Phylogenetic and chemical studies in the potential psychotropic species complex of Psilocybe atrobrunnea with taxonomic and nomenclatural notes.

Five Psilocybe species with unresolved systematic position (P. atrobrunnea, P. laetissima, P. medullosa, P. pelliculosa, and P. silvatica) were investigated using four molecular markers (EF1-α, ITS, LSU, and IGS). Phylogenetic analysis revealed that with the exception of P. laetissima, which is now rightfully classified in the genus Leratiomyces, all investigated species belong to Psilocybe sect. Psilocybe. For the first time, psychotropic compounds psilocin and psilocybin were detected in P. medullosa using gas chromatography-mass spectrometry. On the contrary, neither psilocin, nor psilocybin was detected in P. atrobrunnea and negative results were also obtained from mycelia grown in vitro on tryptamine/tryptophan-amended media. These results strongly suggest that biosynthesis of these alkaloids was lost in P. atrobrunnea. With the exception of minor differences detected in EF1-α marker, all sequences of American and European collections of P. atrobrunnea were identical. On the other hand, a thorough nomenclatural study revealed that the name P. atrobrunnea must be considered dubious; the oldest available candidate name, P. fuscofulva, was therefore adopted. The molecular data suggests that morphologically identical American P. silvatica and European P. medullosa likely represent distinct species; epitypes of both taxa were therefore designated.

Local distribution of ectomycorrhizae-associated basidiomycetes in forest soil correlates with the degree of soil organic matter humification and available electrolytes.

Spatial distribution of ectomycorrhizae-associated basidiomycetes was determined in oakbirch forest using terminal restriction fragment length polymorphism (T-RFLP) analysis. The data were correlated with actual soil humidity, pH, electric conductivity of the soil extract, absorbance A(465) and A(665) of water and alkali soil extracts and with the ratio A(465)/A(665) (parameter A4/A6). Natural non-homogeneity of the soil parameters was used as experimental gradient. Distance-based redundancy analysis of the T-RFLP data (with soil parameters being taken as environmental parameters) provided significant results when ITS1F-terminanted restriction fragments were analyzed. Among other fungi, a Mycena galericulata related fungus was observed to correlate negatively with A4/A6, indicating its association with highly humified soil organic matter. Positive association of other, unidentified fungi with A4/A6 was also observed. Several other unidentified fungi negatively correlated with electric conductivity of the soil extract. The results may explain nonhomogeneity of the spatial distribution of the fungi associated with ectomycorrhizae as a result of their interaction with non-homogeneous soil environment.

Microbial chlorination of organic matter in forest soil: investigation using 36Cl-chloride and its methodology.

Chloride, which comes into the forest ecosystem largely from the sea as aerosol (and has been in the past assumed to be inert), causes chlorination of soil organic matter. Studies of the chlorination showed that the content of organically bound chlorine in temperate forest soils is higher than that of chloride, and various chlorinated compounds are produced. Our study of chlorination of organic matter in the fermentation horizon of forest soil using radioisotope 36Cl and tracer techniques shows that microbial chlorination clearly prevails over abiotic, chlorination of soil organic matter being enzymatically mediated and proportional to chloride content and time. Long-term (>100 days) chlorination leads to more stable chlorinated substances contained in the organic layer of forest soil (overtime; chlorine is bound progressively more firmly in humic acids) and volatile organochlorines are formed. Penetration of chloride into microorganisms can be documented by the freezing/thawing technique. Chloride absorption in microorganisms in soil and in litter residues in the fermentation horizon complicates the analysis of 36Cl-chlorinated soil. The results show that the analytical procedure used should be tested for every soil type under study.

Influence of mineral and organic fertilization on soil fungi, enzyme activities and humic substances in a long-term field experiment.

