“In vitro” biofilm formation by Penicillum frequentans strains on sandstone,granite, and limestone

The appearance of crusts or patinas on surfaces of stone monuments are indicative signs of weathering. In many cases, microorganisms are mainly responsible for stone decay, giving rise to the formation of characteristic biodeteriorative patinas, called biofilms. In the present work, in vitro biofilm formation on sandstone, limestone, and granite block samples by Penicillium frequentans strains isolated from two Spanish cathedrals is demonstrated. Spore suspensions of P. frequentans strains were inoculated on each block sample of unaltered stone material cited above. Biofilms of 1–2 mm thickness were formed on each of the three rock samples, and analysed by means of Fourier-transform infrared spectroscopy, scanning electron microscopy and X-ray mapping (energy-dispersive X-ray spectrometry, KEVEX system). In the three cases, biofilms were principally composed of: fungal mycelium, mineral particles released from the stony substratum, and newly formed organic salts such as oxalate and citrate. These biofilms enhance and accelerate the deteriorative process of rocks due to the loss of stone material (biopitting and mineral grains captured by mycelium) and to alteration of the mineral crystalline networks (cation release by organic acids). Correspondence to: G. Gómez-Alarcón     

Biomass and organic acids in sandstone of a weathering building: Production by bacterial and fungal isolates

Ten fungal and nine bacterial strains were isolated from a weathering sandstone building. Their growth, organic acid production, and acidification capacity were assessed in culture under nutritional conditions similar to those in situ. Biomass (10–50 nmol phospholipid-PO₄g⁻¹) within the rock was small compared to soils. The isolated organisms were able to produce high amounts of those acids found in the sandstone, but acid production did not cause a drastic reduction in culture pH. It is suggested that the importance of acidification in microbial degradation of sandstone has been overestimated and that, under in situ pH and nutritional conditions, cation chelation by microbially produced organic acid anions may be more relevant to the weathering process.     

Manganese and iron oxidation by fungi isolated from building stone

Acid and nonacid generating fungal strains isolated from weathered sandstone, limestone, and granite of Spanish cathedrals were assayed for their ability to oxidize iron and manganese. In general, the concentration of the different cations present in the mineral salt media directly affected Mn(IV) oxide formation, although in some cases, the addition of glucose and nitrate to the culture media was necessary. Mn(II) oxidation in acidogenic strains was greater in a medium containing the highest concentrations of glucose, nitrate, and manganese. High concentrations of Fe(II), glucose, and mineral salts were optimal for iron oxidation. Mn(IV) precipitated as oxides or hydroxides adhered to the mycelium. Most of the Fe(III) remained in solution by chelation with organic acids excreted by acidogenic strains. Other metabolites acted as Fe(III) chelators in nonacidogenic strains, although Fe(III) deposits around the mycelium were also detected. Both iron and manganese oxidation were shown to involve extracellular, hydrosoluble enzymes, with maximum specific activities during exponential growth. Strains able to oxidize manganese were also able to oxidize iron. It is concluded that iron and manganese oxidation reported in this work were biologically induced by filamentous fungi mainly by direct (enzymatic) mechanisms.Correspondence to: G. Gomez-Alarcon.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

An RNA-Sequencing Study of the Genes and Metabolic Pathways Involved in Aspergillus niger Weathering of Potassium Feldspar

