OSIP1 is a self-assembling DUF3129 protein required to protect fungal cells from toxins and stressors.

Secreted proteins are key players in fungal physiology and cell protection against external stressing agents and antifungals. Oak stress-induced protein 1 (OSIP1) is a fungal-specific protein with unknown function. By using Podospora anserina and Phanerochaete chrysosporium as models, we combined both in vivo functional approaches and biophysical characterization of OSIP1 recombinant protein. The P. anserina OSIP1Δ mutant showed an increased sensitivity to the antifungal caspofungin compared to the wild type. This correlated with the production of a weakened extracellular exopolysaccharide/protein matrix (ECM). Since the recombinant OSIP1 from P. chrysosporium self-assembled as fibers and was capable of gelation, it is likely that OSIP1 is linked to ECM formation that acts as a physical barrier preventing drug toxicity. Moreover, compared to the wild type, the OSIP1Δ mutant was more sensitive to oak extractives including chaotropic phenols and benzenes. It exhibited a strongly modified secretome pattern and an increased production of proteins associated to the cell-wall integrity signalling pathway, when grown on oak sawdust. This demonstrates that OSIP1 has also an important role in fungal resistance to extractive-induced stress.

Visualization of Fungi During Wood Colonization and Decomposition by Microscopy: From Light to Electron Microscopy.

Fungi are the principal decomposers of wood together with xylophage insects and, as such, have a central role in nutrient cycling of forest ecosystems. These fungi are also envisaged as promising tools for converting wood and waste of wood industries into chemicals, as alternative to fossil chemicals. At the same time, wood decomposers pose a threat to wooden building materials and are intensively fought. As a consequence, intense researches have been conducted over the past 50 years to identify the fungi responsible for wood decomposition, the mechanisms by which they do so, the wood properties involved in resistance or sensitivity to attacks and ways to preserve woods. Many tools are now available to study fungal colonization of wood, including: "omics" techniques, enzymatic assays, spectrometry, etc. However, all these approaches provide bulk information and the data obtained by these methods contain no information on the localization of fungi, the stage of decomposition of the wood and the potential interactions between microorganisms. In these regards, microscopy approaches provide complementary information that can strengthen conclusions. The present chapter describes a diverse range of microscopy approaches, from simple bench light microscopy to confocal and electron microscopies, to shed light on the way fungi colonize wood tissues.

Microwave-Assisted Synthesis of Pd Nanoparticles into Wood Block (Pd@wood) as Efficient Catalyst for 4-Nitrophenol and Cr(VI) Reduction.

Palladium (Pd) nanoparticle catalysis has attracted increasing attention due to its efficient catalytic activity and its wide application in environmental protection and chemical synthesis. In this work, Pd nanoparticles (about 71 nm) were synthesized in aqueous solution by microwave-assisted thermal synthesis and immobilized in beech wood blocks as Pd@wood catalysts. The wood blocks were first hydrothermally treated with 10% NaOH solution to improve the internal structure and increase their porosity, thereby providing favorable attachment sites for the formed Pd nanoparticles. The stable deposition of Pd nanoparticle clusters on the internal channels of the wood blocks can be clearly observed. In addition, the catalytic performance of the prepared Pd@wood was investigated through two model reactions: the reduction of 4-nitrophenol and Cr(VI). The Pd@wood catalyst showed 95.4 g-1 s-1 M-1 of normalized rate constant knorm and 2.03 min-1 of the TOF, respectively. Furthermore, Pd nanoparticles are integrated into the internal structure of wood blocks by microwave-assisted thermal synthesis, which is an effective method for wood functionalization. It benefits metal nanoparticle catalysis in the synthesis of fine chemicals as well as in industrial wastewater treatment.

Comparative Copper Resistance Strategies of Rhodonia placenta and Phanerochaete chrysosporium in a Copper/Azole-Treated Wood Microcosm.

Copper-based formulations of wood preservatives are widely used in industry to protect wood materials from degradation caused by fungi. Wood treated with preservatives generate toxic waste that currently cannot be properly recycled. Despite copper being very efficient as an antifungal agent against most fungi, some species are able to cope with these high metal concentrations. This is the case for the brown-rot fungus Rhodonia placenta and the white-rot fungus Phanerochaete chrysosporium, which are able to grow efficiently in pine wood treated with Tanalith E3474. Here, we aimed to test the abilities of the two fungi to cope with copper in this toxic environment and to decontaminate Tanalith E-treated wood. A microcosm allowing the growth of the fungi on industrially treated pine wood was designed, and the distribution of copper between mycelium and wood was analysed within the embedded hyphae and wood particles using coupled X-ray fluorescence spectroscopy and Scanning Electron Microscopy (SEM)/Electron Dispersive Spectroscopy (EDS). The results demonstrate the copper biosorption capacities of P. chrysosporium and the production of copper-oxalate crystals by R. placenta. These data coupled to genomic analysis suggest the involvement of additional mechanisms for copper tolerance in these rot fungi that are likely related to copper transport (import, export, or vacuolar sequestration).

