Effects of liming on ectomycorrhizal community structure in relation to soil horizon and tree hosts

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The forest tree decline in the 1980’s

At the beginning of the 1980’s, many forests of central europe were confronted with symptoms of needle yellowing and defoliation. Immediately attributed to acid rain and thus to industrial pollution, this forest decline was widely covered by the media, and has been a subject of sharp debate for the scientific community for a decade, until liming was proved to be an efficient way to restore tree health.
First symptoms appeared in czechoslovakian spruce forests damaged by acid rain because of the high quantities of SO2 produced by lignite combustion (Bonneau, 2007). These symptoms gradually reached Bavaria, Ardennes (Nys, 1989) and then Schwartzwald and the Vosges, and were attributed to SO2 production in these industrial territories. In 1989, 37% of the 190 000 ha of softwood were affected by this forest disease (Richter & Nageleisen, 2007). Nevertheless, monitoring of air pollution in the Vosges revealed that the quantities of SO2 were at least 2 times lower than the toxicity threshold (Bonneau, 2007). The cause of tree decline was thus not yet identified.
An alternative hypothesis was that defoliation was the consequence of 1976 drought. Indeed, spruce defoliation and needle yellowing were observed in dense tree stands growing on superficial substrates (i.e mountain soils). But the delay of several years between drought and tree phenology impeded prevented from establishing clear relationships between the two phenomenons (Lévy & Becker, 1987).
A third possible cause of this forest decline was identified at the same time. Foliar analyses of yellowing spruces in Schwartzwald revealed strong deficiencies in magnesium (Zöttl & Mies, 1983). Tree magnesium deficiency causes yellowing of the needle tip or yellowish spots between veins, with brutal transition to the green colour, followed by brownish colour or by desiccation of the needle (Bonneau, 1995). These foliar nutrient imbalances were also correlated with soil exchangeable calcium and magnesium. In the same way, deperishment was more frequent in nutrient-poor sandstone and granite bedrocks (Landmann & Nageleisen, 2001; Nechwatal & Oswald, 2003). Moreover, the natural rain acidity in the Vosges forests (pH between 4.3 and 5.4) leads to the drainage of divalent cations linked to soil colloids and their replacement by aluminium, certain forms of which are toxic for plants. Many of these deperishing forest stands had cation losses of 2% of Mg and 0.5% of Ca per year (Dupouey et al., 1998). Analyses of time series also demonstrated a progressive soil acidification (Göttlein, 1992; Hallbäcken and Tamm, 1986).
The cause of forest decline was thus attributable to a combination of these three factors. Actually, most of the Mg in young needles comes from surface humus mineralization. A drought stops this mineralization and the required Mg is transferred from 1-year-old needles to the younger ones. Moreover, in acidic soils, mineral layers have very low retention abilities of Mg derived from humus. Thus, the 1976 drought caused strong Mg deficiencies in the young needles and mineral nutrition was declined in the following years because of the progressive loss of Mg from the soil due to acid rain and soil acidification (Bonneau, 2007).

