trees increased following colonization by the ectomycorrhizal fungus Paxillus involutus. This effect was very clear in the fine roots of Pinus sylvestris, Picea abies, and Betula pendula, the most common forest trees in Finland. The nonmycorrhizal fine roots had a clearly different composition of archaeal 16S rRNA genes in comparison to the mycorrhizal fine roots. In the phylogenetic analysis, the 1.1c crenarchaeotal 16S rRNA gene sequences obtained from the fine roots formed a well-defined cluster separate from the mycorrhizal ones. Alnus glutinosa differed from the other trees by having high diversity and detection levels of Crenarchaeota both on fine roots and on mycorrhizas as well as by harboring a distinct archaeal flora. The similarity of the archaeal populations in rhizospheres of the different tree species was increased upon colonization by the ectomycorrhizal fungus. A minority of the sequences obtained from the mycorrhizas belonged to Euryarchaeota (order Halobacteriales).

Increasing oxygen deficiency changes rare and moderately abundant bacterial communities in coastal soft sediments.

Coastal hypoxia is a major environmental problem worldwide. Hypoxia-induced changes in sediment bacterial communities harm marine ecosystems and alter biogeochemical cycles. Nevertheless, the resistance of sediment bacterial communities to hypoxic stress is unknown. We investigated changes in bacterial communities during hypoxic-anoxic disturbance by artificially inducing oxygen deficiency to the seafloor for 0, 3, 7, and 48 days, with subsequent molecular biological analyses. We further investigated relationships between bacterial communities, benthic macrofauna and nutrient effluxes across the sediment-water-interface during hypoxic-anoxic stress, considering differentially abundant operational taxonomic units (OTUs). The composition of the moderately abundant OTUs changed significantly after seven days of oxygen deficiency, while the abundant and rare OTUs first changed after 48 days. High bacterial diversity maintained the resistance of the communities during oxygen deficiency until it dropped after 48 days, likely due to anoxia-induced loss of macrofaunal diversity and bioturbation. Nutrient fluxes, especially ammonium, correlated positively with the moderate and rare OTUs, including potential sulfate reducers. Correlations may reflect bacteria-mediated nutrient effluxes that accelerate eutrophication. The study suggests that even slightly higher bottom-water oxygen concentrations, which could sustain macrofaunal bioturbation, enable bacterial communities to resist large compositional changes and decrease the harmful consequences of hypoxia in marine ecosystems.

Corrigendum: Effects of Organic Pollutants on Bacterial Communities Under Future Climate Change Scenarios.

[This corrects the article DOI: 10.3389/fmicb.2018.02926.].

Trends in bacterial and fungal communities in ant nests observed with Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Next Generation Sequencing (NGS) techniques-validity and compatibility in ecological studies.

Microbes are ubiquitous and often occur in functionally and taxonomically complex communities. Unveiling these community dynamics is one of the main challenges of microbial research. Combining a robust, cost effective and widely used method such as Terminal Restriction Fragment Length Polymorphism (T-RFLP) with a Next Generation Sequencing (NGS) method (Illumina MiSeq), offers a solid alternative for comprehensive assessment of microbial communities. Here, these two methods were combined in a study of complex bacterial and fungal communities in the nest mounds of the ant Formica exsecta, with the aim to assess the degree to which these methods can be used to complement each other. The results show that these methodologies capture similar spatiotemporal variations, as well as corresponding functional and taxonomical detail, of the microbial communities in a challenging medium consisting of soil, decomposing plant litter and an insect inhabitant. Both methods are suitable for the analysis of complex environmental microbial communities, but when combined, they complement each other well and can provide even more robust results. T-RFLP can be trusted to show similar general community patterns as Illumina MiSeq and remains a good option if resources for NGS methods are lacking.

Species-specific synergistic effects of two plant growth-promoting microbes on green roof plant biomass and photosynthetic efficiency.