Salt accumulation and effects within foliage of Tilia × vulgaris trees from the street greenery of Riga, Latvia.

Green infrastructures within sprawling cities provide essential ecosystem services, increasingly undermined by environmental stress. The main objective in this study was to relate the allocation patterns of NaCl contaminants to injury within foliage of lime trees mechanistically and distinguish between the effects of salt and other environmental stressors. Using field material representative of salt contamination levels in the street greenery of Riga, Latvia, the contribution of salt contaminants to structural and ultrastructural injury was analyzed, combining different microscopy techniques. On severely salt-polluted and dystrophic soils, the foliage of street lime trees showed foliar concentrations of Na/Cl up to 13,600/16,750 mg kg-1 but a still balanced nutrient content. The salt contaminants were allocated to all leaf blade tissues and accumulated in priority within mesophyll vacuoles, changing the vacuolar ionic composition at the expense of especially K and Ca. The size of mesophyll cells and vacuoles was increased as a function of NaCl concentration, suggesting impeded transpiration stream. In parallel, the cytoplasm showed degenerative changes, suggesting indirect stress effects. Hence, the lime trees in Riga showed tolerance to the dystrophic environmental conditions enhanced by salt pollution but their leaf physiology appeared directly impacted by the accumulation of contaminants within foliage.

Do tree rings record changes in soil fertility? Results from a Quercus petraea fertilization trial.

Through the variations in their dimension, density, anatomy or isotopes composition, tree rings have provided invaluable proxies to evaluate past changes in the environment. Whereas long-term records of changes in soil fertility are particularly desired for forest ecosystem studies, the use of the chemical composition of tree rings as potential marker is still controversial. Dendrochemistry has sometimes been considered as a promising approach to study past changes in soil chemistry, whereas some authors stated that element translocations in the wood preclude any possibility of reliable retrospective monitoring. Here, we aimed at testing whether the wood elemental content of fertilized oaks (Quercus petraea) differed from control trees >30 years after a NPKCaMg fertilization and, if so, if the date of fertilization could be retrieved from the ring analysis. The contents in N, Mg, P, K, Ca and Mn were measured for each of the 43 sampled trees and in every ring of the 58-year long chronology with a non-destructive method coupling a Wavelength Dispersive Spectroscope (WDS) with a Scanning Electron Microscope (SEM). The results showed significantly higher contents in Ca and lower contents in Mn in fertilized compared to control trees. However, there was no difference in elemental content between the rings of the fertilized trees built in the 20 years before and those built after fertilization. Thus, whereas the effect of fertilization on increasing ring width was dramatic, immediate and relatively short-lasting, the elemental composition of the entire ring sequence was impacted, precluding the dating of the event. These results question the possibility to reconstruct long-term changes in soil fertility based on dendrochemistry.

Bacterial biofilm formation on the hyphae of ectomycorrhizal fungi: a widespread ability under controls?

Ectomycorrhizal (ECM) fungi establish symbiosis with roots of most trees of boreal and temperate ecosystems and are major drivers of nutrient fluxes between trees and the soil. ECM fungi constantly interact with bacteria all along their life cycle and the extended networks of hyphae provide a habitat for complex bacterial communities. Despite the important effects these bacteria can have on the growth and activities of ECM fungi, little is known about the mechanisms by which these microorganisms interact. Here we investigated the ability of bacteria to form biofilm on the hyphae of the ECM fungus Laccaria bicolor. We showed that the ability to form biofilms on the hyphae of the ECM fungus is widely shared among soil bacteria. Conversely, some fungi, belonging to the Ascomycete class, did not allow for the formation of bacterial biofilms on their surfaces. The formation of biofilms was also modulated by the presence of tree roots and ectomycorrhizae, suggesting that biofilm formation does not occur randomly in soil but that it is regulated by several biotic factors. In addition, our study demonstrated that the formation of bacterial biofilm on fungal hyphae relies on the production of networks of filaments made of extracellular DNA.

The coastal environment affects lead and sodium uptake by the moss Hypnum cupressiforme used as an air pollution biomonitor.

Several studies suggest that potential competition exists between marine cations and heavy metals for binding sites on the cell wall of mosses. This competition would impact the heavy metal concentration measured in mosses by biomonitoring programs, which may underestimate air pollution by heavy metals in a coastal environment. In the present study, we aim to identify possible mechanisms affecting lead uptake by mosses in a coastal environment, specifically, the competition between lead (Pb2+) and sodium (Na+) for binding sites in Hypnum cupressiforme (Hc). We also compared the response of continental and coastal Hc populations to Pb2+ exposure by immersing the moss samples in artificial solutions that comprised six experimental treatments and subsequently locating and quantifying Pb2+ and Na+ using the sequential elution technique and X-ray microanalyses with a scanning electron microscope. We demonstrated that high concentrations of Pb2+ prevented Na+ from binding to the cell wall. We also examined the effect of the salt acclimation of Hc on Pb2+ and Na+ accumulation. Coastal Hc populations accumulated more Na and less Pb than continental Hc populations in all treatments. Moreover, our results showed treatment effects on the intra/extracellular distribution of Na+, as well as site. This feedback on the influence of salt stress tolerance on Pb2+ uptake by mosses requires further study and can be investigated for other heavy metals, leading to a better use of mosses as biomonitoring tools.