Forest liming as a method for restoring the health of declining forest stands

Calci-Magnesian amendments are used as a forest management practice to compensate for cation depletion by spreading Ca and Mg directly over forest soils. Calcium and magnesium can be applied as pure compounds or mixes between CaCO3, Ca(OH)2, CaO, CaSO4 or MgSO4, depending on the chemical properties of the forest soil (Bonneau, 1995). Lime can be applied by hand, by towed blowers or by helicopter (Bonneau, 1995) and has routinely been used in Germany in the mid 1980’s at the doses of 3 to 4 t/ha (Worken & Brumme, 1997). To date, such liming has been relatively rare in France and limited to forest experiments, especially in the Ardennes, Vosges (Renaud et al., 2000) and Normandie. In all cases, lime application restored tree mineral nutrition on a long term basis. The effects of calcareous amendments on forest soils are now well documented and can be classified into 6 categories: *impact on soil biology: Biological modifications are concentrated into the upper soil layers, where liming has more influence on soil acidity. Increased respiration is observed in humus. Earthworm abundance, bacterial biomass and microbial NH4 and NO2 oxidizers are stimulated by liming (Anderson, 1998), at the detriment of fungi (Kreutzer, 1995). Nevertheless, liming can cause an increase of root rot, decrease mycorrhizal vitality, and shifting the structure of root system, towards a slight reduction of fine root density in the topsoil layer and an increase of the root density in the 5-15cm. The root turnover is also reduced after liming. The nematode community tends to shift from a mycophagous to a bacteriophagous one (Arpin, 2007).
*impact on soil chemistry: Liming increases the pH of soil solutions in the upper layers and induces the deprotonation of -COOH groups of organic humic compounds (Kreutzer, 1995). It leads to higher cation exchange capacity and thus Ca and Mg retention in soils colloids. The replacement of the detrimental acidic cation Al3+ by Ca2+ and Mg2+ in the ion exchange complex and its elimination from the upper layers by leaching reduces exchangeable acidity, concentration of exchangeable Al3+ and thus Al toxicity (Ingerslev, 1997). Liming reduces the soils solution concentration of Mn, Cd and Zn due to their precipitation as oxides, and increased the total soil concentration of Fe and Cu, which form stable complexes with organic matter. The change in soil chemistry could be accompanied by nitrate leaching in populations which are not able of revitalization. The mineralization rates increase with biological activity (Huber et al., 2006).
*soil structure: Liming, by stimulating biological activity, and especially earthworms, which incorporate organic matter to a depth of 20cm (Judas et al., 1997), increases top soil aeration and thus improves soil structure and soil water retention capacity (Schack-Kirchner & Hildebrand, 1998).
*humus: calcareous amendments transform mor or moder into mull-type humus (Kreutzer, 1995; Renaud et al., 2000).
*tree nutrition: The nutritional status of limed trees, as shown by the concentrations of elements in leaves or needles, indicates improved Ca (Huber et al., 2006) or Ca-Mg nutrition (Belkacem et al., 1992; Renaud et al., 2000).
*vegetation: Liming reduces the germination rate of acidophilic species but stimulated that of widespread ones (Olsson & Kelner, 2002). The ground flora, including the moss community, shifts from acidophilic to more nitrophilic in limed areas (Hallbäcken & Zhang, 1998), and this effect is dose-dependant (Dulière et al., 2000). However, when liming is provided at lower doses, the acidophilic species are conserved.
Liming is now once again a topical subject for forest management as a tool to improve the production of wood biomass, because of its mild impact on the diversity of forest ecosystems, and its relatively low cost (around 9% of the cost of the total sylviculture costs: Bonneau, 1995).

The ectomycorrhizal fungi

The fine roots of most of the tree of economical interest in the temperate and boreal forests (pine, fir, oak, beech, spruce) are symbiotic with a large group of Asco- and Basidiomycete fungi. The fungal partner forms with the root a mixed organ called ectomycorrhiza (ECM), characterized by a mycelial mantle which covers the root tip, and more diffused emanating mycelium (Smith & Read, 1997). The symbiotic association allows to explore a higher soil volume via the extraradical mycelium, and transfers water and nutrients to the root (Garbaye and Guehl, 1997). The same tree can be associated with many ECM species, and all of those are more or less specialized and complementary for nutrient uptake and translocation. The functional diversity of the ECM community is thus a critical parameter for the ability of the tree population to forage for limiting soil resources and to adapt to environmental disturbances.
Ectomycorrhizal fungi assimilate C, N and P compounds, and nutrients available in soil as Ca, Mg, K and translocate them to the roots through the extensive vegetative mycelium (Smith & Read, 1997). ECMs are obviously involved in nutrient uptake by the tree because they cover the majority of fine roots where the tree absorbs nutrients, and thus all nutrients have to transit through fungal mantle, via active or passive mechanisms. These nutrients are sometimes sparingly available in temperate forest soils, and can be from organic (i.e included in organic matter) or inorganic (i.e contained in mineral particles) origin.