Microbial transformation of potassium feldspar to produce organic composite potassium fertilizer is recognized to be an important method of effective use of the huge reserves of low grade K⁺-bearing rock in China. The mechanism underlying microbial weathering of silicate minerals is still unclear, and this is an obstacle to practical methods of application. To thoroughly understand the molecular mechanism responsible for the weathering of potassium feldspar by Aspergillus niger at a molecular level, high-throughput RNA-sequencing (RNA-seq) and treatment with different potassium sources (cultured in Czapek medium with soluble K⁺ or potassium feldspar) were used to investigate the differentially expressed genes of A. niger associated with potassium feldspar weathering and the related metabolic pathways. A series of differentially expressed genes related to the synthesis and transportation of organic acids, polysaccharides, and proteins (enzymes) were found to be closely associated with the K⁺ released from minerals through bioinformatic analysis. In addition, 12 genes that showed apparent expression differences by RNA-seq analysis and are relevant to organic acid synthesis, protein modification, maintenance of cellular homeostasis, and material transportation, were selected to be further verified using RT-qPCR. Compared to the fungal samples cultured with soluble K⁺, those with potassium feldspar have certain genes that are more up-regulated, such as the genes for Na⁺,K⁺-ATPase (447.6 multiples), cystathionine beta-synthase (5.6 multiples), cysteine synthase (9 multiples), and glutathione synthase (3.5 multiples). The analysis indicates that A. niger weathering of potassium feldspar is due to the synergistic effect of many factors including the up-regulation of certain genes and activation of related metabolite pathways. The research improves our understanding of the mechanisms of microbial weathering of silicate minerals.     

Penicillium frequentans population dynamics on peach fruits after its applications against brown rot in orchards

Aims: The study provides useful information on the temporal population dynamics of the biological control agent, Penicillium frequentans, after its applications against brown rot in orchards. Methods and Results: Population dynamics of P. frequentans were studied on peach flower and fruit surfaces after different field treatments. Eight experiments were carried out in commercial peach orchards in Spain, over four growing seasons from 2002 to 2005. Six different formulated P. frequentans conidia were applied four to six times from blossom to harvest and P. frequentans population sizes were monitored using conidial numbers and colony forming units (CFU) per flower or fruit. A consistent population of P. frequentans, ranging from 10⁵ to 10⁶ number of conidia or 10³ to 10⁴ CFU of P. frequentans per flower or fruit, was obtained. Colonization of peach surfaces by all P. frequentans formulation are similar and it appears to follow a general pattern: (i) higher colonization of fruits at preharvest than on the flowers at bloom; (ii) high populations just after treatments, especially after preharvest treatments; and (iii) a slight decline between treatments, especially in cool and moist springs. The exponential model was the most appropriate for fitting and comparing the P. frequentans dynamic populations on peaches and nectarines over time. The linearization of the P. frequentans population curve was essential to determine dynamic population and for population levels forecast. A positive relationship between number of blossom and preharvest applications, temperature, relative humidity and dynamic of P. frequentans population applied on peaches was studied using a multiple regression model. Conclusions: Blossom and preharvest applications were the most important spray times for obtaining the highest population of P. frequentans on peach surfaces. Significance and Impact of the Study: The study provides useful information on dynamic P. frequentans population and its effects on the brown rot biocontrol.     

Fungal degradation of calcium-, lead- and silicon-bearing minerals

The aim of this study was to examine nutritional influence on the ability of selected filamentous fungi to mediate biogenic weathering of the minerals, apatite, galena and obsidian in order to provide further understanding of the roles of fungi as biogeochemical agents, particularly in relation to the cycling of metals and associated elements found in minerals. The impact of three organic acid producing fungi (Aspergillus niger, Serpula himantioides and Trametes versicolor) on apatite, galena and obsidian was examined in the absence and presence of a carbon and energy source (glucose). Manifestation of fungal weathering included corrosion of mineral surfaces, modification of the mineral substrate through transformation into secondary minerals (i.e. crystal formation) and hyphal penetration of the mineral substrate. Physicochemical interactions of fungal metabolites, e.g. H⁺ and organic acids, with the minerals are thought to be the primary driving forces responsible. All experimental fungi were capable of mineral surface colonization in the absence and presence of glucose but corrosion of the mineral surface and secondary mineral formation were affected by glucose availability. Only S. himantioides and T. versicolor were able to corrode apatite in the absence of glucose but none of the fungi were capable of doing so with the other minerals. In addition, crystal formation with galena was entirely dependent on the availability of glucose. Penetration of the mineral substrates by fungal hyphae occurred but this did not follow any particular pattern. Although the presence of glucose in the media appeared to influence positively the mineral penetrating abilities of the fungi, the results obtained also showed that some geochemical change(s) might occur under nutrient-limited conditions. It was, however, unclear whether the hyphae actively penetrated the minerals or were growing into pre-existing pores or cracks.     