Changes in microfungal communities, fungal activities and humic substances (HS) in agricultural soils kept under different fertilization regimes were observed and their causal relationships were investigated in a long-term field experiment. Fertilization did not change the abundance of HS-utilizing microfungi and, except for organic amendment alone, total culturable microfungi were also unaffected by this factor. Organic fertilization increased activities of manganese peroxidase (MnP) and proteinase, but decreased endo-1,4-beta-glucanase activity compared to the corresponding control without organic fertilization. In soils treated with mineral fertilizers, the activities of MnP, endo-1,4-beta-glucanase and proteinase were higher than in control without any mineral treatment. Both the aromaticity of fulvic acid and the molar mass of humic acid was lower in soil with organic fertilization, which may be a result of oxidative degradation mediated by higher MnP activity observed in treatments with organic fertilization.

Humic acid-like material from sewage sludge stimulates culture growth of ectomycorrhizal fungi in vitro.

Significant effects of humic acid-like material (HALM) extracted from sewage sludge on dry matter production of cultures of ectomycorrhizal basidiomycetes were found in vitro. Mycelial growth of the majority of isolates tended to increase in the presence of the HALM and this effect was significant for 6 isolates. Strongest stimulation was observed in the case of Amanita muscaria, Leccinum aurantiacum and Lactarius deterrimus. The results suggest that the HALM can be used as an additive to media for cultivation of ectomycorrhizal basidiomycetes.

Fluorescence spectroscopy: a tool to characterize humic substances in soil colonized by microorganisms?

The ability of eight soil microfungal species, Alternaria alternata, Clonostachys rosea f. rosea, Exophiala cf. salmonis, Fusarium cf. coeruleum, Fusarium redolens, Paecilomyces lilacinus, Penicillium canescens and Phoma sp., and two known basidiomycete humic acid (HA) degraders, Trametes versicolor and Phanerochaete chrysosporium, to modify fluorescence properties of fulvic acids (FA) and/or HAs was determined. Effects of minerals and/or glucose on the modifications were examined. FA purified on polyvinyl-polypyrrolidone (PVPP) chromatography column was used. Purification of FA on PVPP column removed the low-molar-mass FA-structural components and excess of extractant (NaOH) used during FA preparation. Excitation spectra of FA entering the purification, purified FA and the removal solution indicate that organic compounds rich in carboxylic groups dominate in the removal solution and higher content of phenolic groups is a characteristic of purified FA. Many microfungal species shifted the emission maximum (measured at 470 and 468 nm of excitation wavelength) of FA, and also HA to longer wavelengths. The opposite effect (shift of the HA emission maximum to shorter wavelengths) of microfungi was observed for HA complemented by glucose. Depending on the presence of glucose in the medium, most microfungi changed also the shape of the emission spectra of HA and FA and the excitation spectra of FA. HA excitation spectrum measured at 590 nm of emission wavelength was significantly affected by the presence of glucose. Mineral ions caused a minor shift in the position of excitation maximum (measured at 590 nm of emission wavelength) toward longer wavelengths.

Organic and mineral fertilization, respectively, increase and decrease the development of external mycelium of arbuscular mycorrhizal fungi in a long-term field experiment.

Effects of long-term mineral fertilization and manuring on the biomass of arbuscular mycorrhizal fungi (AMF) were studied in a field experiment. Mineral fertilization reduced the growth of AMF, as estimated using both measurements of hyphal length and the signature fatty acid 16:1omega5, whereas manuring alone increased the growth of AMF. The results of AMF root colonization followed the same pattern as AMF hyphal length in soil samples, but not AMF spore densities, which increased with increasing mineral and organic fertilization. AMF spore counts and concentration of 16:1omega5 in soil did not correlate positively, suggesting that a significant portion of spores found in soil samples was dead. AMF hyphal length was not correlated with whole cell fatty acid (WCFA) 18:2omega6,9 levels, a biomarker of saprotrophic fungi, indicating that visual measurements of the AMF mycelium were not distorted by erroneous involvement of hyphae of saprotrophs. Our observations indicate that the measurement of WCFAs in soil is a useful research tool for providing information in the characterization of soil microflora.