Organic nutrient uptake by ECMs

In forest soils, the most abundant source of nutrients is plant litter, composed of cellulose, hemicellulose and lignin, which encloses a high diversity of compounds containing a lot of elements required for plant nutrition. The degradation of these macromolecules is mostly achieved by fungi, through the secretion of powerful cell-wall bound hydrolytic enzymes (Kirk & Cullen, 1998; Cullen & Kersten, 2005), and, to a lesser extent, bacteria (Gramss et al., 1999). Saprotrophic fungi are not the sole organisms involved in nutrient cycling in forest soils: ECM fungi also play a major role and present high similarities with saprotrophs in their active foraging for nutrients (Leake & Boddy, 2002). Indeed, ECMs are capable of producing cell-wall bound enzymes to scavenge nutrients from organic matter (Dighton, 1983), even if in lower quantities than saprotrophic fungi. In comparison with leaf saprotrophic basidiomycetes, lower cellulase, cellobiohydrolase and similar phosphomonoesterase activities were produced by ECM fungi in pure cultures (Colpaert & Van Laere, 1996). In the same way, ECMs produced low but significant activities of phenol-oxidases compared with saprotrophic fungi (Bending & Read, 1997). Moreover, there is obvious evidence of polyphenol-oxidases (PPO) secretion by ECMs (Burke & Cairney, 2002). Moreover, comparing DNA sequences between ECMs and white-rot fungi revealed that the genes coding for ligninolytic enzymes (lignin and manganese peroxidases, laccase multicopper oxidases) were widespread in a large taxonomic range of ECM fungi (Chen et al., 2001 ; Luis et al., 2004). The ECM fungi are also involved in cuticle, plant cell wall, phenol and tannin degradation (Caldwell et al., 1991; Bending & Read, 1997; Günther et al., 1998). These results suggest that ECM fungi express many ‘decomposing abilities’ (Read & Perez-Moreno, 2003).
Among enzymes produced by ECM fungi, endoglucanases, cellobiohydrolases, and ß-glucosidases can convert cellulose into glucose. Endoglucanases hydrolyses internal bonds in cellulose chains. Cellobiohydrolase continues the hydrolysis progressively at both ends of existing chains and those created by endoglucanases, and forms cellobiose, a molecule composed of two glucoses linked in ß 1-4. ß-glucosidases cleaves the cellobiose into two glucoses (Kirk & Cullen, 1998). The degradation of hemicellulose is more complex and involves eight different enzyme families depending on the molecule forming the unit of the hemicellulose polymer. Concerning lignin degradation, the lignin-degrading systems needs powerful oxidative enzymes because of C-C links and ether bonds. The three main groups of lignin degrading enzymes are lignin peroxidases, manganese peroxidases and laccases (Kirk & Cullen, 1998). Genes coding for these three enzyme families and close to those of the white-rot fungi Phanerochaete chrysosporium (lignin peroxidase) and Pleurotus ostreatus (manganese peroxidase) are present in the genome of the ECM fungus Laccaria bicolor, suggesting the potential ability of this fungus to degrade lignin. Polyphenol oxidase activities have been detected in pure cultures of the ECM fungi Suillus bovinus, Pisolithus tinctorius, and Paxillus involutus (Burke & Cairney, 2002) and manganese peroxidase in Tylospora fibrillosa (Chambers et al., 1999).
The low availability of N is a characteristic of forest ecosystems dominated by ECM trees (Smith & Read, 1997). Moreover, the concentration of the soluble forms of N in soils (ammonium and nitrate) is very low; more than 95% of the soil N is in organic form in temperate forest ecosystems (Chalot & Brun, 1998). The ECM symbiosis is thus of critical importance for tree N nutrition because ECMs can have access to N from the two main organic sources abundant in forest humus: chitin and proteins. Protease activities have been reported for some ECM species: Suillus variegatus, S. bovinus, Piloderma croceum, Pisolithus tinctorius, Cenococcum geophilum, Amanita rubescens, Lactarius subdulcis, Hebeloma crustuliniforme, Paxillus involutus, and Thelephora terrestris (Smith & Read, 1997). Proteases includes a variety of enzymes: those secreted by the ECM fungus Cenococcum geophilum belong to the serine protease family. In the same way, chitnolytic enzymes render available organic N incorporated into fungal or insect chitin (chain of N-acetylglucosamine, containing one atom of N per monomer) (Lindahl & Finlay, 2006), and N-acetyl-glucosaminidase activities have already been reported for excised ECM root tips (Pritsch et al., 2004).
Enzyme assays using fluorescent or colorimetric substrates have been developed in order to measure enzyme activities of excised ECMs (Courty et al., 2005) or in situ in root systems (Dong et al., 2007).