Bewuchsuntersuchungen auf Natursteinsubstraten im Gezeitenbereich des Nordsylter Wattenmeeres: Sessile und hemisessile Tiere

Six different types of test substrates, arranged in order of texture from smooth (Solnhofen limestone, Bunter sandstone, and basalt) to rough surfaces (Middle Triassic limestone, granite, and basaltic lava), were exposed in tidal zone of the wadden sea near the harbour of List (Island of Sylt, North Sea). The test substrates were fixed to panels at the midtide to high-water level, the midtide to low-water level, and 75 cm below the latter (sublittoral level). Animal settlement was primarily influenced by abiotic factors at the two higher levels. In the sublittoral zone, however, influences of biotic factors (competitors and predators) predominated. Therefore, the physical quality of the chosen substrates more decisively affected the growth of settling animals above the midtide to low-water mark than in the deeper zone. At the midtide to high-water levelBalanus balanoides only settled on the rough surface of the Middle Triassic limestone and in the troughs of the Bunter sandstone; barnacles attached to the even surface of the latter, were destroyed by wave beating. They died on the rough surface of the dark-coloured granite, when this stone was warmed during low-water. At the midtide to low-water level, the barnacles survived best on the rough surface of the Middle Triassic limestone and the granite. Only the larvae ofB. crenatus andB. improvisus preferred to settle on the even surface of the Bunter sandstone and the basalt; but there they died from wave beating or desiccation. The surface of the basaltic lava, formed by sharp-edged pores, was unsuitable for settling of barnacles. Colonies of Hydrozoa covered each substrate with a somewhat rough surface structure, even the basaltic lava. At the sublittoral level, differences in settling between the single substrates disappeared more and more. Nevertheless, the three species of barnacles showed the same preferences in settling, as they did at the higher levels. In August,Asterias rubens destroyed all barnacles and thus restricted the lower limit of barnacle settlement at the low-water mark. The distribution of epibiotic organisms is dependent upon the density of their living substrates, directly attached to the stone surfaces. During the short time of their growth, hemisessile young,Mytilus edulis settled on thready forms like algae, or on raised areas like the top of barnacle shells. Therefore, young mussels could be found on stones, that already carried a compact cover ofEnteromorpha sp. or a dense settlement of adult barnacles. The polychaetePolydora ciliata rarely burrowed directly into stony substrates (Middle Triassic limestone, Solnhofen limestone). It settled primarily between barnacle shells where it was sheltered from wave beating and at the same time profited from the current produced by the filter-feeding organs of the barnacles. The density of this polychaete was directly proportional to the density of the barnacles. AfterA. rubens had destroyed the barnacles in the sublittoral zone,P. ciliata disappeared too. At the low-water mark, however, theP. ciliata population between living and active barnacles increased. Hence, the lower limit ofP. ciliata — as those of barnacles and mussels — was fixed by this predator.

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Herrn Professor W. E. Ankel zur Vollendung des 80. Lebensjahres in Dankbarkeit gewidmet.     