Hyphal growth and mycorrhiza formation by the arbuscular mycorrhizal fungus Glomus claroideum BEG 23 is stimulated by humic substances.

Effects of humic substances (humic acid or fulvic soil extract) or saprophytic microorganisms (Paecilomyces lilacinus and an unidentified actinomycete) on growth of mycelium and mycorrhiza formation by Glomus claroideum BEG23 were studied in a hydroponic system. Humic substances stimulated root colonization and production of extraradical mycelium by the mycorrhizal fungus. Both humic and fulvic acids tended to decrease populations of culturable bacteria and fungi in the cultivation system, indicating a moderately antibiotic activity. The addition of saprophytic microorganisms able to use humic substances to the cultivation system further stimulated the development of the mycorrhizal fungus. However, stimulation of G. claroideum was also observed when the saprophytic microorganisms were heat-killed, suggesting that their effect was not linked to a specific action on humic substances. The results indicate that humic substances may represent a stimulatory component of the soil environment with respect to arbuscular mycorrhizal fungi.

Microbiological aspects of determination of trichloroacetic acid in soil.

Soils have been shown to possess a strong microbial trichloroacetic acid (TCA)-degrading activity. High TCA-degradation rate was also observed during soil extraction with water. For correct measurements of TCA levels in soil all TCA-degrading activities have to be inhibited immediately after sampling before analysis. We used rapid freezing of soil samples (optimally in liquid nitrogen) with subsequent storage and slow thawing before analysis as an efficient technique for suppressing the degradation. Frozen soil samples stored overnight at -20 degrees C and then thawed slowly exhibited very low residual TCA-degrading activity for several hours. Omitting the above procedure could lead to the confusing differences between the TCA levels previously reported in the literature.

Trichloroacetic acid in Norway spruce/soil-system. II. Distribution and degradation in the plant.

Independently from its origin, trichloroacetic acid (TCA) as a phytotoxic substance affects coniferous trees. Its uptake, distribution and degradation were thus investigated in the Norway spruce/soil-system using 14C labeling. TCA is distributed in the tree mainly by the transpiration stream. As in soil, TCA seems to be degraded microbially, presumably by phyllosphere microorganisms in spruce needles. Indication of TCA biodegradation in trees is shown using both antibiotics and axenic plants.

Response of saprotrophic microfungi degrading the fulvic fraction of soil organic matter to different N fertilization intensities, different plant species cover and elevated atmospheric CO2 concentration.

The response of the cenosis composition of soil saprotrophic microfungi able to utilize the fulvic fraction of soil organic matter to increased concentration of atmospheric carbon dioxide, plant species cover quality and different levels of nitrogen fertilization was determined under field conditions in a free-air carbon dioxide enrichment experiment. Twenty-nine species of microfungi were isolated from the tested soil. The effects of CO2 enrichment and plant species cover were not significant. Nitrogen fertilization was identified as the only significant factor inducing changes in the abundance of soil microorganisms. This was reflected in a relatively low value of quantitative Sørensen similarity index on comparing fertilized and unfertilized treatments and in 2-way ANOVA of total CFU counts. Some differences were observed in species diversity between the two variants of all treatments. No association between microfungi and the factors under study was found by using the Monte Carlo Permutation test in redundancy analysis.

Effect of Chalara longipes on decomposition of humic acids from Picea abies needle litter.

The effect of the saprotrophic ascomycete Chalara longipes on the decomposition of humic acids was determined in a cultivation experiment. The fungus was incubated in liquid cultures in a full-strength (F system) and an organic nitrogen-free medium (F-N system), both amended with pure humic acids isolated from spruce forest litter. Fungal biomass production was highest in the F system with humic acids and lowest in the F-N system, the effect of organic nitrogen and humic acids being significant (p < 0.05). The presence of organic nitrogen seems to be essential for growth. The fungus utilized humic acids; molar mass distribution in media obtained by gel-permeation chromatography showed decrease in the humic acids fraction. Decolorization of the media reached 75 and 64 % in the F and F-N systems, respectively. The molar mass of humic acids probably also decreased as estimated from the increase in the absorbance A465/A665 (A4/A6) ratio. It is assumed that the utilization of humic acids may be mediated by the production of organic acids (as their components) and production of some oxidative enzymes.