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Inorganic nutrient uptake by ECMs (contribution to a multi-author review in preparation for Soil Biology and Biochemistry)

Besides nitrogen, a major tree-growth limiting element (Aerts, 2002), forest trees also need inorganic nutrients of mineral origin such as PO43-, Ca2+, Mg2+, Fe3+, K+. Ectomycorrhizal fungi contribute significantly to the uptake of these ions (Smith & Read, 1997), most of which derive from the dissolution of soil mineral particles: apatite contains phosphorus, feldspars, micas and hornblendes provide Ca, Mg and K, while Fe3+ is found in mica and in secondary minerals such as iron oxides (Landeweert et al., 2001). The ability of the ECM fungi to dissolve minerals was reported for identified and unidentified mycorrhizal isolates (Table 1; Paris et al., 1996; Mahmood et al., 2001). Pure cultures of some of these fungal species appeared capable of dissolving tri-calcic phosphorus or biotite. Evidence of the fungal impact on the dissolution of minerals was also described for seedlings infected by ECM fungi. The transformation of chlorite and mica into clay minerals, as well as the dissolution of apatite, was reported for seedlings infected with Piloderma croceum and Suillus variegatus, respectively (Arocena et al., 2004; Wallander, 2000a). In the same way, an experiment with tree seedlings showed that the ECM fungus Paxillus involutus significantly contributed to the dissolution of muscovite (source of K) and increased root potassium contents (van Schöll et al., 2006a). Interestingly, ectomycorrhizal fungi associated with seedlings have also been shown to recover phosphorus from apatite (Wallander et al., 2000a) and potassium from biotite and microcline (Wallander & Wickmann, 1999).
The relevance of these mechanisms at the ecosystem scale has been stressed by Blum et al. (2002) who showed that a large part of the calcium used by the trees was mobilized from apatite by ECM fungi. Another manifestation of fungal mineral dissolution could be the formation of smooth tunnels of constant diameter in feldspar and hornblende grains (Hoffland et al., 2002). These tunnels are often colonized by fungal hyphae. Because feldspar tunnel density is positively correlated with ECM density, Hoffland et al. (2003) emitted the hypothesis that ectomycorrhizal hyphae were involved in mineral dissolution. Nevertheless, the contribution of tunneling to weathering is probably low because it occurs mainly in very old soils and for a very low fraction of total soil minerals (Smits et al., 2005).
However, elements stored in mineral particles are not readily available for microorganisms. ECM fungi, as many bacteria and plants, need to release them through complexolysis and acidolysis. Complexolysis is a variant of hydrolysis where negatively charged molecules attach to mineral cations through electrostatic and covalent forces (Haas et al., 2006). This reaction leads to the solubilization of nutrients from the mineral surface. Acidolysis (i.e the destructuration of the crystallin structure of the mineral via the action of protons) also releases mineral nutrients in the soil solution. Organic acids of low molecular weight (LMWOAs) are considered as the main agents of mineral dissolution, because of their dual acidifying and complexing properties (Ochs et al., 1994; Barker et al., 1998). A significant part of the LMWOAs in soils are components of the root exudates sensu lato (Jones, 1998), suggesting an important role of the fungal symbionts in the case of ectomycorrhizal trees. Oxalate is one of the most widespread and abundant organic acids in forest soils (Jones et al., 2003). The secretion of oxalate has been reported for various ECM species (Table 1), such as Paxillus involutus, Piloderma spp., and Suillus spp., in contrast to Hebeloma cylindrosporum and Amanita muscaria which do not produce this acid (Casarin et al., 2003; Rosling et al., 2004). Because they do not produce oxalate crystals in presence of Ca, the ECM fungi Tylospora fibrillosa, Cenococcum geophilum and Thelephora terrestris are also suspected not to produce oxalate. However, there is a high diversity of ECM fungi for which oxalate production was observed in pure culture, in symbiotic association with seedlings or as hyphal mats or ectomycorrhizal root tips root tips in forest soils (Table 1).