In vitro weathering of phlogopite by ectomycorrhizal fungi

The ways in which ectomycorrhizal fungi benefit tree growth and nutrition have not been fully elucidated. Whilst it is most probably due to improved soil colonization, it is also likely that ectomycorrhizal fungi could be directly involved in nutrient cycling of soil reserves. This study assessed access by two species of ectomycorrhizal fungi to soil nonexchangeable K⁺ reserves. The incubation of ectomycorrhizal fungi in bi-compartment Petri dishes with phlogopite led to cation exchange reactions and to crystal lattice weathering. Paxillus involutus COU led to irreversible phlogopite transformations, while Pisolithus tinctorius 441 led to reversible ones. Simultaneous depletion in K⁺ and Mg²⁺ led to an enhanced weathering of phlogopite by P. tinctorius 441. The observation of phlogopite evolution shows that some specific Al³⁺ immobilization occurred under P. tinctorius 441. The data suggest that these bio-weathering mechanisms could be related to the release of fungal organic acids or other complex forming molecules.     

Plant-driven weathering of apatite - the role of an ectomycorrhizal fungus

Ectomycorrhizal (EcM) fungi are increasingly recognized as important agents of mineral weathering and soil development, with far‐reaching impacts on biogeochemical cycles. Because EcM fungi live in a symbiotic relationship with trees and in close contact with bacteria and archaea, it is difficult to distinguish between the weathering effects of the fungus, host tree and other micro‐organisms. Here, we quantified mineral weathering by the fungus Paxillus involutus, growing in symbiosis with Pinus sylvestris under sterile conditions. The mycorrhizal trees were grown in specially designed sterile microcosms in which the supply of soluble phosphorus (P) in the bulk media was varied and grains of the calcium phosphate mineral apatite mixed with quartz, or quartz alone, were provided in plastic wells that were only accessed by their fungal partner. Under P limitation, pulse labelling of plants with ¹⁴CO₂ revealed plant‐to‐fungus allocation of photosynthates, with 17 times more ¹⁴C transferred into the apatite wells compared with wells with only quartz. Fungal colonization increased the release of P from apatite by almost a factor of three, from 7.5 (±1.1) × 10^?10 mol m^?2 s^?1 to 2.2 (±0.52) × 10^?9 mol m^?2 s^?1. On increasing the P supply in the microcosms from no added P, through apatite alone, to both apatite and orthophosphate, the proportion of biomass in roots progressively increased at the expense of the fungus. These three observations, (i) proportionately more plant energy investment in the fungal partner under P limitation, (ii) preferential fungal transport of photosynthate‐derived carbon towards patches of apatite grains and (iii) fungal enhancement of weathering rate, reveal the tightly coupled plant–fungal interactions underpinning enhanced EcM weathering of apatite and its utilization as P source.     

A comparison between Ca and Sr cycling in forest ecosystems

In favourable conditions, the ⁸⁷Sr/⁸⁶Sr isotope ratios of the Sr delivered by rain and soil mineral weathering differ. Assuming that Ca and Sr behave similarly in forest ecosystems, several authors have used the ⁸⁷Sr/⁸⁶Sr variation in forest compartments to calculate the contribution of rain and mineral weathering to Ca fluxes and pools. However, there are a number of experimental reports showing that Ca and Sr may behave differently in the soil and in the plant. We have tested this Ca–Sr analogy in the field by measuring the variation of Sr and Ca concentrations, fluxes and pools in spruce, beech and maple stands on granite, sandstone and limestone. Results show that (1) variations of Ca and Sr concentrations are generally correlated at each level of the ecosystems. (2) In spruce on acid soils, a preferential uptake of Ca over Sr occurs (Aubure spruce Sr/Ca = 0.8×10⁻³; soil exchangeable Sr/Ca between 2 and 6×10⁻³). On calcareous soils, a preferential uptake of Sr over Ca by spruce may occur. (3) In spruce and beech on acid and calcareous soils, a preferential translocation of Ca over Sr from roots to leaves occurs ((Sr/Ca) in leaves was between 10 and 90% of that in roots). (4) The biological cycling of Ca and Sr leads to an enrichment of the upper soil layers in Ca and Sr. Compared to Sr, Ca accumulates in the upper layer of acid soils because Ca cycling through litterfall is favoured over Sr cycling, and possibly because of the selectivity of acid organic exchangers for Ca. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthesis‐based microbial communities on two churches in northern Germany: Weathering of granite and glazed brick

