César Nicolás

Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors

Summary Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose‐degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition ‘toolbox’ has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

Involutin Is an Fe3+ Reductant Secreted by the Ectomycorrhizal Fungus Paxillus involutus during Fenton-Based Decomposition of Organic Matter

ABSTRACT Ectomycorrhizal fungi play a key role in mobilizing nutrients embedded in recalcitrant organic matter complexes, thereby increasing nutrient accessibility to the host plant. Recent studies have shown that during the assimilation of nutrients, the ectomycorrhizal fungus Paxillus involutus decomposes organic matter using an oxidative mechanism involving Fenton chemistry (Fe2+ + H2O2 + H+ → Fe3+ + ˙OH + H2O), similar to that of brown rot wood-decaying fungi. In such fungi, secreted metabolites are one of the components that drive one-electron reductions of Fe3+ and O2, generating Fenton chemistry reagents. Here we investigated whether such a mechanism is also implemented by P. involutus during organic matter decomposition. Activity-guided purification was performed to isolate the Fe3+-reducing principle secreted by P. involutus during growth on a maize compost extract. The Fe3+-reducing activity correlated with the presence of one compound. Mass spectrometry and nuclear magnetic resonance (NMR) identified this compound as the diarylcyclopentenone involutin. A major part of the involutin produced by P. involutus during organic matter decomposition was secreted into the medium, and the metabolite was not detected when the fungus was grown on a mineral nutrient medium. We also demonstrated that in the presence of H2O2, involutin has the capacity to drive an in vitro Fenton reaction via Fe3+ reduction. Our results show that the mechanism for the reduction of Fe3+ and the generation of hydroxyl radicals via Fenton chemistry by ectomycorrhizal fungi during organic matter decomposition is similar to that employed by the evolutionarily related brown rot saprotrophs during wood decay.

Tracing Changes in the Microbial Community of a Hydrocarbon-Polluted Soil by Culture-Dependent Proteomics

Hydrocarbon contamination may affect the soil microbial community, in terms of both diversity and function. A laboratory experiment was set-up, with a semi-arid control soil and the same soil but artificially contaminated with diesel oil, to follow changes in the dominant species of the microbial community in the hydrocarbon-polluted soil via proteomics. Analysis of the proteins extracted from enriched cultures growing in Luria-Bertani (LB) media showed a change in the microbial community. The majority of the proteins were related to glycolysis pathways, structural or protein synthesis. The results showed a relative increase in the complexity of the soil microbial community with hydrocarbon contamination, especially after 15 days of incubation. Species such as Ralstonia solanacearum, Synechococcus elongatus and different Clostridium sp. were adapted to contamination, not appearing in the control soil, although Bacillus sp. dominated the growing in LB in any of the treatments. We conclude that the identification of microbial species in soil extracts by culture-dependent proteomics is able to partially explain the changes in the diversity of the soil microbial community in hydrocarbon polluted semi-arid soils, but this information is much more limited than that provided by molecular methods.

Chemical-Structural Changes of Organic Matter in a Semi-Arid Soil After Organic Amendment

A 9-month incubation experiment using composted and non-composted amendments derived from vine pruning waste and sewage sludge was carried out to study the effects of the nature and stability of organic amendments on the structural composition of organic matter (OM) in a semi-arid soil. The changes of soil OM, both in the whole soil and in the extractable carbon with pyrophosphate, were evaluated by pyrolysis-gas chromatography and chemical analyses. By the end of the experiment, the soils amended with pruning waste exhibited less organic carbon loss than those receiving sewage sludge. The non-composted residues increased the aliphatic-pyrolytic products of the OM, both in the whole soil and also in the pyrophosphate extract, with the products derived from peptides and proteins being significantly higher. After 9 months, in the soils amended with pruning waste the relative abundance of phenolic-pyrolytic products derived from phenolic compounds, lignin and proteins in the whole soil tended to increase more than those in the soils amended with sewage sludge. However, the extractable OM with pyrophosphate in the soils amended with composted residues tended to have higher contents of these phenolic-pyrolytic products than that in non-composted ones. Thus, despite the stability of pruning waste, the composting of this material promoted the incorporation of phenolic compounds to the soil OM. The pyrolytic indices (furfural/pyrrole and aliphatic/aromatic ratios) showed the diminution of aliphatic compounds and the increase of aromatic compounds, indicating the stabilization of the OM in the amended soils after 9 months. In conclusion, the changes of soil OM depend on the nature and stability of the organic amendments, with composted vine pruning waste favouring humification.

Type and quantity of organic amendments determine the amount of carbon stabilized in particle-size fractions of a semiarid degraded soil

ABSTRACT A 9-month lab experiment was carried out with three different amendments (vine pruning wastes, PW; composted vine pruning wastes, cPW; and sewage sludge, SS) added at three different rates (90, 180, and 240 t ha−1, dry weight) in order to test whether the type or the quantity of the amendments applied to a semiarid, degraded soil determined the Corg accumulation in its particle-size fractions (coarse sand, 200–2,000 µm; fine sand, 63–200 µm; silt, 2–63 µm; and clay, 0.1–2 µm). All amendments, independently of their C/N ratios, resulted in similar Corg content and accumulation in coarse sand and silt-sized fractions after 9 months. In the clay-sized fraction, enrichment in Corg produced the incorporation of particles from this particle-size fraction into the silt-sized fraction. Likewise, increasing the application rates of the amendments led to larger Corg contents into the particle-size fractions of all amended soils except for the clay-sized fraction. The application of SS resulted in lower basal respiration-to-Corg ratios in the clay-sized fraction than the application of PW and cPW, suggesting a higher protection of the Corg in the SS treatment. These results indicate that organic amendments from woody plants with C/N ratios higher than 30, such as PW, favor Corg accumulation in the fine sand-sized fractions. Furthermore, our findings suggest that the application rate of such amendments, rather than the C/N ratios and amendment origin (from sludge or woody plants), is the key factor for promoting Corg accumulation in the silt-sized fractions of semiarid degraded soils.

Genomics and Spectroscopy Provide Novel Insights into the Mechanisms of Litter Decomposition and Nitrogen Assimilation by Ectomycorrhizal Fungi

The majority of nitrogen in forest soils is found in organic form, primarily as proteins. This nitrogen is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. To capture the nitrogen, the fungi must at least partly disrupt the recalcitrant organic matter–protein complexes within which the nitrogen is embedded. In this review, we will describe how a combination of spectroscopic methods and transcriptome analyses has provided novel insights into the mechanisms by which the ectomycorrhizal fungus Paxillus involutus decomposes organic matter when acquiring nitrogen from plant litter. The observed chemical changes were consistent with a hydroxyl-radical attack, involving Fenton chemistry similar to that of saprophytic brown-rot fungi. Unlike the saprophytic fungi, P. involutus did not show any expression of genes encoding extracellular enzymes needed to metabolize the released carbon. We suggest that these activities have been lost in ectomycorrhizal fungi as an adaptation to symbiotic growth on host photosynthate. Indeed experiments have shown that the decomposition of plant litter and assimilation of nitrogen are triggered by the addition of glucose. In contrast, the addition of ammonium, the most abundant inorganic N form in forest soils, had relatively minor effects of the decomposition of litter material by P. involutus. The data suggest that the expression of the decomposition and nitrogen assimilation processes can be tightly regulated by the host carbon supply. Finally, the prospects of using novel spectroscopic methods and transcriptomic data to identify specific transcripts or chemical signatures that can be used as biomarkers for probing the activity of mycorrhizal fungi in the field are discussed.