Table of contents :

INTRODUCTION GENERALE 
Le dépérissement forestier des années 1980 14 Le chaulage : une méthode de restauration des forêts dépérissantes
La symbiose ectomycorrhizienne
L’assimilation des nutriments organiques par les ECMs 24 L’assimilation des nutriments minéraux par les ECMs
Objectifs de la thèse
GENERAL INTRODUCTION 
The forest tree decline of the 1980’s
Forest liming as a method for restoring the health of declining forest stands
The ectomycorrhizal fungi 50 Organic nutrient uptake by ECMs
Inorganic nutrient uptake by ECMs 58 Aims of the thesis
References
Forest liming durably impact the communities of ectomycorrhizal and fungal epigeous fruiting bodies
Abstract
Introduction
Material & Methods 96 Results
Discussion
References
Effects of liming on ectomycorrhizal community structure in relation to soil horizon and tree hosts
Abstract
Introduction
Material & Methods
Results
Discussion
References
CHAPTER II. CONSEQUENCES OF LIMING ON POTENTIAL ACTIVITIES OF ORGANIC MATTER DEGRADATION BY ECMS 
Abstract
Introduction
Material & Methods
Results
Discussion
References
CHAPTER III. CONSEQUENCES OF LIMING ON POTENTIAL ACTIVITIES OF MINERAL WEATHERING BY ECM COMMUNITIES 206
Simple microplate assays to measure iron mobilization and oxalate secretion by ectomycorrhizal tree roots
Abstract
Introduction
Microplate test n°1 : Determination of complexed iron mobilized by ECMs using the CAS
Microplate test n°2 : Determination of free iron trapped by ECMs using the Ferrospectral ® reagent
Microplate test n°3 : Determination of oxalate secretion by ECMs
Comparisons between LMWOAs and siderophores for iron chelation using the CAS assay References
Oxalate secretion and iron mobilization by beech ectomycorrhizas: influences of liming
Abstract
Introduction
Material & Methods
Results
Discussion
References
CHAPTER IV. CONSEQUENCES OF LIMING ON ELEMENTAL COMPOSITION OF ECTOMYCORRHIZAE 
Abstract
Introduction
Material & Methods
Results
Discussion
References
CHAPITRE V. SYNTHESE GENERALE ET PERSPECTIVES 
1. Conclusions générales à propos de l’étude effectuée à Humont
Résumé des résultats
Résultats et discussion
2. Quel est le degré de variablilté inter-sites des effets du chaulage sur les communautés d’ECMs ?
Caractérsiqtiques du site d’échantillonnage
Résultats et discussion
CHAPTER V. GENERAL SYNTHESIS AND PERPECTIVES 
1.General conclusions from the work in Humont
Review of the results 318 Results and discussion
2. What is the degree of site-specificity in the effect of liming on ECM communities?
Site and tree stand characteristics 334 Results and discussion
References 

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