Microbial communities on two churches in Schleswig‐Holstein (northern Germany) were characterized using cultural methods as well as light and scanning electron microscopy. Dilution‐plate counts of fungi [10⁶/g dry weight (gdw) stone] and heterotrophic bacteria (10⁵/gdw) were as high as those reported for soils. Counts of algae (10⁶/gdw) were somewhat higher than those for soils. Biomass expressed as phospholipid phosphate concentration (PLP; 115–137 nmol PLP/gdw) was nearly 10 times that of Antarctic sandstone, at least twice that in an architectural red sandstone, and approached that found in soils. Amounts of chlorophyll a were higher than those for the Antarctic sandstone (known to contain algae and cyanobacteria) and were at the lower end of the range reported for soils. The high biomass is supported by photosynthetic carbon input, and hypotheses are presented that describe the role of these epi‐ and endolithic communities in the weathering of their substrata.     

Weathering of Granite from the Damma Glacier Area: The Contribution of Cyanogenic Bacteria

The dissolution potential of five cyanogenic bacteria was studied at 25°C during 32 days using granite material from the Damma glacier (Central Alps, Switzerland) as the sole source of nutrients. The bacterial species Pseudomonas fluorescens and Pseudomonas sp. CCOS 191 were the most effective to exudate various organic acids and consequently mobilized Fe. The molecular mechanisms include both, proton-promoted and ligand-promoted dissolution, preferentially at pH below 5 and in the pH range between 5.0 and 5.8, respectively. In addition, bacterially produced cyanide plays a minor role through the formation of soluble hexacyanoferrate complexes. To our knowledge, this study is the first that reveals the direct measurement of metal-cyanide complexes formed during biotic granite weathering.     

NAD+/NADH homeostasis affects metabolic adaptation to hypoxia and secondary metabolite production in filamentous fungi*

Filamentous fungi are used to produce fermented foods, organic acids, beneficial secondary metabolites and various enzymes. During such processes, these fungi balance cellular NAD⁺:NADH ratios to adapt to environmental redox stimuli. Cellular NAD(H) status in fungal cells is a trigger of changes in metabolic pathways including those of glycolysis, fermentation, and the production of organic acids, amino acids and secondary metabolites. Under hypoxic conditions, high NADH:NAD⁺ ratios lead to the inactivation of various dehydrogenases, and the metabolic flow involving NAD⁺ is down-regulated compared with normoxic conditions. This review provides an overview of the metabolic mechanisms of filamentous fungi under hypoxic conditions that alter the cellular NADH:NAD⁺ balance. We also discuss the relationship between the intracellular redox balance (NAD/NADH ratio) and the production of beneficial secondary metabolites that arise from repressing the HDAC activity of sirtuin A via Nudix hydrolase A (NdxA)-dependent NAD⁺ degradation.     

In vitro studies on the effects of fungicides on beneficial fungi of peach twig mycoflora

In vitro studies were carried out to investigate a possible integrated use of chemical and biological means to control the peach twig blight pathogen,Monilinia laxa. Three fungal antagonists ofM. laxa (Penicillium purpurogenum, Penicillium frequentans andEpicoccum nigrum) and six fungicides (vinclozolin, iprodione, thiram, captan, benomyl and thiophanate-methyl) were used in the study. Sensitivity of the fungal isolates to the fungicides was determined in vitro by calculating ED50 values. Benomyl and thiophanate-methyl were the most fungitoxic compounds and captan was the least fungitoxic.M. laxa andP. purpurogenum were the most sensitive to all chemicals tested, whileE. nigrum andP. frequentans presented bigger differences in their sensitivity to chemicals compared toM. laxa. E. nigrum was consistently less sensitive to benomyl (ED50=2.26 ppm), thiophanate-methyl (ED50=9.61 ppm) and vinclozolin (ED50=3.89 ppm) than the other fungi.P. frequentans was less sensitive to captan, vinclozolin, iprodione, thiophanate-methyl and thiram thanM. laxa (8, 7, 5, 4 and 2 times respectively). These results suggest thatE. nigrum andP. frequentans could be successfully used in an integrated control programme that combines biological and chemical methods.     