Long-term fertilization affects the abundance of saprotrophic microfungi degrading resistant forms of soil organic matter.

The effect of mineral and organic fertilization on the occurrence of soil microorganisms was determined in a field experiment. The colony-forming unit counts of saprotrophic microfungi, when estimated on a silicate gel medium containing fulvic acid as a sole carbon source, increased significantly with increasing doses of mineral and organic fertilization. Partial correlation analysis indicated that, unlike bacteria and actinomycetes, microfungi utilizing fulvic acid were significantly associated with soil organic carbon. No significant effects on bacteria and microfungi counted on common microbiological media were observed but counts of actinomycetes increased in a manured soil extensively fertilized by a mineral fertilizer. Fulvic acid utilizing microfungi, which are associated with areas rich in organics, play possibly the main role in mineralization of resistant forms of soil organic matter.

Trichloroacetic acid in Norway spruce/soil-system. I. Biodegradation in soil.

Trichloroacetic acid (TCA) as a phytotoxic substance affects health status of coniferous trees. It is known as a secondary air pollutant (formed by photooxidation of tetrachloroethene and 1,1,1-trichloroethane) and as a product of chlorination of humic substances in soil. Its break-down in soil, however, influences considerably the TCA level, i.e. the extent of TCA uptake by spruce roots. In connection with our investigations of TCA effects on Norway spruce, microbial processes in soil were studied using 14C-labeling. It was shown that TCA degradation in soil is a fast process depending on TCA concentration, soil properties, humidity and temperature. As a result, the TCA level in soil is determined by a steady state between uptake from the atmosphere, formation in soil, leaching and degradation. The process of TCA degradation in soil thus participates significantly in the chlorine cycle in forest ecosystems.

The effect of fulvic acids on the toxicity of lead and manganese to arbuscular mycorrhizal fungus Glomus intraradices.

The effect of fulvic acids (FA) on arbuscular mycorrhizal (AM) fungi and on the toxicity of lead and manganese toward these symbionts were demonstrated in vitro. Incubation of root segments colonized with the AM fungus Glomus intraradices in undiluted fraction of FA (813 mg/L carbon) decreased an outgrowth of intraradical hyphae. Diluted FA solutions (< 271 mg/L C) did not influence the proportion of root segments bearing proliferating hyphae; solution containing 27.1 mg/L C even increased the proliferation. A decrease of heavy metal toxicity toward the fungus was observed when FA (81.3 mg/L C) were added to the solutions containing higher concentrations (< or = 100 mumol/L) of Mn and Pb; the positive effect of FA was not significant at higher concentrations of metals (0.5 mmol/L). A short-term cultivation of six different saprophytic microorganisms--three actinomycetes and three filamentous fungi--on the FA media (81.3 mg/L C) did not result in a modification of hyphal proliferation from the root segments subsequently incubated in these media.

Magnesium ions alleviate the negative effect of manganese on Glomus claroideum BEG23.

The phytotoxicity of excessive levels of manganese (Mn), an essential micronutrient, can be alleviated significantly by a high supply of magnesium (Mg) ions to plants. A similar interaction of these two elements in the development of arbuscular mycorrhizal (AM) fungi has been verified in two experimental systems. In in vitro experiments, an outgrowth of hyphae from excised, surface-disinfected root segments colonised with Glomus claroideum BEG23 was measured after 5 days incubation in liquid media. When only Mn ions were present in the media at higher concentrations (>0.05 mM), the growth of hyphae from root segments was reduced significantly. Addition of magnesium sulphate to the incubation solution reduced the inhibitory effects of Mn on hyphal growth. Alleviation of Mn toxicity by Mg ions observed in in vitro experiments was verified also for the symbiotic association between G. claroideum and maize as a host plant in a hydroponics sand culture experiment.