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells. While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

Ecological succession leads to chemosynthesis in mats colonizing wood in sea water.

Chemosynthetic mats involved in cycling sulfur compounds are often found in hydrothermal vents, cold seeps and whale falls. However, there are only few records of wood fall mats, even though the presence of hydrogen sulfide at the wood surface should create a perfect niche for sulfide-oxidizing bacteria. Here we report the growth of microbial mats on wood incubated under conditions that simulate the Mediterranean deep-sea temperature and darkness. We used amplicon and metagenomic sequencing combined with fluorescence in situ hybridization to test whether a microbial succession occurs during mat formation and whether the wood fall mats present chemosynthetic features. We show that the wood surface was first colonized by sulfide-oxidizing bacteria belonging to the Arcobacter genus after only 30 days of immersion. Subsequently, the number of sulfate reducers increased and the dominant Arcobacter phylotype changed. The ecological succession was reflected by a change in the metabolic potential of the community from chemolithoheterotrophs to potential chemolithoautotrophs. Our work provides clear evidence for the chemosynthetic nature of wood fall ecosystems and demonstrates the utility to develop experimental incubation in the laboratory to study deep-sea chemosynthetic mats.

Developmental priming of stomatal sensitivity to abscisic acid by leaf microclimate.

Plant water loss and CO2 uptake are controlled by valve-like structures on the leaf surface known as stomata. Stomatal aperture is regulated by hormonal and environmental signals. We show here that stomatal sensitivity to the drought hormone abscisic acid (ABA) is acquired during leaf development by exposure to an increasingly dryer atmosphere in the rosette plant Arabidopsis. Young leaves, which develop in the center of the rosette, do not close in response to ABA. As the leaves increase in size, they are naturally exposed to increasingly dry air as a consequence of the spatial arrangement of the leaves, and this triggers the acquisition of ABA sensitivity. Interestingly, stomatal ABA sensitivity in young leaves is rapidly restored upon water stress. These findings shed new light on how plant architecture and stomatal physiology have coevolved to optimize carbon gain against water loss in stressing environments.

Liming in a beech forest results in more mineral elements stored in the mantle of Lactarius subdulcis ectomycorrhizas.

Liming is a forest practice used to counteract forest decline induced by soil acidification. It consists of direct Ca and Mg input in forest soil and restores tree mineral nutrition, but also causes drastic changes in nutrient availability in soil. Ectomycorrhizal (ECM) fungi significantly contribute in nutrient uptake by trees, and can recover them through organic acid secretion or through enzymatic degradation of organic matter. The symbiotic fungi use their extraradical mycelium for nutrient uptake, and then store them into the ECM mantle. In this study we measured how liming influences element contents in the mantle of Lactarius subdulcis ECMs, an abundant and particularly active in oxalate and laccase secretion in beech stands. For this purpose we used SEM observation coupled with energy- (EDX) and wavelength-dispersive-X-ray microanalyses (WDX). Results showed that ECM mantles of this species presented significantly higher Ca, Mg, Mn, K, Si, Al and Fe contents in limed plots. The nutrient amounts of L. subdulcis ECMs were significantly different between individuals for all the elements, showing a differential storage ability between individuals. The storage role of the ECM mantle can be interpreted in two different ways: i) a detoxification role for Al or heavy metals and ii) an increased potential nutrient resource by the fungus, which can benefit the tree.

Biomonitoring of traffic-related nitrogen oxides in the Maurienne valley (Savoie, France), using purple moor grass growth parameters and leaf (15)N/(14)N ratio.

Effects of traffic-related nitrogenous emissions on purple moor grass (Molinia caerulea (L.) Moench) transplants, used here as a new biomonitoring species, were assessed along 500 m long transects orthogonal to roads located in two open areas in the Maurienne valley (French Alps). Leaves were sampled during summer 2004 and 2005 for total N-content and (15)N-abundance determination while nitrogen oxides (NO and NO(2)) concentrations were determined using passive diffusion samplers. A significant and negative correlation was observed between plant total N-content, and (15)N-abundance and the logarithm of the distance to the road axis. The strongest decreases in plant N parameters were observed between 15 and 100 m from road axis. They were equivalent to background levels at a distance of about 800 m from the roads. In addition, motor vehicle pollution significantly affected vegetation at road edge, as was established from the relationship between leaf (15)N-abundance, total N-content and road traffic densities.