Mineralogical transformation and neoformation in granite caused by the lichens Tephromela atra and Ochrolechia parella

Tephromela atra and Ochrolechia parella are among the most abundant lichens colonizing granitic monuments in the region of Galicia (northwest Spain). In this work, their interaction with a two-mica granite used in the construction of the Toxosoutos Monastery (Noia, Galicia) was studied, using optical microscopy, scanning electron microscopy (with back-scattered-electron and energy-dispersive X-ray detection), X-ray diffractometry and atomic absorption spectroscopy to evaluate their physical, mineralogical and chemical effects. Both lichens contributed to physical weathering by penetrating intermineral voids and mineral cleavage planes, disaggregating the rock and entrapping the loosened mineral grains in their thalli. Significant chemical and mineralogical weathering also occurred, including depletion of potassium from biotite, transformation of this mica into hydroxyaluminium-vermiculite, and neoformation of whewellite and calcite in the lichen thalli. Neoformation of these calcium minerals on a calcium-poor rock such as granite is noteworthy, and this is the first time calcium carbonate has been detected within a lichen colonizing a granitic rock. Precipitation of the calcium carbonate was attributed to the local pH in the thalli having been raised due to release of sodium from nearby plagioclase during weathering.     

Endolithic Phototrophs in Built and Natural Stone

Lichens, algae and cyanobacteria have been detected growing endolithically in natural rock and in stone buildings in various countries of Australasia, Europe and Latin America. Previously these organisms had mainly been described in natural carbonaceous rocks in aquatic environments, with some reports in siliceous rocks, principally from extremophilic regions. Using various culture and microscopy methods, we have detected endoliths in siliceous stone, both natural and cut, in humid temperate and subtropical climates. Such endolithic growth leads to degradation of the stone structure, not only by mechanical means, but also by metabolites liberated by the cells. Using in vitro culture, transmission, optical and fluorescence microscopy, and confocal laser scanning microscopy, both coccoid and filamentous cyanobacteria and algae, including Cyanidiales, have been identified growing endolithically in the facades of historic buildings built from limestone, sandstone, granite, basalt and soapstone, as well as in some natural rocks. Numerically, the most abundant are small, single-celled, colonial cyanobacteria. These small phototrophs are difficult to detect by standard microscope techniques and some of these species have not been previously reported within stone.     

Pioneering fungi from the Damma glacier forefield in the Swiss Alps can promote granite weathering

Fungi were isolated from fine granitic sediments, which were collected at 15 sampling points within a 20 m × 40 m area in front of the Damma glacier in the central Swiss Alps. From the 45 fungal isolates grown on nutrient-rich agar media at 4 °C, 24 isolates were selected for partial sequencing and identification based on the small subunit ribosomal DNA. Sequencing data revealed that the isolated fungi represented three fungal phyla and 15 species. The weathering potential of 10 of the 15 fungal species was tested with dissolution experiments using powdered granite material (<63 μm). The results showed that the zygomyceteous species Mucor hiemalis, Umbelopsis isabellina and Mortierella alpina dissolved the granite powder most efficiently due to the release of a variety of organic acids, mainly citrate, malate and oxalate. In particular, the high concentrations of Ca, Fe, Mg and Mn in the solutions clustered well with the high amounts of exuded citrate. This is the first report on fungi that were isolated from a non-vegetated glacier forefield in which the fungi's capabilities to dissolve granite minerals were examined.     