Rhizophagus irregularis, an arbuscular mycorrhizal fungus, and Bacillus amyloliquefaciens, a bacterium, are microorganisms that promote plant growth. They associate with plant roots and facilitate nutrient absorption by their hosts, increase resistance against pathogens and pests, and regulate plant growth through phytohormones. In this study, eight local plant species in Finland (Antennaria dioica, Campanula rotundifolia, Fragaria vesca, Geranium sanguineum, Lotus corniculatus, Thymus serpyllum, Trifolium repens, and Viola tricolor) were inoculated with R. irregularis and/or B. amyloliquefaciens in autoclaved substrates to evaluate the plant growth-promoting effects of different plant/microbe combinations under controlled conditions. The eight plant species were inoculated with R. irregularis, B. amyloliquefaciens, or both microbes or were not inoculated as a control. The impact of the microbes on the plants was evaluated by measuring dry shoot weight, colonization rate by the arbuscular mycorrhizal fungus, bacterial population density, and chlorophyll fluorescence using a plant phenotyping facility. Under dual inoculation conditions, B. amyloliquefaciens acted as a "mycorrhiza helper bacterium" to facilitate arbuscular mycorrhizal fungus colonization in all tested plants. In contrast, R. irregularis did not demonstrate reciprocal facilitation of the population density of B. amyloliquefaciens. Dual inoculation with B. amyloliquefaciens and R. irregularis resulted in the greatest increase in shoot weight and photosynthetic efficiency in T. repens and F. vesca.

Effects of Organic Pollutants on Bacterial Communities Under Future Climate Change Scenarios.

Coastal ecosystems are highly dynamic and can be strongly influenced by climate change, anthropogenic activities (e.g., pollution), and a combination of the two pressures. As a result of climate change, the northern hemisphere is predicted to undergo an increased precipitation regime, leading in turn to higher terrestrial runoff and increased river inflow. This increased runoff will transfer terrestrial dissolved organic matter (tDOM) and anthropogenic contaminants to coastal waters. Such changes can directly influence the resident biology, particularly at the base of the food web, and can influence the partitioning of contaminants and thus their potential impact on the food web. Bacteria have been shown to respond to high tDOM concentration and organic pollutants loads, and could represent the entry of some pollutants into coastal food webs. We carried out a mesocosm experiment to determine the effects of: (1) increased tDOM concentration, (2) organic pollutant exposure, and (3) the combined effect of these two factors, on pelagic bacterial communities. This study showed significant responses in bacterial community composition under the three environmental perturbations tested. The addition of tDOM increased bacterial activity and diversity, while the addition of organic pollutants led to an overall reduction of these parameters, particularly under concurrent elevated tDOM concentration. Furthermore, we identified 33 bacterial taxa contributing to the significant differences observed in community composition, as well as 35 bacterial taxa which responded differently to extended exposure to organic pollutants. These findings point to the potential impact of organic pollutants under future climate change conditions on the basal coastal ecosystem, as well as to the potential utility of natural bacterial communities as efficient indicators of environmental disturbance.

Heterogeneity of fungal and plant enzyme expression in intact Scots pine-Suillus bovinus and -Paxillus involutus mycorrhizospheres developed in natural forest humus.