Organic fertilization changes the response of mycelium of arbuscular mycorrhizal fungi and their sporulation to mineral NPK supply.

The synergetic effect of organic (cow manure) and mineral fertilization on the development arbuscular mycorrhizal (AM) fungi was demonstrated. The length of AM mycelium and sporulation were used as sensitive markers of the physiological state of soil AM fungal population. In manured treatments, both parameters increased in proportion with increasing mineral fertilization. In unmanured soil, the opposite trend was observed for the length of AM hyphae, which decreased with increasing mineral fertilization. Correlation analysis showed the dependence of length of AM hyphae and sporulation on soil available phosphorus. The correlation was negative in soil with no mineral fertilization and positive in soil supplied with luxury doses of mineral fertilizer.

Soil Saprotrophic Microfungi Associated with Roots of Calamagrostis epigeios on an Abandoned Deposit of Toxic Waste from Smelter Factory Processing Pyrite Raw Materials.

Saprotrophic microfungi associated with roots of Calamagrostis epigeios growing in industrial waste contaminated with high concentrations of heavy metals (manganese, iron, zinc) were isolated on three different media. The isolates were obtained from three plots colonized by plant cenoses, representing three different succession series of plant cover: initial, intermediate, and advanced. A total of 60 species of saprotrophic microfungi were found in the roots during three samplings. In addition, unidentified dark and white sterile mycelia were frequently isolated. Trichoderma koningii was frequent in the roots formed under the advanced phytocenose. The abundance of dark sterile mycelium gradually decreased with progressing succession of the plant cover. Chrysosporium merdarium was rarely observed in the roots under the intermediate and advanced plant cenoses. Multivariate redundancy analysis showed that the effect of succession stage of phytocenoses on incidence of saprotrophic microfungi in mycocenoses present in roots was statistically significant. When the similarity of root mycocenoses of the three plots was studied using the similarity coefficients, the results proved that the similarities between the initial and intermediate or intermediate and advanced mycocenoses were higher than that between the initial and advanced mycocenoses. This corresponds to a different speed of succession of the mycocenoses. The results suggest that the dark sterile mycelia, abundant at slowly developing plots, play a dominant role during the first stages of succession where the plant cover is poor.

Effect of spermine on proliferation of hyphae of Glomus fistulosum, an arbuscular mycorrhizal fungus, in maize roots.

Effects of two oligoamines, putrescine and spermine, on proliferation of intraradical hyphae in surface disinfected root segments were studied under axenic conditions in vitro. No significant effects of putrescine were observed. Spermine significantly stimulated hyphal growth at a concentration of about 1.5 mumol/L. High concentration (> 150 mumol/L) caused a strong inhibition of hyphal growth and of the percentage of root segments bearing proliferating hyphae. DL-alpha-difluoromethylornithine, a metabolic inhibitor of polyamine synthesis, caused a significant inhibition of proliferation of the hyphae only in the presence of 2 mumol/L spermine.

Effect of soil bacteria on hyphal growth of the arbuscular mycorrhizal fungus Glomus claroideum.

The effect of 46 bacterial strains isolated from tilled and non-tilled soils collected at 3 localities on the growth of intraradical hyphae of the arbuscular mycorrhizal (AM) fungus Glomus claroideum was demonstrated. A larger number of stimulatory bacterial isolates was obtained from tilled soils, but the bacteria showing the strongest stimulation of hyphal growth were isolated from a soil that had not been cultivated. Isolates obtained from hyphae of AM fungi showed no substantial stimulatory effects, but produced more uniform effects on hyphal growth than the isolates of bacteria obtained from soil. Bacterial cenoses present in 3 different soils differ significantly in their effects on AM fungi.