Effects of organic and inorganic salts on docosahexaenoic acid (DHA) production by a locally isolated strain of Thraustochytrium sp. T01

Abstract

The traditional source for docosahexaenoic acid (DHA) i.e. fish oil is currently being replaced by microbial sources due to the unpleasant odor and the risk of chemical contamination of fish. Thraustochytrium sp., marine microalgae-like protist is a known source of DHA. In our previous study, we reported a high yielding strain, T01, of Thraustochytrium sp. for DHA production isolated from the mangroves of South India. This strain shows promising yields of biomass and DHA. Shake flask study of T01 yielded 6.17?±?0.04 gL^?1 of DHA. In the present work, we report the effects of organic and inorganic salts on DHA production. Addition of organic salts such as sodium acetate, pyruvate, citrate and malate led to increase in the DHA content in T01 strain. The DHA content increased by 40–46% on addition of sodium salts of organic acids, while inorganic phosphates increased DHA by 33%. The total lipid content also increased (28–33%) with salts of organic acids and 28% with phosphate, but not as much as the increase in DHA. Addition of all the salts together did not show a significant increase in lipid and DHA contents as compared to the addition of individual salts.     

Use of strontium isotopes and foliar K content to estimate weathering of biotite induced by pine seedlings colonised by ectomycorrhizal fungi from two different soils

Pinus sylvestris seedlings, colonised by ectomycorrhizal (EM) fungi from either of two different soils (untreated forest soil and a limed soil from a clear cut area), were grown with or without biotite as a source of K. The biotite was naturally enriched in ⁸⁷Sr and the ratio of ⁸⁷Sr/ ⁸⁶Sr in the plant biomass was estimated and used as a marker for biotite weathering and compared to estimates of weathering based on foliar content of K. Different nutrient regimes were used to expose the seedlings to deficiencies of K with and without an application of nitrogen (NH₄NO₃) in excess of seedling demand. The seedlings were grown for 220 days and the elemental composition of the shoots were analysed at harvest. The EM colonisation was followed by analysing the concentration of ergosterol in the roots and the soils. Bacterial activity of the soil was estimated by the thymidine incorporation technique. The concentration of organic acids in the soil solution was measured in the soil in which seedlings colonised by EM fungi from the untreated forest soil were grown. It was found that seedlings colonised by EM fungi from untreated forest soil had taken up more K in treatments with biotite addition compared to seedlings colonised by EM fungi from the limed forest soil (p<0.05). Seedlings from untreated forest soil had larger shoots and contained more K when grown with biotite compared to KCl as K source, indicating that biotite had a stimulatory effect on the growth of these seedlings which was not related to K uptake. Seedlings from the limed soil, on the other hand, had similar foliar K content when grown with either biotite or KCl as K source. The larger uptake of K in seedlings from untreated forest soil was not an effect of a more developed EM colonisation of the roots since seedlings from the limed soil had a higher ergosterol concentration both in the soil and in the roots. Nutrient regimes had no significant influence on the total uptake of K but the ⁸⁷Sr/ ⁸⁶Sr isotope ratio in the plant biomass indicated that seedlings grown with excess nitrogen supply had taken up proportionally less Sr from the biotite (1.8% of total Sr content) compared to seedlings grown with a moderate nitrogen supply (5.0%). Furthermore, seedlings grown with excess nitrogen supply had a reduced fungal colonisation of roots and soil and bacterial activity was lower in these soils. The ⁸⁷Sr/ ⁸⁶Sr ratio in the plant biomass was positively correlated with fungal colonisation of the roots (r ²=0.98), which may indicate that the fungus was involved in releasing Sr from the biotite. Uptake of K from biotite was not related to the amount of organic acids in the soil solution. Oxalic acid was positively related to the amount of ergosterol in the root, suggesting that oxalic acid in the soil solution originates from the EM symbionts. The accuracy of the estimations of biotite weathering based on K uptake by the seedlings in comparison with the ⁸⁷Sr/⁸⁶Sr isotope ratio measured in the shoots is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.