Isozyme expression of nutrient-mobilizing and defence-related enzymes were examined in different functional components of intact Scots pine (Pinus sylvestris L.) ectomycorrhizal root systems developed in natural unsterilized forest humus. Scots pine seedlings colonized by Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) strains were grown on humus in two-dimensional Perspex⌖ microcosms to allow development of complete ectomycorrhizal root systems. Soluble proteins from uncolonized short roots, whole mycorrhizal root tips or dissected mantle and core fractions, fungal strands and the outermost soil colonizing fine hyphae were subjected to native polyacrylamide gel electrophoresis and stained to detect esterase, acid phosphatase, peroxidase and different polyphenoloxidase isozyme activities. Tissue-specific esterase isozymes were detected in all components and most, including unique Hartig net isozymes, were of fungal origin. High fungal acid phosphatase activities were detected in mycorrhiza and fine hyphae of S. bovinus, supporting earlier findings of active phosphatase activity at the fungal interface in the Hartig net region and in the fine hyphal margins of extramatrical mycelium actively colonizing the humus. All compartments in the P. involutus mycorrhizosphere had weaker acid-phosphatase activities. Peroxidases formed a large part of the soluble-protein content in non-mycorrhizal short roots. The amount of peroxidases, on a tissue f. wt basis, was similar in mycorrhizal and non-mycorrhizal roots, but differential isozyme expression was detected, indicating a change in root peroxidase activities following mycorrhiza formation. The expression of DL-3,4-dihydroxyphenylalanine metabolizing polyphenol oxidase enzymes was reduced in mycorrhizas compared with non-mycorrhizal short roots. In the extramatrical mycelial components, fine hyphae expressed the highest polyphenoloxidase activity. P. involutus displayed some polyphenol-oxidizing isoenzymes not detected in S. bovinus systems. Laccase activity was not detected in the plant and fungal components examined. It is concluded that enzyme activities and isozyme expression are differentially regulated in the different functional components of Scots pine mycorrhizospheres.

Suillus bovinus glutamine synthetase gene organization, transcription and enzyme activities in the Scots pine mycorrhizosphere developed on forest humus.

• Glutamine synthetase (GS) expression and activity is of central importance for cellular ammonium assimilation and recycling. Thus, a full characterization of this enzyme at the molecular level is of critical importance for a better understanding of nitrogen (N) assimilation in the mycorrhizal symbiosis. • Genomic and cDNA libraries of Suillus bovinus were constructed to isolate the GS gene, glnA, and corresponding cDNAs. The transcription initiation site was identified and transcription and enzyme activities were characterized in pure culture mycelium and mycorrhiza, and extramatrical mycelium samples harvested from Scots pine-Suillus bovinus microcosms grown on forest humus. • Pure culture mycelium, mycorrhiza and extramatrical mycelium all exhibited equivalent levels of GS transcription, translation and enzyme activities. However, levels of transcription and enzyme activity did not correlate as a large majority of detectable transcripts showed specific 5'-end truncation. • Our data suggest that GS is constitutively expressed and not directly affected by environmental conditions of the symbiotic N uptake. Any changes in the intracellular ammonium level are most likely handled by regulatory flexibility of GS at enzyme level.

Oxalate-metabolising genes of the white-rot fungus Dichomitus squalens are differentially induced on wood and at high proton concentration.

Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes.

Identification of nitrogen mineralization enzymes, L-amino acid oxidases, from the ectomycorrhizal fungi Hebeloma spp. and Laccaria bicolor.

Amino acids are major nitrogen sources in soils and they harbour a central position in the nitrogen metabolism of cells. We determined whether Hebeloma spp. and Laccaria bicolor expressed the enzyme L-amino acid oxidase (LAO), which catalyses the oxidative deamination of the alpha-amino group of L-amino acids. We measured LAO activities from the mycelial extracts of seven laboratory-grown fungal strains with three methods, and we measured how LAO activities were expressed in one Hebeloma sp. strain grown on four nitrogen sources. Hebeloma spp. and L. bicolor converted L-phenylalanine, but not D-phenylalanine, to hydrogen peroxide, 2-oxoacid, and ammonia, suggesting that they expressed LAO enzymes. The enzymes utilized five out of seven tested L-amino acids as substrates. LAO activities were maximal at pH 8, where Michaelis constant (Km) values were 2-5mm. The LAO of Hebeloma sp. was expressed on every nitrogen source analysed, and the activities were the highest in mycelia grown in nitrogen-rich conditions. We suggest that LAO is a mechanism for cellular amino acid catabolism in Hebeloma spp. and L. bicolor. Many soil bacteria and fungi also express LAO enzymes that have broad substrate specificities. Therefore, LAO is a potential candidate for a mechanism that catalyses nitrogen mineralization from amino acids at the ecosystem level.